Cancer Letters, 61 (1991) 35 -43 Elsevier Scientific Publishers Ireland
35 Ltd.
Tumor localization of monoclonal antibodies renal carcinoma in a xenograft model A.K. Guha”, T. Ghose”, M. Mammena Departments
of “Pathology,
M. Singh”,
bNuclear
Medicine
J. Aquinob,
against human
A.H. Blair’, S.J. Lunera and
and ‘Biochemistry,
Dalhousie
University.
Halifax.
N.S.
B3H
4H7
[Canada) (Received
2 July 1991)
(Accepted
9 September
1991)
Summary
ingly used to target chemotherapeutic agents or radionuclides selectively towards cancer cells [1,2]. Metastatic renal cell carcinoma (RCC) is a relentless, progressive disease that is hardly affected by any form of current therapy including chemotherapy [3]. We have produced several monoclonal antibodies against human RCC associated antigens [4]. We have also demonstrated that when these MABs are linked to the antimetabolite methotrexate (MTX) [5,6], or to MTX containing small unilamellar vesicles, the conjugated antibodies bind to human RCC cell lines such as Caki-1 and are more effective inhibitors of Caki-1 cells in vitro [7] or Caki-1 cells grown as ascites tumors in nude mice [8] compared to equimolar amounts of the drug alone or the drug linked to a non-specific immunoglobulin. However, to be clinically useful, tumorselective localization of these MABs in vivo is essential. Using intrarenal xenografts of Caki- 1 cells in nude mice, we demonstrate that the two of our MABs tested i.e. DAL K29 and DAL K45 and their respective F(ab)* fragments indeed localize in the tumor xenografts.
We investigated the localization of intravenously injected DAL K45 and DAL K29, two monoclonal antibodies (MABs) against human renal cell carcinoma (RCC), and their F(abj2 fragments in nude mice bearing intratenal transplants of the RCC line Caki-I. More of the MABs or their F(ab)g specifically localized in the tumor than in any normal tissue with the exception of blood. Compared to parent MABs, F(ab)+ were cleared faster from all tissues. In serum, the MABs and F(ab)+ showed a single radioactive peak reimmunoreactiuiry. taining partial DAL K45-F(ab)* showed the highest tumor:normal tissue localization ratios and the most distinct gamma-camera image at 24 h. Keywords: monoclonal antibodies; tumor localization; tumor imaging; human renal carcinoma Introduction Monoclonal antibodies tumor associated antigens
(MAB) to human are being increas-
Correspondence to: T. Ghose, Department Dalhousie University, Sir Charles Tupper Halifax,
N.S. B3H 4H7.
0304-3835/91/$03.50 Published .znd Printed
Materials and Methods Tumor and monoclonal antibodies The Caki-1 cell line, originally derived from a skin metastasis of renal cell carcinoma from
of Pathology, Medical Bldg.,
Canada.
0 1991 Elsevier Scientific Publishers in Ireland
Ireland
Ltd
36
an adult Caucasian male [9] and the nonspecific mouse IgGi-producing myeloma line ATCC TIB9 were obtained from the Human Tumor Cell Bank through the American Type Culture Collection (Rockwell, MD). A subline of Caki-1 cells was developed by serial transplantation of Caki-1 cells subcapsularly into the kidney of adult female, athymic (nu/nu) BALB/c mice (Life Science Inc., St. Petersburg, FL.) so that intrarenal inoculation of 2 x lo6 Caki-1 cells produced progressive tumors in 100% of inoculated mice. For intrarenal inoculation, a longitudinal incision, approximately 1 cm long, was made in the skin of the mice under anesthesia, the kidney was exposed after dissection of the perirenal fat and tumor cells were inoculated subcapsularly using a 25 gauge needle. Tumors could be palpated in the left kidney approximately 15 - 20 days after tumor inoculation and weighed about 500 mg at the conclusion of the imaging studies. MAB DAL K29 reacted with 8/9 renal carcinomas in our laboratory and precipitated molecules with molecular masses of 118 kDa and 150 kDa from extracts of surface-labeled Caki-1 cells. MAB DAL K45 reacted with 4/6 RCCs tested and precipitated 177-kDa and 150-kDa antigens from surface-labeled Caki-1 cells. The details of the specificity and reactivity of these two MABs have been reported [4]. The non-specific murine monoclonal IgGi and the MABs were isolated by precipitation with caprylic acid [lo] from the ascites fluid of BALB/c mice (Life Science Inc.) intraperitoneally inoculated with lo7 myeloma or appropriate hybridoma cells. Polyacrylamide gel electrophoresis (PAGE) of the purified preparations revealed a homogeneous IgG band and a very small amount of contaminating albumin. F(ab)z fragments of the different IgGi preparations were obtained by digestion with pepsin (Sigma, St. Louis, MO) at 37OC, (pH 4.2) at a pepsin protein ratio of 1:33. The optimal duration of digestion for each of the IgGi preparations was determined by prior studies. F(ab)z fragments were isolated using chromatography with a pre-
