Radioimmunodetection: technical problems and methods of improvement

European Journal of Surgical Oncology 1999; 25: 529–539

Radioimmunodetection: technical problems and methods of improvement I. G. Sergides, R. C. T. Austin and M. C. Winslet Royal Free Hospital, London, UK

Radioimmunodetection (RAID) is a technique which uses radiolabelled antibodies to visualize tumours, taking advantage of antigens preferentially expressed by malignant tissue. Gamma radiation emitted by radioisotopes can be detected using an external gamma camera (RAID), or intraoperatively with a hand-held Geiger counter (radioimmunoguided surgery, RIGS). RAID has significant inherent problems. Many have been overcome as a result of nearly 50 years of research, and others still remain as obstacles precluding the routine use of the technique. This article summarizes the technical limitations of RAID and outlines the relative successes of the methods evolved to overcome them.  1999 Harcourt Publishers Ltd

Key words: adult; radioimmunodetection; technical.

Introduction A range of agents can be targeted to tumours using antibodies specific for antigens preferentially expressed by malignant tissue. The most experience with this approach has been gained with the use of radio-isotopes, leading to the technique known as radioimmunolocalization (RIL). After performing a skin test to exclude allergy to the preparation, a radio-isotope labelled antibody is administered to a given patient. Once localized to the tumour tissue, the radio-isotope (and hence the sites of malignancy) can be detected with an external gamma camera—a process known as immunoscintigraphy or radioimmunodetection (RAID) (Fig. 1). Alternatively, lower energy isotopes can be detected using a hand-held Geiger counter during an operative procedure: radioimmunoguided surgery (RIGS). Although this conceptually simple technique has been investigated and refined for almost 50 years it still has inherent limitations. These are listed below and possible methods of overcoming these limitations with their relative successes are then discussed. RAID involves the use of anti-tumour antibodies. These antibodies are specific for tumour associated antigens (TAAs) produced by a particular malignancy. TAAs have minimal or no expression in benign adult tissues and hence are used as target molecules for this procedure. Some TAAs, such as SM3 for ovarian1 and breast cancer2 and PR1A3 for colorectal cancer, are epithelial surface antigens lying on the inner surfaces of cells, thus being exposed to the circulation only by neoplastic architectural disruption. Another group are the oncofetal antigens such as carcinoembryonic antigen (CEA) and alpha-fetoprotein Correspondence to: Prof. M. C. Winslet, Department of Surgery, 9th Floor, Royal Free Hospital, Pond Street, Hampstead, London NW3 2QG, UK. 0748–7983/99/050529+11 $12.00/0

(AFP). CEA is expressed in many malignant conditions, but can be elevated in the blood of patients with benign and inflammatory lesions of certain organs, whereas AFP is primarily raised in germ cell and hepatocellular carcinomas. Other TAAs include tumour-associated glycoprotein-72 (TAG-72) which is not expressed to a significant amount by normal tissue excepting secretory phase endometrium and transitional colonic mucosa. TAG-72 is, however, produced by 95% of colorectal cancers, 70% of breast cancers and 90% of ovarian cancers.

Technical limitations of RAID Antibody RAID was originally carried out using affinity-purified polyclonal antibodies (Pabs). These are essentially diverse cocktails of antibodies which require laborious purification before they can be used. In 1975 Milstein and Kohler3 described a method of preparing large numbers of specific monoclonal antibodies (Mabs). It was thought that the single epitope restriction of Mabs would make it possible to target tumours more accurately and with minimal crossreactivity. However this only eliminates non-specific binding if the relevant epitope is unique to the tumour, and polyclonal serum can be as epitope-specific as monoclonal serum though at the cost of losing the highest affinity antibody. Mabs are currently not entirely sensitive for malignant tissue. For example, Mab B72.3 recognizes TAG-72 and has been used extensively for the detection of several malignancies including breast, lung, ovarian and colorectal. However B72.3 does not react with up to 20% of colorectal cancers and so immediately a sensitivity below 80% is inherent in the technique.4 Immunoglobulins are large  1999 Harcourt Publishers Ltd

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Educational section Radiolabelled Mab

Cancer cell

Gammacamera

Fig. 1. The principles of radioimmunolocalization.

molecules and so have difficulty in penetrating large tumour masses, especially if poorly vascularized and in the early stages of the malignant process. Furthermore, as the movement of macromolecules through the interstitial space is dependent upon pressure gradients between the vascular and extravascular spaces, the relatively high interstitial pressure at the centre of most tumours opposes antibody migration towards the tumour matrix.5

