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154 Biochimica etBiophysicaActa, 762 (1983) 154-164 Elsevier Biomedical Press

BBA 11126

IMPROVED RADIOIMMUNODETECTION OF TUMOURS USING LIPOSOME-ENTRAPPED ANTIBODY

GILLIAN M. BARRATT a, BRENDA E. RYMAN a, RICHARD H.J. BEGENT b, PATRICIA A. KEEP b, FRANCES SEARLE b JOAN A. BODEN b and KENNETH D. BAGSHAWE b

a Department of Biochemistry, Charing Cross Hospital Medical School (University of London) and h Department of Medical Oncology, Charing Cross Hospital, Fulham Palace Road, Hammersmith, London W6 8RF (U.K.)

(Received October 21st, 1982)

Key words: Liposome entrapment," Tumor detection," Antibody

The discrimination of radioimmunodetection of tumours is reduced by the presence of circulating radiolabelled antibody (primary antibody). We have prepared liposomes containing an antibody to the primary antibody (secondary antibody), with the intention of complexing and delivering to the liver primary antibody which is not associated with the tumour. In mice bearing xenografts of human tumours which secrete the marker carcinoembryonic antigen (CEA), liposomally entrapped secondary antibody was able to reduce the blood levels of 1251-1abelled anti-CEA within 2 h, without reducing the amount of anti-CEA bound to the tumour. We therefore suggest that the use of liposomally entrapped secondary antibody would improve the diagnostic potential of radioimmunodetection of tumours and their metastases.

Introduction

Radioimmunodetection is a rapidly advancing branch of cancer diagnosis [1]. It involves administration of a radiolabelled (usually 131I-labelled) antibody to a tumour product, for example, carcinoembryonic antigen (CEA), human chorionic gonadotrophin (human CG) or a-foetoprotein, followed by detection of the gamma emitting antibody by external scintigraphy. The location of the antigen-secreting primary tumour or metastases will be indicated by a concentration of radioactivity at these sites. Tumours of human patients which secrete CEA [2-4], human CG [5,6] and a-foetoprotein [7-9] have been localised by this technique. In addition, antibodies to other tumour products have been localised in animal models [10-12]. However, there are still problems to be overcome. In particular, at the time of scanning, 24-72 h after antibody administration, a large proportion of the radiolabelled antibody is still in

the circulation of the patient, making a simple ~31I camera image difficult to interpret. One solution which has been adopted [13] is to give the patient 99mTc-labelled albumin, and also free [99mTc]pertechnetate, and to obtain a second image, that of 99mTc distribution, corresponding to the distribution of a substance which is not concentrated by any particular organ. The technetium scan is then subtracted from the iodine scan to obtain a picture of the specific distribution of the antibody. Tumour localisation by the subtraction method has yielded results which correlate well with other methods of tumour detection [2-9], but problems of interpre- tation are encountered due to artifacts produced by different distribution of the two radionuclides in normal tissues and the different energies of the radionuclides.

We have had some experience of liposomes as agents for the clearance of unwanted substances from the circulation. The use of liposomally-entrapped chelating agents to treat metal overload

0167-4889/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

has been investigated with some success [14]. We have also shown that liposomes containing antibody to digoxin can accelerate the clearance of excess drug from the circulation of experimental animals, while this was not observed when free anti-digoxin antibody was given [15-16]. The basis of both these techniques is that intravenously ad- ministered liposomes are taken up rapidly by the reticulo-endothelial system [17,18]. We therefore decided to use the same approach for the removal of circulating antibodies to tumour products. In order to achieve this it was necessary to entrap in liposomes an antibody to the radiolabelled antibody (this will be referred to as entrapped secondary antibody) and administer this after the labelled antibody (referred as primary antibody) and before scanning.