equilibrated Sephacryl S200 column [ 111. PAGE of the different F(ab)s revealed prominent F(ab), bands with minor smaller molecular weight contaminants. Protein was determined by the Lowry method [12]. Radioiodination Carrier free lz51 or i3iI (DuPont New England Nuclear) were incorporated into appropriate IgGr or F(ab)s preparations using chloramine T (Sigma, St. Louis, MO) as described before [13]. Free iodine was separated from the protein bound iodine using PD-10 Sephadex G25 columns. 1311and ‘*‘I activities were counted using appropriate windows of a gamma emission counter (Nuclear Chicago, IL). Determination of the immunoreactive fraction (IRF), the number of binding sites on Caki-1 cells and the affinity of binding Determination of the IRF according to the method of Lindmo and co-workers 114,151 was carried out by incubating varying numbers of cells (between lo5 and 107) with a constant amount of labeled antibody (typically 0.3 pg). Determination of the number of binding sites for the MABs and the apparent I&,,,, was carried out by incubating a constant number of cells (usually 1 x 10’) with varying amounts of labeled antibody in a total volume of 100 ~1 in glass tubes (12 x 77 mm pre-coated with a 1% BSA solution in phosphate buffered saline (PBS)). After 2 h at 4OC, the unbound radioactivity was removed by washing four times with 2.0 ml of 0.1% BSA plus 0.02% sodium azide in PBS at 4OC. Washing was carried out in the same tubes with centrifugation for 3 min in a Beckman serafuge. Radioactivity associated with the cell pellet was determined. Control incubations, either of cells with a labeled non-specific IgG of the same subclass or of cells with labeled specific MAB plus an excess of unlabeled specific MAB showed that less than 5% of the bound radioactivity was non-specifically bound even at the highest levels of MAB in incubation mixtures. Appropriate corrections were introduced in the curve fitting of the binding data. The Ligand pro-
37
gram of Munson and Rodbard [16] as adapted for a PC by MacPherson [17] was used to calculate the number of binding sites per cell and the apparent K,,,,. Values for the total amount of MAB radioactivity added (dpm) were multiplied by the immunoreactive fraction for that radioiodinated MAB preparation to correct for inactive MAB in the preparation. Gamma camera imaging and determination of distribution of radioiodine-labeled MA& in Caki-1 xenografted nude mice Tumor-bearing mice in groups of 3 were each given intravenous injections of approximately 50 PC1 of 1311-labeled MABs or their F(ab)* fragments and 2 PC1 of 1251 labeled MMG or its F(ab)* fragment. Mice were given Lugol’s iodine in drinking water starting 3 days before administration of radiolabeled agents. Animals under pentobarital anesthesia (35 mg/kg, intraperitoneally) were scanned every 24 h with a large field-of-view gamma scintillation camera interfaced to a PDP 11/34 computer. For assay of radioactivity in tissues, groups were killed at 24, 48 and 96 h after administration of the radiolabeled agents. A 0.5 ml sample of blood was obtained by cardiac puncture from each anesthetized mouse. The axillary vessels were then severed and blood was flushed out of the organs by injection of warm PBS into the heart. Aliquots of tissues were washed in PBS, blotted dry, weighed and individually digested in concentrated sulfuric acid. Radioactivity was determined in duplicate aliquots using at least two different dilutions of each tissue digest. In the analysis of tissues from the mice given both 1311and 1251 labeled preparations, appropriate windows were used for separate assays of 1311and 1251 activites, corrections being made for the counts contributed by 1311to the rz51 window. The total body count of radioactivity was measured using a Mediac Dose Calibrator (Nuclear, Chicago, IL) at 8-h intervals up to a maximum of 96 h to obtain the biological halflife. Radioactivity measurements on the animals were corrected for decay of radioactivity
HPLC
assay of MABs and mouse sera HPLC assays were performed using a BlO-SILO column (BioRad, CA) on 50 ~1 samples of serum obtained from nude mice at 24 and 48 h post injection. Samples of sera were diluted 1:l with PBS. The eluting buffer contained 0.05 M sodium sulfate plus 0.02 M sodium dihydrogen phosphate (pH 6.8). One milliliter fractions were collected at a rate of 1 fraction per min and counted for radioactivity associated with each of the iodine isotopes. The radioactivity data were plotted versus fraction number and the recovery of radioactivity calculated. A sample of the original serum was also counted so the recovery of radioactivity from HPLC could be related to the original serum. For determination of antitumor antibody activity in mouse serum, sera from antibody injected mice were incubated with a large number of Caki-1 cells (i.e. lo7 cells) to provide an excess of antigen-binding sites. After incubation at 37OC for 1 h, the cells were washed and the amount of cell associated radioactivity was determined. Statistical evaluation The significance of the differences amount of the MABs and their fragments that localized in the tumor grafts and various normal tissues was mined by Student’s t-test.