Antigen Circulating Many TAAs expressed by tumours are secreted into the bloodstream where they may theoretically interfere with RAID. Circulating antigen may neutralize antibody by occupying its binding sites or causing large antibody–antigen complexes to form with diminished capacity to diffuse into tumours. Alternatively, complexes may actively be taken up by tumours either by random accumulation of macromolecules or by intratumour phagocyte entrapment of complexes, but these are both non-specific effects. Immune complexes are often taken up by the reticulo-endothelial system hence reducing the availability of antibody for tumour binding, but this may also have some advantages as it can reduce the non-specific blood-pool background count. Animal and human studies have shown that antibody clearance alters after binding to CEA.6,7 Studies in mice carrying tumour xenografts secreting different amounts of CEA and injected with radioactive indium (111In) and iodine (125I) labelled antibodies revealed that raised levels of CEA are associated with a decrease in the levels of circulating radioactivity and at the highest levels of CEA a decrease in tumour uptake is seen.7 The radio-isotopes are deposited in the liver and spleen as shown by high 111In levels. A process of dehalogenation rapidly removes the 125I from the antibody and so there is increased urinary excretion of the isotope (59% for 125I as opposed to 8% for 111In).7 Furthermore, an excess of CEA prevents clearance of immune complexes from the blood stream. It has been suggested that an excess of antibody in the formation of antibody–antigen complexes causes exposure of galactose residues which leads to specific uptake by hepatic galactose receptors. This does not happen in antigen excess.8

Circulating antigen can complicate RAID protocols. When low levels of monoclonal immunoglobulin (IgG) against melanoma p97 antigen were intravenously administered most of the radioactivity was found to be complexed to circulating antigen6. Although the authors did not comment on the pharmacodynamic effects, this caused them to increase the dose by 200 times.6 Also, early studies with anti-prostate specific antigen (PSA) antibodies failed when the serum PSA levels were high due to complex formation. Other clinical studies have shown that circulating antigen has only little effect on RAID, especially in the case of CEA. Even a CEA blood titer of >2000 ng/ml with about 50% complexation does not prevent imaging,9,10 and indeed there is evidence to suggest a positive correlation between circulating CEA levels and the outcome of RAID on colorectal, cervical and lung cancers, though not for ovarian cancers.11–13 Suggested reasons for this include masking of CEA by patient’s autoanti-CEA antibodies, or differential expression of CEA determinants in blood and tumour.14 It has certainly been suggested that circulating CEA is not as haptenic as cell-bound CEA for whatever reason and therefore imaging can still proceed.15 It has been shown in humans that increasing levels of CEA lead to increasing formation of antibody–CEA complexes. In contrast to Goldenberg et al.10 however, some studies have shown no correlation between CEA levels or the number of complexes formed and positive results on immunoscintigraphy.16 A correlation between the blood levels of TAG-72 in patients and the results of RAID has, however, been shown, the sensitivity increasing from 63 to 85% with positive blood levels (>10 U/ml) as opposed to negative levels.17 Cellular Tumour cells have developed evasive mechanisms to prevent immunological attack. Exposure of T and B lymphocytes to TAAs may induce clonal selection, allowing immunocompetent effector cells to proliferate, leading to clonal anergy and downregulation of immunogenic capacity.18,19 In this way disseminated extracellular TAA may induce T and B cell tolerance. One characteristic of the majority of tumour cell populations, especially solid tumours, is the heterogeneous expression of surface antigens. This intratumoural variation takes place at

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Educational section (3) Minimal TAA expression by normal tissue

(1) Fc binding

(2) Non-specific binding Normal tissue

Fig. 2. Mechanisms of increased background count due to non-specific uptake of antibody by normal tissue.