For the preliminary experiments we worked with xenografts of human colorectal carcinoma which secrete carcinoembryonic antigen. The primary antibody used in scanning, anti-CEA, was raised in a goat. The secondary antibody which was entrapped in liposomes was derived from a horse antiserum raised to goat IgG. Firstly we set out to discover whether sufficient secondary antibody could be entrapped in liposomes in an active form to bind the amount of primary antibody given in radioimmunodetection studies without the dose of liposomes becoming too great. Secondly we wished to see whether such liposomes could influence the distribution of labelled primary antibody in vivo. Finally, we needed to investigate the effect of liposomes containing secondary antibody on the concentration of radio-labelled primary antibody in the vicinity of the antigen-secreting tumour.

Materials and Methods

Primary antibody. Goat primary antibody (designated PKIG, D2; [19]) was raised to carcinoembryonic antigen (CEA), immunopurified and absorbed against normal colon, liver, spleen and serum [20], and the anti-CEA y-globulins labelled with 1251 in ice by the chloramine-T technique, after Hunter and Greenwood [21].

Secondary antibody. Horse antiserum raised to goat 7-globulin was a gift of Burroughs Wellcome. This was purified to a y-globulin fraction by pre-

155

cipitation with 50% saturated ammonium sulphate followed by ion-exchange chromatography on DEAE-Sephadex A50 (Pharmacia), y-globulin being eluted with 10 mM phosphate buffer, pH 7.4, [22]. This preparation is referred to as 'secondary antibody'. By radioimmunoassay (see be- low) 10-30% of the original immunological activity was retained in the ,{-globulin fraction. For initial measurements of protein entrapment in liposomes secondary antibody was radioactively labelled with 125I as described for the primary antibody.

Radioimmunoassay. Assays for the immunological activity of secondary antibody and liposomes containing secondary antibody were carried out using the Kemtek automated radioimmunoassay system [23].

Preparation of liposomes. Liposomes were prepared by the method of Gregoriadis and Ryman [17,18]. Egg phosphatidylcholine, made in our laboratory by the method of Dawson [24], cholesterol (Sigma) and phosphatidic acid (Lipid Products) in chloroform solution, were mixed in the molar ratio 9 : 9 : 2 and evaporated to dryness. [3H]cholesterol, when used was added to the lipid mixture. For each 100 ~tmol of lipids used 1 ml of 5 mM phosphate-buffered saline, pH 7.4, containing 10 mg horse-anti-goat IgG, was added and a liposome suspension was formed and allowed to swell overnight. For control liposomes, phosphate-buffered saline alone was added. The liposomes were sonicated in an ice bath using a MSE Soniprep probe sonicator, giving 10 bursts of 30 s at 8/~m peak-to-peak amplitude interspersed with 30 s cooling for every 1 ml of liposomes to be processed. 4 ml was the largest volume sonicated at one time. After sonication the preparation was allowed to stand for 1 h at room temperature, and liposomes were separated from non-entrapped IgG by gel chromatography on Sepharose 6B-CL (Pharmacia) in phosphate-buffered saline. Lipo- some-rich fractions were pooled and the entrapment of IgG estimated either by radioactive count- ing or by chemical determination of protein and cholesterol. Before administration to animals the liposomes were concentrated to 100 t~mol lipids per 1 ml suspension by bulk dialysis against Sep- hadex G50.

Labelling of the lipid membrane with the

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y-emitter 99mTc was achieved using the method of Richardson et al. [25]. 1 ml of the concentrated liposome suspension and 99mTc as pertechnetate in 1 ml saline were added simultaneously to 0.5 ml 3 mM SnC1 z in a sealed vial. The vial was shaken and left to stand for 30 min. Since the efficiency of attachment of 99mTC to the liposomes is reported to be almost 100% [25], no separation stage was employed and the liposomes were injected into animals immediately.

Estimation of the protein content of IgG-containing liposomes was performed by the method of Lowry et al. [26] as modified by Heath et al. [27], using sodium deoxycholate to disrupt the liposomes prior to assay. Cholesterol assays on liposomes were carried out using the ferric chloride method [28]. From the results the total lipid in the sample could be estimated.

For electron microscopy of liposomes, a drop of the suspension, after dilution with saline if necessary, was placed on a clean glass slide. A carbon coated copper grid was floated first on the liposome preparation and next on a drop of 2% phos- photungstic acid. The grid was then dried by touching on filter paper, viewed with a transmis- sion electron microscope and photographed at an original magnification of x 87 000.