in the F(ab), xenodeter-
Results Characterization of DAL K45, DAL K29 and their F(ab), moieties with respect to binding to Caki- 1 cells Table I shows the number
of sites on Caki-1 cells available for binding to DAL K45, K29 and their F(ab)* moieties. The immunoreactive fraction (IRF) of all DAL K29 and its F(ab), preparations was 270.75; those of DAL K45 and DAL K45 F(ab)z were ~0.50. The fraction of the MAB that localized in different tissues (Figs. la-d) show the fractions of injected radioactivity that localized in different tissues. The highest tumor localization
38 Table 1. MAB or F(ab),
No. of binding sites per Caki- 1 cells (x 105)
K,,,, (M” x 109)
K45 K45 F(ab), K29 K29 F(ab),
1.2 1.2 10.4 10.7
27 2.4 0.2 0.15
Binding of MABs DAL K29, DAL K45 and their F(ab), fragments to human renal cell carcinoma Caki-1 cells. Values for the total number of binding sites per cell and the K,,, were determined as described in Materials and Methods.
FRACTION MEAN
VALUES
OF K29 THAT OF TISSUES
FROM
LOCALIZED 3 NUDE
MICE
FRACTION PEACENTAGE
% OF K29 F(ab)2 MEAN
VALUES
THAT
OF TISSUES
FROM
LOCALIZED 3 NUDE
MICE
PERCENTAGE
PERCENTAGE
MEAN
was found after injection of MAB DAL K45, i.e. 2.5% of the injected dose which remained virtually unchanged during the 96 h period of observation. In contrast, only 0.9% of the injected DAL K29 localized in the tumor. This was observed at 48 h after injection and declined to approximately 0.5% of the injected dose at 96 h. As regards F(ab), fragments, approximately 1.3% of the injected dose of K45 F(ab)s localized in the tumor at 24 h and this declined to 0.9% at 48 h. The corresponding figures for K29 F(ab), were, respectively, 1.5 and 0.4% of the injected dose. Irrespective of whether labeled IgG or F(ab)s moieties were administered, the highest tumor localization was achieved at 24 h after injection. How-
VALUES
OF K45 OF TISSUES
THAT FROM
LOCALIZED 3 NUDE
MICE
% OF K45 MEAN
VALUES
F(ab)2
THAT
OF TISSUES
FROM
LOCALIZED 3 NUDE
MICE
PEACENTAGE
Fig. 1. Fractions of injected radioactivity that localized in different tissues at various times after intravenous of 1311-labeled MAB DAL K29, DAL K45 or their F(ab), fragments.
injection
39
ever, at 48 h the amounts of intact K29 and K45 IgG in the tumor xenograft remained virtually unchanged, but the amounts of the corresponding F(ab)z moieties were reduced to l/3 at 48 h. Both K29 and K29 F(ab)zshowed the highest hepatic and splenic localization at 48 h and the lowest at 96 h. There was no difference between K29 IgG and K29 F(ab)z with respect to the proportion of injected dose that localized in liver and spleen. Compared to K29 IgG, a higher proportion of K45 IgG showed hepatic and splenic localizaton that remained virtually unchanged during the 96 h period of observation. The proportion of K45 F(ab)z that localized in the liver and spleen was approximately half that of the parent IgG. Compared to all the normal
TUMOR
MEAN
TISSUE
RATIOS
K29
TUMOR
! NORMAL
/ NORMAL
TISSUE
RATIOS
K45
TUMOR
/ NORMAL
MEANVALUESOF 9 7
Tumor to normal tissue ratios of localization Excluding whole blood and serum, both DAL K29 and K45 showed tumor to normal tissue (T:N) ratio above 1.0 at all the times of assay (Fig. 2a,c). The T:N ratios were generally higher after injection of DAL K45 than after the injection of DAL K29. This includes the T:N kidney ratios of localization. Compared to the intact DAL K29 IgG, higher T:N ratios were obtained with the F(ab)z moieties at 24 h, but the ratios went down considerably at 48
I NORMAL
VALUES OF TISSUES FROM
TUMOR
tissues (except blood or serum), there was significantly more tumor localization of K45 IgG at 96 h (P < O.Ol), K45 F(ab)z at 48 h (P < 0.005) and K29 F(ab)z at 24 h (P < 0.05).
TISSUES FROM
3 NUDEMICE
MEAN
MEANVALUESOF
3 NUDEMICE
RATIOS
10 9 8 7 6 5 4 3 2 1 0
6 5 4 3 2 , 0
TlME;DAyS)
Fig. 2.