immunohistochemical, morphological and molecular biological levels. Cells which express little or no antigen may escape detection due to their failure to bind the Mab. Therefore the sub-optimal sensitivity of Mabs is due partly to a combination of the immune system’s tolerance of the tumour and tumour heterogeneity.20,21 Background radioactivity After RIL the ratio of radioactivity taken up by the tumour to that in the surrounding tissues can be calculated and is called the tumour to background ratio (T/B). The higher the T/B, the better the contrast. A high background level of radiation due to radioactivity in normal tissues reduces the T/B which reduces the success of imaging. Normal tissues accumulate radioactivity in several ways. Non-specific uptake Antibodies can bind to normal tissues in a non-specific manner. This does not have to occur at the binding site of the antibody, the stem (Fc fragment) can also be bound (Fig. 2). Antibody may also be nonspecifically taken up by areas of inflammation. Metabolism Concentration of radiolabelled antibody occurs at the sites of processing and excretion. These sites include the liver, the kidney, the bladder and the reticulo-endothelial system. Therefore, tumours in the kidney, for example, which both metabolizes small antibody fragments and excretes the radiolabel, will be obscured. Circulation Administered radioactivity remains in the circulation, hence large volumes of blood such as those found in the heart and aorta cause a high background count. This is largely dependent on the plasma half-life of the Mab and the radiolabel. Unconjugation The radio-isotope can become unconjugated from the antibody and free radio-isotope may be concentrated at the above sites. Free radioactive iodine can be concentrated in the thyroid gland, and to avoid this patients are treated with potassium iodide before administration of the radioantibody.

HAMA

Immune response

Fig. 3. Human anti-mouse antibody (HAMA) response to Fc portion of Mab.

Radiolabel A radiolabel with a long half-life and which emits high-energy gamma radiation will decrease the T/B. Some radiolabels are more susceptible to unconjugation as mentioned above. Shed antigen If an antigen is not fixed to the tumour but appears in the lymphatics, nodal involvement may be misdiagnosed.1 The consequence of this and the above effects is to increase the background count, hence reducing the efficacy of RAID. Human anti-mouse antibodies Almost all of the Mabs used in RAID are of the IgG class and are murine in origin. The appearance of human antimouse antibodies (HAMA) occurs in 30–50% of patients after one dose of murine Mab and this figure can rise to over 90% with repeated administrations. This response is to the Fc portion of the antibody (Fig. 3). A significant HAMA response will limit the efficacy of RAID in several ways.22 The binding of HAMA to murine antibody could affect antibody function, shorten its half-life, enhance the

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clearance of antibody or possibly induce an allergic serum sickness reaction thus inhibiting its use. In a study by AbdelNabi et al. in 1992,23 40 mg of ZCE-025, an anti-CEA antibody, were injected 4–6 monthly into patients to assess efficacy and safety. HAMA production was found in 30% of patients after one injection increasing to 64% of patients after a second administration. Adverse reactions were noted in 4% of patients with one dose and 16% of patients who received more than one injection. Prophylaxis was given to patients receiving third or further doses. Despite the formation of HAMA no compromise of RAID was found and there was no alteration in the biodistribution of the imaging radioimmunoconjugate.23 The increased risk of reaction on re-exposure has been confirmed with this antibody with three of 17 (18%) patients reacting to repeat injections compared to two of 156 (1%) on first injection.24 However, mechanisms of uptake for antibody–antibody complexes are different from those for antibody–antigen complexes, the former being cleared rapidly in the mouse model,8 which theoretically implies that HAMA could have a greater effect on RAID than circulating antigen.

Improvements in the RAID technique A variety of methods have been developed to counter the inherent flaws in the RAID technique.

Delayed imaging Kinetic differences in specific and non-specific uptake of radioactivity provide an opportunity to image at a time when the T/B is optimal. Background radioactivity falls with time due to excretion and decay of the radioisotope. Tumour radioactivity also falls with time, but not as fast as the background. In an animal model T/B had been reported to rise from 1:1 on the first day post-administration to 9:1 on the 5th day25 and there is a similar effect in patients. Imaging should be carried out when T/B is as high as possible but before absolute tumour levels are too low for accurate detection. Clinically, a gap of 2–4 weeks is usually given between administration of the antibody and RAID, the timing being dependent on the selected antibody, radioisotope and detection method. This unwanted characteristic of RAID may be minimized with the advent of superior imaging agents, which have shorter half-lives as described below.

Background subtraction If the background is labelled using a non-specific antibody the background signal can then be subtracted from the results of RAID.26 Subtraction has led to improved results in patients in whom tumours would not otherwise have been seen,9 though the potential for false results should be remembered.11 The technique also has its disadvantages, such as increasing the dose of radioactivity and complicating the procedure.