Animal experiments. For the experiments in- volving tumour bearing mice, two separate xenograft lines of human CEA-secreting tumours were maintained in outbred nude (nu /nu) mice. These were designated TAF and Pl16 cell lines. P116 is a moderately well differentiated colonic tumour line which has been shown to secrete CEA [29]. The TAF cell line was derived from material from a colo-rectal cancer patient at Charing Cross Hospital, and also secretes CEA. In mice these tumours do not metastasise. Mice were injected with both primary antibody and liposomes via the tail vein under ether anaesthesia. Rabbits were injected via the ear vein and blood samples were taken from the same site.

Results

A. Entrapment and availability of lgG in liposomes Initially the entrapment of secondary antibody

in small negatively-charged vesicles was assessed by the association of 125I-labelled IgG with

[3H]cholesterol in the liposome peak after gel chromatography of the liposome preparation on Sepharose 6B-CL. This gave a protein-to-lipid ratio of 126 + 30/~g//~mol. This represents almost com- plete entrapment of the added protein. In subse- quent experiments the secondary antibody in the liposomes was assessed by the modified Folin assay and compared to cholesterol elution measured by either radioactive tracer lipids or by cholesterol estimations on the column fractions. By this method a mean protein-to-lipid ratio of 53.4 + 31.7 /~g/~mol representing about 50% entrapment, was obtained. The latter figure is almost certainly the correct estimate of entrapment, and the higher values for 125I-labelled protein were due to [125I]iodide dissociating from the protein and becoming encapsulated in liposomes. Fig. 1 shows the elution of secondary antibody and cholesterol on Sepharose 6B-CL in a typical liposome preparation.

An electron micrograph of negatively-stained liposomes containing secondary antibody is shown in Fig. 2A. The mean size of the vesicles is 87 _+ 55 nm, and they are uni- or oligo-lamellar. Some aggregation of the vesicles has occurred with what is possibly released immunoglobulin at the point of association. Similar liposomes that do not con-

NON-ENTRAPPED PROTEIN LIPOSOMESI m

1.00

ml ELU]ED

0. 75

o.5o

3----

0.25

0

500

Fig. 1. Chromatographic separation of liposomes containing secondary antibody from non-entrapped secondary antibody. A sample containing 178 ~mol of lipids as liposomes and 14.4 mg of secondary antibody was loaded onto a Sepharose 6B-CL column, diameter 4 cm, height 42 cm and eluted with 5 mM phosphate, pH 7.4, containing 0.9% (w/v) NaCl. l0 ml fractions were collected. (o) Elution of protein; (C)) Elution of cholesterol.

157

Fig. 2. Electron microcopy of liposomes. A liposomes containing secondary antibody (approx. 50 /~g per/~mol lipids); B liposomes containing no secondary antibody, magnification :,<40900.

tain any entrapped IgG do not show this phe- nomenon (Fig. 2B).

In order to assess the anti-CEA (i.e. primary antibody) binding capacity of the liposomes, various dilutions of a preparation of liposomes containing secondary antibody of a known lipid concentration were incubated with ~25I labelled anti- CEA in the presence of serum. Dilutions of free secondary antibody and empty liposomes were also incubated with labelled anti-CEA. After filtration the amount of complexed anti-CEA in each incubation was counted. Empty liposomes, that is liposomes containing no entrapped protein, at concentrations up to 22 /xmol lipids per ml bound no 125I-labelled anti-CEA in this assay. By comparing the binding of anti-CEA to liposomes containing secondary antibody to the binding of the anti-CEA to known dilutions of secondary antibody the amount of available secondary antibody in liposomes was 4.6 /~g per /~mol lipids. Since the total secondary antibody concentration in liposomes was 53/~g per/~1 lipids, only about 9% of the entrapped secondary antibody is available to bind to its antigen. The rest must presumably be sequestered within the aqueous compart- ments of the liposomes.

B. The effect of liposomes on the clearance of 1251-labelled anti-CEA in normal mice

A group of 30 AKR mice were injected into the tail vein with 15 ~Ci of ~25I-labelled anti-CEA. 24 h later 15 of these received an intravenous

injection of 10 /~mol of lipids as liposomes containing secondary antibody (approx. 500 ~tg protein per mouse). The remaining mice acted as controls. Groups of three treated and three control mice were killed at various times after liposome administration to the treated group, and the radioactivity in blood, liver, spleen and kidney was measured. Table I shows the results. In the control animals the blood levels of 125I-labelled anti-CEA did not fall much over the period 24-48 h after injection. However, 30 min after injection of liposomally-entrapped secondary antibody the blood anti-CEA was reduced to 60% of that in the corresponding control group, and at 2 h after liposome treatment the level was only 26% of that in the control group. This level remained constant up to 24 h after the liposome administration. Treatment with secondary antibody-containing liposomes also promoted the accumulation of anti-CEA in the liver and spleen over the initial 2 h of the experiment. There was little accumulation of anti-CEA in these organs in the absence of liposomes. The amount of radioactivity in the kidneys of treated animals was less than that in the control animals, presumably due to the reduced blood levels of anti-CEA in the liposome-treated group.

In a further experiment 15 mice were injected with IzsI-labelled anti-CEA, and 24 h later with 10 /xmol of lipids as liposomes containing entrapped secondary antibody and externally attached 99mTC. Groups of three animals were killed at various time intervals and the tissues were counted for t25I

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TABLE I

EFFECT OF SECONDARY ANTIBODY IN LIPOSOMES ON THE CLEARANCE OF mSI-LABELLED ANTI-CEA (PRIMARY ANTIBODY) IN MICE

All mice were given intravenous injection of 15 #Ci 125I-labelled anti-CEA (1.5 ~g). 24 h later the treated group was given 10/~mol lipids as small, negatively-charged liposomes containing 500/~g secondary antibody. Groups of three treated and three control mice were killed at various times after liposome injection, and tissues were counted for 125I. Results are % of injected dose per g tissue (mean ± S.D.). T = treated. C = control.

Time mSI-labelled anti-CEA (% injected dose per g tissue) (± S.D.)

Blood Liver Spleen Kidney

30 ~ n T 6.7±1.6 13.6±2.0 26.2±10.1 2.5±0.6 C 11.1±2.7 3.9±0.5 2.6±0.4 3.5±0.3

2 h T 3.0±0.3 14.9±2.1 22.3±3.2 2.0±0.3 C 11.5±3.4 3.8±0.4 2.9±0.6 3.5±0.4

6 h T 2.8±0.7 12.5±1.5 14.3±5.9 2.0±0.4 C 10.7±0.9 3.6±0.2 2.4±0.2 2.7±0.8

18h T 2.5±0.2 4.4±0.3 6.0±0.9 1.3±0.2 C 7.4±0.9 2.0±0.3 1.5±0.2 2.7±0.4

24h T 2.6±0.5 1.5±0.4 6.4±1.4 1.4±0.2 C 10.4±0.5 2.6±0.3 2.0±0.2 2.7±1.1

and 99mTC. The 125I distribution was similar to that described in Table I. Table II shows the 99mTC

distribution in the mice. The liposomes are cleared rapidly from the blood and accumulate in liver and spleen. The 99mTc label persisted in these organs for 18 h. This is probably not associated with intact liposomes, but due to the label being transferred to some component of the cells. This distribution pattern is characteristic of small, negatively-charged liposomes [17,18]. Thus the

presence of liposomes containing secondary antibody had caused a change in the distribution of 125I-labelled primary antibody to one resembling the distribution of the liposomes themselves, and caused a significant reduction in the blood levels of primary antibody.

C. The effect of liposomes on the distribution oJ anti-CEA in tumour-bearing mice

The clearance of blood anti-CEA by liposomes

TABLE II

DISTRIBUTION OF 99mTc-LABELLED LIPOSOMES CONTAINING SECONDARY ANTIBODY IN MICE PRE-TREATED WITH ANTI-CEA

Mice were given 12~I-labelled anti-CEA (1 t~g, 11 #Ci), and 24 h later 10/~mol of lipids as liposomes containing 500/~g secondary antibody and labelled with 99mTc (50 t~Ci/mouse at time of injection). Groups of three mice were killed at intervals.