Second antibody The background count can also be reduced by removing radioactivity from the circulation. This can be achieved by the use of a second antibody active against the RAID antibody, which can be delivered alone or incorporated into liposomes. After the original antibody has had time to bind to tumour tissue, these liposomes can be administered to the patient in order to bind the radioactive antibody remaining in the blood stream. The liposome–antibody complexes are then cleared from the blood by uptake into the reticulo-endothelial system, leading to concentration in the liver and spleen. When the clearance of imaging antibody from the blood is achieved in this fashion there is also a decrease in the concentration of antibodies in tumours. However there is an overall increase in T/B and no adverse effects have been noted.27 Further assessment of this technique28 has revealed a faster clearance of antibody from the serum, with enhanced gamma-camera imaging of the tumour. Radioactivity is initially concentrated in the liver and clears slowly over the following 24 h. It has been shown both in animal and human studies that liposome-entrapped secondary antibody has no advantages over the antibody used alone.29–31 However, whichever approach is used, this technique removes little of the first antibody from the extravascular space which contains at least 50% of the injected dose and consequently the effect is invariably limited.32 Furthermore, the sequestration of antibody by organs of the reticulo-endothelial system induced by this technique greatly increases their exposure to radioactivity and limits the detection of metastases at these sites.

Targeting agents Fragments RAID may be improved by the production of superior targeting agents to deliver radioactivity to tumour sites. As well as using whole antibodies to target tumours, fragments of antibodies retaining their antigenic specificity can be used. These are created by enzymatic removal of the constant or effector region of the whole antibody (known as the Fc portion) to produce Fab′ or F(ab′)2 fragments (Fig. 4). This has several advantages. Fragments have a shorter circulating half-life, being filtered and excreted in increased amounts by the kidney, and due to their smaller molecular weight they can diffuse faster and more deeply into tumours. The latter has been shown using autoradiography.33,34 Not only do fragments clear from the background more quickly, but the loss of the Fc fragment reduces non-specific antibody binding by Fc receptors on cells. It also reduces the immune response to foreign protein. Indeed, as mentioned above, most of the HAMA response is directed against the constant Fc portion. The Fab′, which is the smallest of the three fragments, is monovalent and therefore has a decreased affinity for the tumour antigen. An advantage of monovalency is that Fab′ fragments do not create large circulating immune complexes if they meet tumour antigen in the blood stream. Studies using an immunoglobulin against human mammary tumours implanted into athymic nude mice comparing Fab′, F(ab′)2 and Mab showed that F(ab′)2 cleared from the blood two to

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Educational section H

H L

Fab portion

{

L

S-

-S-

-S-

S-

}

Fc portion

Papain digestion

S-

-S-

Pepsin digestion

-S-

S-

Fab fragments

S-

-S-

-S-S-

-S-

S-

F(ab' )2 fragments

Fig. 4. Proteolytic fragments of antibody. L, light chain; H, heavy chain.

three times faster than Mab and that Fab′ was considerably faster than both. This was felt to be the reason for the decreased background count noted with fragments. In the case of Fab′ the very fast clearance resulted in large amounts of radioactivity being found in the kidney and bladder which meant that F(ab′)2 was considered the better imaging agent.35 Similar experiments using both an anti-CEA antibody and a non-specific control IgG antibody in hamsters grafted with human colon carcinomas have been undertaken. For both control and anti-CEA antibodies Fab′ is cleared from the blood significantly faster than whole antibody with F(ab′)2 occupying an intermediate position. Unfortunately total tumour uptake is also decreased with the speed of clearance giving values of 0.1, 1.1 and 2.6% injected dose per gram of tumour (%ID/g) for Fab′, F(ab′)2 and Mab respectively at 2 days post-injection. Combining these figures to calculate tumour to blood ratios revealed markedly better results for the anti-CEA F(ab′)2 fragments at all time points taken. Specific uptakes calculated using the control antibody are also greater for F(ab′)2. In vivo de-iodination of the 131I radiolabel has been thought to contribute to the high speed of Fab clearance, as well as the monovalency of the fragments and their low molecular weight (50,000 Da). Once again F(ab′)2 was considered to be the best overall imaging agent.26 In summary it appears that the smaller the fragment the faster the excretion and hence the reduction in background, with the qualification that Fabs clear so quickly that the background is increased in areas of excretion such as the kidneys and bladder. However, the faster reduction of circulating antibody fragment appears to lead to a decrease in the total uptake of radiolabel by tumour. This last point has been contested by Harwood et al.36 who showed that if time points were chosen soon enough after injection the amount of fragment localized in tumour was equivalent to that obtained with intact antibody and that it was the faster loss of the fragment

from the tumour site that made it appear that less radioactivity had accumulated. Despite the short half-life and increasing concentration of radioactivity in the urinary tract other researchers have found that Fabs give higher tumour uptakes and better scanning results than F(ab′)2 or Mabs.33 Mathematical modelling confirms that the clearance of Fab′ from the body is 35 times as fast as that for Mab and also shows that Fab disseminates more rapidly into a larger volume of distribution. Catabolism of Fab′ is mainly renal as opposed to Mab which is broken down by the gut. The shorter halflife of Fabs means that they cycle through the interstitium a decreased number of times and therefore advantages gained by the faster reduction in background count may be lost as they have less chance of coming across a tumour.37

Designer antibodies Further improvements in the structure of antibodies are being explored with the production of ‘designer antibodies’ using new technology.38 Progress in molecular cloning at the cDNA level using expression vector systems has enhanced the knowledge of binding sites permitting the engineering of antibody molecules and other immune targeting agents based on TAA epitopes.39 Selected areas of a given antibody can be obtained and recombined to produce customized proteins for tumour targeting, and antibody size can be decreased allowing faster diffusion and deeper penetration into tumours. The affinity of the molecule can also be altered by changing the number of hypervariable (binding) regions that it contains and modification of the Fc effector portion of the antibody may also prove to be useful (for example in changing its function to enhance its ability to activate complement). Antibodies can be fragmented to obtain their Fab′ portions which can then be grouped together as oligomers to avoid the decreased binding ability of Fabs due to their monovalency. These

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oligomers show more uniform penetration of tumours and are cleared more rapidly than F(ab′)2 fragments.40 Antibody specificity is encoded for by V genes. These can be inserted into bacteriophages leading to the expression of functional antibody fragments. The generation of large libraries of these genes has made it possible to produce antibodies in a way that is tailored to clinical needs. Products of this technology include single-chain antigen binding fragments (sFV) consisting of variable heavy (VH) or variable light (VL) domains, and recombinant sFV peptides which are VH and VL domains connected by peptide linkers and replicated in E. coli.41 These agents display the same advantages as those mentioned above as well as the ability to alter affinity for antigen via changes in the antigenbinding region. Nevertheless, it has been a problem to maintain the affinity of the parent molecule in these engineered fragments, which have reduced affinity in vivo. Peptides are also less stable than larger molecules in biological systems. Avoiding HAMA Attempts can be made to reduce the immunogenicity of the antibody and hence reduce the production of HAMA either by making purely human antibodies or chimeric ones in which the murine Fc fragment is replaced by that of a human antibody. Human Mabs have been shown to reduce immunogenicity in patients so that repeated treatment is possible should it be required. Some patients will still inevitably develop HAMA against the murine hypervariable V domain.42 Both human and humanized chimeric antibodies have been shown by some authors to have an increased plasma half-life compared to murine Mabs,43 whilst others report faster clearance from the body due to pharmacodynamic alterations (secondary to changes in glycosylation sites or Ig domains). More efficient binding with radionucleotide chelates and more rapid transportation of human antibodies into the extravascular spaces have also been demonstrated.35,43 Antibody affinity Theoretically, increasing the affinity of antibodies will lead to an increase in uptake and residence time in the tumour. However, they may be less able to penetrate deeply, being strongly bound at the tumour surface, and may suffer more interference due to increased binding to circulating antigen.44 Buchegger et al.33 compared four anti-CEA antibodies of differing affinities and specificities. Of the two Mabs directed against the same CEA epitope the one with the higher affinity had improved results. However the one with the highest affinity to CEA of all (k=18e+10/M) was not the best performer, the best results being achieved by the antibody with the highest specificity.33 There is support for the concept that in general antibodies with high affinities of around 1e+10/M with minimal or no cross-reactivity are the most useful for localization studies6,45 but once again there is no definite answer and further experimentation is required.46 Pre-targeting Systems to separate the individual steps of tumour targeting and introduction of radiolabel have led to significant