Time 99mTc (% injected dose per g tissue) (± S.D.)

Blood Liver Spleen Kidney

30 ~ n 8.8±0.4 31.8±4.5 39.2±2.8 11.7±1.1 2 h 2.7±0.3 35.0±2.1 36.3±6.4 9.5±0.3 5 h 1.1±0.2 30.7±2.0 32.4±3.7 7.1±0.6

18h 0.4±0.05 31.3±4.7 39.2±4.6 3.7±0.8 24h 0.3±0.05 15.8±0.8 16.5±4.0 2.4±0.3

containing secondary antibody is of value in improving the discrimination of gamma scans only if the anti-CEA associated with the tumour is not reduced concomitantly with the blood levels. This was investigated using nude mice bearing xenografts of human CEA secreting colonic cancers. Mice bearing the TAF tumour were injected 12.5 /xCi (1.3 ~tg) of 125I-labelled anti-CEA and, 24 h later, 10/~mol of lipid as liposomes containing 500 /~g of secondary antibody was injected into one group, while the remainder received no injection and acted as controls. Groups of three treated mice and three controls were killed at 30 min, 2 and 6 h after liposome injection. As well as the normal tissues, tumour was taken for measurement of ~25I. Fig. 3 shows the amounts of ~25I-labelled anti-CEA in the blood and tumours of these

1.5

cpm.g.1 1.0 x 10-6 (_+SD) 0.5

0.8

cpm'g ] 0.6 x 10-6 (tSD) 0.4

0.2

Blood radioactivity

1

I 1

Tumour radioactivity

0 I I I I I I 1 2 3 4 5 6

h after liposomes Fig. 3. 125I-labelled anti-CEA distribution in tumour-bearing mice after treatment with liposomes containing secondary antibody. Nude mice bearing xenografts of a human CEA-secreting turnout were given approximately 12 # Ci J25 I-labelled anti-CEA i.v. 24 h before the experiment. Treated animals (e e) received liposomes with entrapped secondary antibody intravenously at 0 h; control animals (© . . . . . . ©) received no treatment. Three mice from each group were killed at ½, 2 and 6 h after treatment, and blood and tumour samples were counted for 125I [401.

159

animals. The blood anti-CEA in the treated mice was significantly lower than that in control animals. However in the tumours there was little difference between the amount of radioactivity found in each group. The ratio of anti-CEA in the tumour to that in the blood had been incrased in the treated animals, at 6 h this ratio was 0.54 in the controls and 1.92 in the liposome-treated animals (Table III). In another experiment mice bearing a different human tumour xenograft, designated Pl16 cells. This line is a poorer secretor of CEA than TAF cells. These mice were given 125I-labelled anti-CEA, and, 24 h later, half received just 3 /~mol of lipids as liposomes containing secondary antibody. In this experiment mice were sacrificed 6 and 24 h after liposomes. In the 6 h group, the results were similar to those obtained with the TAF, i.e., in the treated group blood radioactivity was reduced relative to control, whereas the tumour radioactivity was not affected, thus increasing the tumour :b lood ratio in the treated group. How- ever, 24 h after liposome treatment, the tumour radioactivity was reduced and the tumour:blood ratio was not much increased over controls (Table III). In order to investigate the effect of increasing the liposome concentration, a group of mice bearing TAF xenografts were given 27 /~mol of lipids as liposomes containing secondary antibody after pre-treatment with t25I-labelled anti-CEA as usual. These were killed 24 h after liposome treatment and found to have blood levels of 125I-labelled anti-CEA only 5% of those in the controls. The tumour radioactivity was halved in the treated animals c o m p a r e d to controls , but the tumour : blood ratio was still improved 10-fold. All this data is summarized in Table III, and shows that liposomes containing secondary antibody have potential use for improving the discrimination of radioimmunodetection of tumours.