improvements in T/B and better diagnostic accuracy. The interval between radioisotope administration and imaging can also be reduced, giving better counting statistics which means better imaging.47 Human imaging studies have shown that maximum Mab uptake is achieved in 1 day but, in order to allow background reduction, an interval of several days is normally needed before imaging. Several pretargeting strategies exist. The most researched is the avidin–biotin system. In order to reduce non-specific uptake of radioactive antibody the monoclonal antibody may be labelled with biotin. The antibody binds to the tumour and time is then allowed for the non-specific uptake to be cleared by the reticulo-endothelial system. Radioactive avidin is then injected which localizes the tumour by taking advantage of the high affinity and specificity of avidin for biotin. Threestep procedures have also been performed whereby simple avidin is administered after the injection of biotin-labelled antibody, and then a second radiolabelled biotinylated antibody is added. Initial experiments were performed in rabbits48 and a similar technique (attaching streptavidin to the Mab) has been undertaken in 10 patients with nonsmall cell lung cancer.49 A disadvantage of this approach is that the metabolism of antigen–antibody complexes in the liver limits the detection of hepatic disease. A further method of pre-targeting is the affinity enhancement system or hapten/antibody system. Bispecific antibodies are used to localize radiolabelled bivalent haptens to tumour cells. This technique has produced superior results to direct imaging in the staging of mediastinal non-small cell lung cancer.50 Again an advantage is that successful imaging is obtained immediately after the radiolabelled hapten is injected. Other pre-targeting methods are the DNA/DNA and prodrug/enzyme systems, though theoretically many other ligand/receptor systems exist. Route of administration Compartmental administration may improve the targeting of an antibody. In 43 patients with colorectal cancers who received antibody fragments which for the most part were labelled with iodine (131I), the sensitivity of tumour detection rose from 70% via intravenous injection of antibody to 83% for intraperitoneal administration. The detection of liver metastases rose from 50 to 87% and in 24 patients studied by both routes there was a sharp increase in the tumour to background ratios calculated from the scans. The authors suggest that this may be because the antibody is in more immediate and longer contact with the tumour masses.51 However, even with intraperitoneal injection, antibody is absorbed into the bloodstream and with the exception of palliative treatment of disseminated peritoneal or ovarian tumours most trials have studied intravenous injection. A recent study in nude mice bearing intraperitoneal human colon cancer investigated the intratumoral distribution of antibody after intravenous and intraperitoneal administration. The tumours were targeted directly by radiolabelled Mab and also in two steps by using radiolabelled streptavidin to bind to tumours pre-targeted with biotinylated antibody. Intravenous injection in both experiments resulted in more uniform intratumoural distribution of radiolabel. Radioactivity after

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Educational section intraperitoneal administration was taken up primarily at the margins of the tumour. In the two-step method penetration became deeper with increased doses of streptavidin.52 Dose In an attempt to define the relationship between uptake and amount of antibody administered, human colonic carcinoma-bearing nude mice have been injected with escalating doses of anti-CEA immunoglobulin. In each case approximately 2% of the injected dose was retained by the tumour, which indicated that increasing the dose of antibody will increase the amount of radioactivity in the tumour.29 However, in a dose escalation study in patients using 131Ilabelled B72.3 no effect was seen on half-life, biodistribution or tumour detection with a 100-fold change in antibody dose (0.28–19.2 mg of Mab per patient). The authors suggest that the lack of cross-reactivity with other antigens was responsible, hence avoiding rapid clearance into a saturable pool of other antibody binding11. Equivalent results have been obtained using 99m Technetium (99mTc) labelled antiCEA Mab Fab′ and F(ab′)2 for imaging human colorectal cancers. It has been found that a dose of 1mg was as proficient at targeting tumours as a dose 10 times higher, and is the optimal dose for imaging with both Fab′ and bivalent F(ab′)2. Other authors have found improvements in localization with increases in antibody dose as well as with the imaging agent they used. There does appear to be non-specific uptake especially in the liver which might act as a saturable pool.53 This has led to attempts to give a large dose of unlabelled ‘cold’ antibody, hence blocking the non-specific uptake sites and allowing the radioisotope labelled antibody to bind to tumour. This assumes that the non-specific pools will be blocked without any effect on the tumour which seems unlikely and despite some positive results the success of this method is not at all clear.46 A dramatic rise in the dose of antibody administered may also lead to an increased production of HAMA. Cocktails Diversity of TAA expression in tumours both in terms of the total amount of antigen given and the number of different TAAs expressed has been demonstrated by immunohistochemical staining.54,55 Therefore, attempts to improve the uptake of radioactivity by the target have been attempted with ‘cocktails’ of several radiolabelled antibodies that recognize different epitopes or antigens on the same tumour56 (Fig. 5). Using two different antibodies separately for immunoscintigraphy of colorectal tumours, detection rates of 59 and 66%, respectively, were observed, though when the two Mabs were used in combination this increased so that 10 out of 13 sites (77%) were positive to detection.54 Sardi et al., however, using two different tumour xenografts in an animal model, found that while one cocktail may increase the amount of radioactivity bound to a tumour, in another tumour the antibodies in the same cocktail may compete with each other and reduce the efficacy of the mixture to less than that of one antibody used alone.57 In other words the optimum cocktail would have to be defined for each tumour. This is clearly not practical, especially in