D. Effect of liposomes on clearance of normal goat lgG in rabbits

Seven rabbits were given intravenous injections of 125I-labelled normal goat IgG. 24 h later three were given 30/~mol of lipids as liposomes containing 1.5 mg of secondary antibody. At the same time two rabbits were injected with 30 /~mol of lipids as liposomes containing no entrapped material (empty liposomes). The final two rabbits

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TABLE I11

125I-LABELLED ANTI-CEA DISTRIBUTION IN TUMOUR-BEARING MICE AFTER TREATMENT WITH LIPOSOMES CONTAINING SECONDARY ANTIBODY.

Mice were given 125I-labelled anti-CEA (0.1-2.0/~g, 1-20 ~tCi), and 24 h later treated mice received liposomes (3-27 ~mol lipid) containing secondary antibody (50/~g/~mol lipid) at the dose indicated in the table. Control mice received no second injection.

Tumour Dose of Time Tissue radioactivity (cpm/g) Ratio of tumour radio- line liposomes after (% of that in untreated activity to blood radio-

(/~mol liposome controls) activity lipids) injection

Blood Tumour Control Treated

P116 3 6 h 30 N.S. a 122 N.S. 0.30 1.22 24 h 39 < 0.05 64 N.S. 0.20 0.32

TAF 10 30 min 56 < 0.002 82 N.S. 0.50 0.73 2 h 34 < 0.01 140 < 0.01 0.40 1.69 6 h 30 < 0.05 107 N.S. 0.54 1.92

27 24 h 5 < 0.01 51 < 0.05 0.53 5.57

a Statistics refer to a two sample Student's t-test comparing the values to untreated controls. n.s., not significant.

were given no second injection and acted as con- trois. Blood samples were taken at intervals before and after l iposome injection and counted for 125I. The temperatures of the rabbits were taken before

and after liposomes. The mean temperature of the antibody-liposome-treated group rose by 0.7°C after liposome injection. However the mean temperature of the empty liposome group fell by

TABLE IV

THE EFFECT OF LIPOSOMES ON THE CLEARANCE OF mSI-LABELLED NORMAL GOAT lgG IN RABBITS

Seven rabbits were given 4 p.g of ~25I-labelled normal goat IgG. 24 h later blood was taken to give 0 h radioactivity, then three rabbits received 30 ~mol of lipids as liposomes containing 1.5 mg of secondary antibody, two more received 30/tmol of lipids as liposomes containing only phosphate-buffered saline (empty liposomes) and two received no second injection (controls). Blood was taken from the ear at intervals, n.d., = not determined.

Treatment Rabbit Radioactivity in blood. (% of that Group no. at 0 h)

2 h 24 h 48 h

Control 1 96.3 64.3 51.4 2 80.6 65.9 56.6 Mean +s.d. 88.5+ 11.1 65.1 + 1.1 54.0_+3.7

Empty 1 83.0 62.7 36.3 Liposomes 2 90.3 55.9 39.3

Mean + s.d. 86.7 + 5.2 59.3 + 4.8 37.8 + 2.1 N.S. a N.S. P < 0.05

Liposomes 1 62.7 50.9 33.9 containing 2 57.9 47.5 28.8 secondary 3 72.6 57.6 n.d. antibody Mean + s.d. 64.4+ 7.5 52.0_+ 5.1 31.4+ 3.6

N.S. P < 0.05 P < 0.05

a Statistics refer to a two-sample Student's t-test for the mean versus the mean in the control group.

0.1°C, and that of the control group rose by 0.9°C over the same period. The rabbits were observed for 21 days after liposome injection, and did not show any adverse effects due to the administration of lipid and foreign protein. The dose of liposomes per kg was the same as the calculated to be necessary to achieve clearance of circulating anti-CEA in patients receiving antibody scans for the detection of tumours.

The effect of liposomes on the levels of goat IgG in the blood is shown in Table IV. 2 h after liposome injection the control and empty liposome treated groups had 87-89% of the original radioactivity remaining in the blood, whereas the group treated with liposomes containing secondary antibody had only 64% of the pre-liposome level in their blood, although this result was not significant. At 24 and 48 h after treatment, the group given entrapped secondary antibody continued to have significantly lower blood levels of the [125I]IGG than the controls. In the animals treated with empty liposomes, the blood levels of [~25 I]IgG were lower than the controls, but higher than in the animals treated with liposomes containing secondary antibody, and, at 48 h after liposomes, was not significantly different from the group treated with full liposomes. After 48 h the remaining [J25I]IgG was cleared at a similar rate in all three groups (Table IV). Thus, these results confirm that secondary antibody entrapped in liposomes is able to clear primary antibody from the circulation. In addition, liposomes containing no antibody have also be shown to accelerate the clearance of labelled IgG from the blood. This may be due to non- specific absorbtion of some of the protein onto liposomes, although empty liposomes do not bind anti-CEA appreciably in vitro. These results therefore confirm those reported for mice, that the administration of secondary antibody in liposomes causes an immediate redistribution of primary an~ tibody from the circulation to the liver and spleen.

Discussion

The entrapment of horse anti-goat IgG in small negatively-charged liposomes reported in (A) represents about 50% of the added protein and i s higher than would be expected for a solute in this type of liposome and indicates that some associa-

161

tion with the outer membranes occurs. This was also the observation of Huang and Kennel [30] who saw increased entrapment of antibodies in sonicated liposomes as sonication time increased. It is tempting to postulate that the hydrophobic Fc portion of the IgG molecule becomes inserted into the lipid bilayer, but similar results have been obtained using (Fab)12 fragments [30]. Given that this association occurs it is difficult to explain why so little of the immunoglobulin is available to bind to its antigen. It may be that most of the IgG in the membrane is orientated incorrectly for binding, or that most is associated with the internal membranes of the liposomes, since Fig. 2A shows that many of the liposomes are oligolamellar. In addition, sonication, even when carried out at low temperature may cause some denaturation of the protein and hence loss of immunological activity. The low antigen binding means that the lipid doses calculated to be necessary for clearance of anti-CEA in patients are high, 600/tmol or about 360 mg of lipid.

More efficient entrapment of IgG within liposomes might be achieved by the chemical coupling of the antibody molecules to the surface of the liposome. In this way more of the active sites might be available to liposomes. Much research has been devoted to the attachment of antil~odies to liposomes with a view to targetting these vesicles to specific cells and tissues, and a number of successful methods are to be found in the litera- ture. These included glutaraldehyde coupling [31], the more complicated disulphide linkage used by Leserman and co-workers [32], and methods pub- lished by Papahadjoupoulos' laboratory [27,33]. In general, such antibody linkages have only been tested for their ability to promote liposome association with specific cell lines in culture, and the stability of the coupling in vivo has not been determined. In the project we have described, the large scale preparation and the stringent require- ments for sterility and non-toxicity necessary for liposome administration to humans may render a complicated chemical procedure impractical. How- ever we are critically evaluating various modes of linkage with our antibody system.

The amount of protein to be entrapped for a given immunological response could be reduced 10-fold if the crude IgG fraction of secondary

162

antibody could be affinity-purified to specific immunoglobulin, as is done for the primary antibody [20]. This would reduce the dose of liposomes which would have to be given to clear a given amount of primary antibody. The lipid dose might also be reduced by using reverse phase vesicles, prepared by the method of Szoka and Papa- hadjopoulos [34] to entrap the secondary antibody. Experiences with anti-digoxin antibody in our laboratory suggest that these large oligolamellar vesicles will encapsulate a large proportion of added antiserum and that up to 50% of this is available to bind antigen, particularly if the phos- pholipid phosphatidylethanolamine is included in the liposomes (Parvez, N., Barratt, G. and Ryman, B.E., unpublished data).