Cancer cell

Fig. 5. Cocktail of Mabs attached to a cancer cell.

respect of the fact that tumours show differences in antigen expression even between metastases within the same patient.58 Other authors have indicated that the use of cocktails simply dilutes out the best antibody leading to a result which is the average of the uptake of all the antibodies used.59 Finally a cocktail of Mabs is similar to a polyclonal serum with fine control of the components and as yet Pabs have not been shown to be superior to Mabs over 15 years of experience.44 Imaging New methods of imaging also improve the results achieved with planar scanning and help to make up for low tumour antibody concentration. Tomoscintigraphy, or single photon emission computed tomography (SPECT), helps to determine the three-dimensional localization of Mab uptake16 and has been shown to increase detection of tumour sites from 43 to 94%.60 RAID is a physiological imaging modality, whereas CT and MRI define anatomical changes. Methods have been developed to fuse the images from SPECT antibody imaging and CT scans enabling the anatomical localization of focal areas of antibody uptake, thus enhancing the specificity of SPECT and the sensitivity of CT.61 On the whole, this is presently an experimental technique and limited to centres with the appropriate equipment. However, a recent study describes low-cost reliable diagnostic results in 11 out of 11 patients with pelvic tumours by fusing the image data from SPECT and MRI on a personal computer.62 Labels There has been a progression in the quality of radioisotope used in RAID. The development of antibody fragments with shorter half-lives and advances in labelling methods have enabled the use of superior metallic radioisotopes.

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Differences in the pharmacokinetics of the radioisotopes used to label Mabs leads to alterations in both the amount and location of background uptake. The choice of labelling method is also important. Labelling an immunoglobulin with a radioisotope must not lower its specificity or reactivity. 131-iodine (131I) This was the first and still is an extensively used isotope. It is easily available and cheap. Iodine can be attached to proteins relatively easily without altering their behaviour. Its half-life of 8.1 days is appropriate for RAID but it releases a high-energy photon (364 KeV) which, being poorly detected by gamma-cameras, also requires the use of a high-energy collimator. This reduces the sensitivity. Beta particles are also emitted, increasing the radiation dose to the patient and limiting the amount that can be injected. The body can remove iodine from antibodies in a process known as dehalogenation. This causes an increased renal output of radioactivity, the iodine being filtered and excreted in the urine. However, this does reduce the exposure of the liver, spleen and bone marrow. For this reason imaging with iodine has been found to be preferable in the abdomen when compared to indium (vide infra) in a multicentre clinical trial in patients with CEA producing tumours.51,63 There are a variety of methods of attaching iodine to antibodies of which the Iodogen technique has been shown to reduce the reactivity of immunoglobulins the least.64 Percentages of antibody accumulating in tumours in nude mice are higher when labelled by the Iodogen technique as compared with other methods.54 123-iodine (123I) This releases relatively less beta energy, allowing an increased dose to the patient. Its energy (159 KeV) is also ideal for detection with a gamma-camera. As it is made in a cyclotron, it is more expensive and less available and it cannot be used with technetium (99mTc) for background subtraction as they have overlapping energies. Its biodistribution is the same as that of 131I. In a study by Delaloye et al., 23 out of 24 colorectal tumours were imaged using Fab′ fragments labelled with 123I but the short physical half-life (13.3 h) and rapid elimination of the conjugate by the urinary tract may be limitations to its use.65 125-iodine (125I) This has a half-life of 60 days. The gamma energy of only 35 KeV leads to a lower background but is too weak to penetrate tissues adequately. It is therefore appropriate for use with a hand-held probe, and is the most commonly used isotope for radioimmunoguided surgery (RIGS). 111-indium (111In) This releases photons of two energies, 241 and 171 KeV, in abundance leading to good detection. It also has a relative lack of high-energy beta particles. Its half-life is suitable (2.83 days) and it is readily available and becoming less expensive. It requires chelation to antibodies and may complex to transferrin once injected. This may be the reason that it is accumulated by the reticuloendothelial system, especially the liver (making detection of liver secondaries difficult) and bone marrow (causing a relatively high background count). Accumulation in the reticulo-endothelial system is one of the main problems with

this isotope. It also accumulates in the colon requiring the use of laxatives prior to screening. Immunoglobulins are labelled with indium via a chelating agent which is attached to the antibody via a linking molecule. The choice of this molecule has a strong influence on the biodistribution of the indium and the selection of the appropriate linking molecule should reduce some of the disadvantageous nonspecific uptake of indium. For example the disulphide linker leads to a particularly fast clearance of indium from both the liver and the whole body. Unlike radioiodines, the 111In labelled complexes are relatively stable in vivo. Technetium-99m (99mTc) Over 70% of nuclear medicine procedures currently use this isotope. It releases gamma energy at 140 KeV with practically no alpha or beta emissions, giving the most favourable patient dosimetry results. It is widely available and inexpensive. Its short halflife of only 6 h means that it is only really suitable for use with fragments. Initial difficulties with the conjugation of 99m Tc to antibodies have been resolved.46 Other less widely used isotopes also exist and the range of radiolabels that could be used is really only confined by the parameters of synthetic chemistry. Targets The heterogeneity of TAA expression has restricted the success of RAID. Certain compounds termed ‘biological response modifiers’ (BRMs) are able to alter the surface TAA expression thus leading to better Mab binding to tumour (Fig. 6). The list of these agents includes human interferons (a and c), interleukins, cytokines and transforming growth factors such as granulocytemacrophage colony-stimulating factor (GM-CSF). The majority of the research involving BRMs has taken place with a view to progress in therapy because many BRMs are able to enhance tumour cell killing, such as c-interferon and GM-CSF which boosts complement-dependent cell lysis (ADCC).66 Also, BRMs can induce and amplify class I and II MHC antigens, as well as augment the expression of TAA. A reasonably low intraperitoneal dose of recombinant c-interferon has been shown to enhance the expression of TAG-72 and CEA on tumour cells aspirated from patients with ovarian cancer and ascites.67 The majority of studies on RAID have used TAAs as the antibody target. More recently, target molecules have been defined which are coded for by proto-oncogenes which may be more intimately associated with the neoplastic state than tumour-associated differentiation antigens like CEA. This may therefore make it possible to target cells at a certain position along the de-differentiation pathway towards malignant change. The level of expression of oncogeneencoded antigens may also indicate the sensitivity of the tumour to adjuvant therapy, hence antibodies used against these targets to image tumours may also give information about further treatment options.68 Conclusion RAID has both its detractors69,70 and those who urge caution in its application.71 On the other hand, many of its limitations

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Educational section Increased antibody uptake

BRM (e.g. interferon)

Cancer cell

Fig. 6. Effect of biological response modifiers (BRM) on tumour cells.

have been overcome by the use of manipulations such as those described above. The technique is reported to have a side-effect rate 10 times less than that for contrast media,72 although less experience is available with the former. The future for RAID is promising yet it remains to be seen whether these improvements have been great enough to allow it to take its place as a complementary investigation in the management of cancer.

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Scholarships The European Society of Surgical Oncology announces three scholarships of BEF 100,000 each The scholarships are intended for the workshop “Methods in Clinical Cancer Research” organised by the Federation of European Cancer Societies, American Association for Cancer Research and American Society of Clinical Oncology. The meeting is an intensive workshop for European junior clinical oncologists of all disciplines to learn the essentials of clinical trials design and will take place in June 2000. The applicant should be a member of the European Society of Surgical Oncology, a junior surgical oncologist under training or practising in the first years as a specialist. A curriculum vitae with a statement of planned research activities and a recommendation from the head of the department is required. Applications should be sent to: The Education Committee, European Society of Surgical Oncology, Rue He´gerBordet 1, B-1000 Brussels, Belgium. The European Society of Surgical Oncology announces two scholarships of BEF 50,000 each The scholarships are intended to support a 2 to 3 week visit to a European institution for a professional education and/or training within a field relevant to surgical oncology. Participation to a course relevant to education within the field of surgical oncology can also be supported. Congress travel will not be included. The application should contain the following: motivation; information about current position and professional education plan; a letter of recommendation from Head of the Department; letter of invitation from the host institution or course curriculum; and statement of research activities and publication. The applicant should be a member of ESSO. Priority will be given to junior doctors seeking education and training within developing fields. Applications will be evaluated twice yearly on 15 April and 15 October. Applications should be sent to: Associate Professor Lars Holmberg, Department of Surgery, University Hospital, S-751 85 Uppsala, Sweden. Fax: +46 18 556808.