The experiments reported here demonstrate clearly that liposomes containing secondary antibody can alter the distribution of radiolabelled primary antibody in experimental animals, and that the circulating concentration of primary antibody is significantly reduced. Equally importantly, we have shown that these liposomes do not reduce the concentration of antibody in the antigen- secreting tumours for the first 6 h after administration (Table III). When [3H]cholesterol was included in the liposomes the amount of isotope found in the tumour was too low to be measured accurately. This result does not augur well for the use of liposomes as drug carriers to tumours. The reduction in tumour-associated primary antibody observed 24 h after liposome administration (Ta- ble III) may be due to re-equilibration with a reduced blood pool. In all the experiments performed on xenograft-bearing mice, liposomes containing secondary antibody increased the ratio of tumour-bound anti-CEA to circulating anti-CEA above that observed in controls.

The results obtained in rabbits confirmed the observation that liposomes containing secondary antibody would reduce circulating anti-CEA and also gave information about the toxicity of the liposomes. Although secondary antibody-containing liposomes were not entirely non-pyrogenic, the rabbits suffered no permanent ill effects. Another possible complication of the use of liposomes in this way is that liposomes have been reported to act as good immunological adjuvants to protein antigens [35]. However, intravenously injected

liposomes have only weak adjuvant properties particularly when negatively charged and' rich in cholesterol [36] and we do not feel that this would be a problem in practice.

The success of the animal work has led to a small trial in patients [37] which has yielded en- couraging results. Five patients with suspected metastases of colorectal cancer were given liposomes containing secondary antibody (8-32 mg protein and 80-300 /~mol lipid per patient) 24 h after injection of 131I-labelled primary antibody and external scintigraphy was carried out before liposomes and 2-48 h later. In three cases discrimination was improved by the use of such liposomes [37]. Thus liposomes containing secondary antibody clearly have a role to play in radioimmunodetection of tumours by reducing the levels of radioactively labelled primary antibody in the circulation without depleting the amount of primary antibody in the vicinity of the tumour.

This technique may be extended to the radioimmunodetection of other tumour types, e.g., those secreting human chorionic gonadotrophin or a- foetoprotein, simply by changing the nature of the secondary antibody entrapped within the liposomes. In the future monoclonal antibodies, derived from rat or mouse hybridomata, will be used in this type of scanning. We are preparing rabbit and sheep antibodies to mouse IgG for entrapment in liposomes and use in the localisation of colorectal carcinoma using radioactively labelled mouse monoclonal antibodies. Primary antibodies radioactively labelled to high specific activity could be used in the thereapy of tumours by providing a high concentration of radioactivity in the region of the neoplasm [38]. As in radioimmunodetection the removal of antibody from the circulation is important so as to avoid irradiation of normal tissues and we are investigating the use of liposomes for this purpose. However, since liposomes will carry the labelled antibody to the reticuloendothelial system, the additional radiation dose to these organs may outweigh the removal of radioactivity from the circulation and this must be assessed carefully in experimental models.

Alternatively, the primary antibody might be complexed to a toxic compound such as the A chain of ricin [39]. By similar strategies the removal of non-tumour associated antibody-toxin

complexes might be achieved by adminis t ra t ion of

l iposomes conta in ing secondary ant ibody.

Liposome-induced clearance may have m a n y

appl icat ions where unwanted substances have to

be removed from the blood. The clearance of toxic

metals, and of digoxin overload, has been men-

tioned. An obvious collorary to the work we have

described is the t reatment of au to immune disease where unwanted endogenous ant ibodies are cur-

rently removed by electrophoresis. However, in the rad io immunode tec t ion of tumours only microgram

quanti t ies of ant ibodies are injected, and even so

large doses of l iposomes are necessary to effect

clearance. For the removal of endogenous antibody, or of therapeutic levels of drugs, the amoun t

of lipid necessary to encapsulate sufficient b ind ing sites may be prohibit ively large. The l imiting fac-

tor for l iposome-induced clearance will always be

the efficiency of en t rapment of an t ibody or other b ind ing agent within the lipid vesicles and the

b ind ing affinity of the agent so entrapped.

Acknowledgements

The authors would like to acknowledge the

Cancer Research Campaign and the Medical Re- search Council for their f inancial backing. Miss

Margaret Caldecourt prepared the electron micro- graphs, Mr. Christopher More the pure lipids and Mrs. Angela Wheatley the manuscript . We are

grateful to all these people for their assistance.

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