RATIOS
K29
TISSUE
RATIOS
K45 Flab)2
TISSUES FROM
F(ab)Z
3 NUDEMICE
3 NUDE MICE
RATIOS
i-
i
TISSUE
VALUES OF TISSUES FROM
Tumor to normal tissue ratios of localization of radioactivity labeled DAL K29, DAL K45 or their F(ab)z fragments.
i
1 TIME [DAYS)
at various times after intravenous
injection of 13*1-
40
and 96 h (Fig. 2b). In contrast, high T:N values were maintained by K45 F(ab)s for at least 48 h (Fig. 2d).
Specificity index of localization The specificity index of localization (SPIL i.e. tumor:normal tissue ratio of the specific antibody/tumor:normaI tissue ratio of an isotype matched non-specific IgG) was > 1 with respect to most tissues (Fig. 3) indicating that the localization of the MABs and their F(ab), moieties was not due to non-specific factors such as increased permeability of tumor vasculature. The SPIL for K45 and K45 F(ab), were higher compared to K29 and K29 F(ab),. SPECIFICITY MEAN
VALUES
RATIOS
OF TISSUES
FROM
Clearance of radioactiuity The t1,2 of whole body radioactivity corresponded closely with the tlj2 of radioactivity in whole blood or serum. The t1,2 of i311labeled K45, K29, K45 F(ab)s and K29 F(ab)2 were respectively 42, 12, 11 and 8 h. Both the MABs and their F(ab)s fragments cleared slower from the tumor xenograft than from any normal tissue. Gamma camera imaging Of the agents tested, the most distinct tumor images were produced by K45 F(ab), at 24 h (Fig. 4), followed in order by K45 at 96 and 48 h, K29 at 96 h and then K29 F(ab), at 48 h. HPLC assay of mouse sera On HPLC assay, the two MABs and their
OF K29 3 NUDE
SPECIFICITY
MICE
RATIOS
MEAN
100
VALUES
RATIOS
OF TISSUES
OF K29 F(ab)2
FROM
3 NUDE
MICE
RATIOS
,_______________.............................. m ..__...____...._.--.-----_................., 10 _ _ __
._ _
1
I
SPECIFICITY MEAN
VALUES
RATIOS
OF TISSUES
FROM
OF K45 3 NUDE
SPECIFICITY
MICE
MEAN
AATl”S
100
VALUES
2
4
RATIOS OF K45 OF TISSUES
FROM
3 NUDE
F(ab)2 MICE
RATIOS
Fig. 3. Specificity indices of localization (i.e. T:N ratio of localization of the specific MAB or F(ab),/T:N ratio of localization of an isotype matched non-specific Ig or its F(ab), fragment) of DAL K29, DAL K45 and their F(ab), fragments.
41
Fig. 4.
A large field-of-view gamma camera image of an intrarenal human xenograft bearing nude mouse 24 h after intravenous injection of 50 j&i of i3’I bound to the F(ab)s moiety of MAB DAL K45. The scan of *311 distribution was obtained at 364 KeV with a 20% window, stored in a PDP 11/34 computer and then displayed in a linear color scale from dark blue (lowest activity) to yellow (highest activity), Localization of radioactivity in excess of the normal tissue background can be seen in the renal cancer xenograft in the right kidney (arrow).
F(ab)z fragments, showed a single radioactive protein peak that corresponded exactly with control radiolabeled MABs or their F(ab), fragments. When sera from the mice obtained at 24 or 48 h after intravenous administration of the labeled MAB or their F(ab)z fragments were incubated with Caki-1 cells, approximately 50% of the radioactivity could be absorbed out by Caki-1 cells indicating that 50% of the radiolabeled preparations in circulation had retained antibody activity. Discussion
There was no difference MAB IgGs and their
between the parent respective F(ab)z
fragments as regards the number of sites on Caki-1 cells to which they could bind. K45 intact IgG showed the highest tumor localization even though fewer binding sites were available to it compared to K29. Our preliminary experiments had demonstrated that the amounts of the two MABs and their F(ab)z fragments injected per mouse were below the saturation dose for the tumor. Therefore the higher tumor localization of tl,z K45 IgG and the prolonged maintenance of this high level of tumor localization may be due to its higher affinity and/or its longer biological tt,z compared to K29 IgG and the two F(ab)* moieties. Consistent with our previously reported observation on radioiodine-labeled antimelanoma MABs [US], the results of HPLC assay of the sera from the mice given radioiodine labeled IgG or F(ab)z demonstrate that, after intravenous administration of radiolabeled MABs or their little dissociated free F(ab)z fragments, radioiodine or radioiodine-containing small catabolic fragments are detectable in serum during the 96 h period of observation even though there is partial loss of antibody activity in the circulating labeled immunoglobulins and their F(ab)z fragments. Among factors that determine the effectiveness of antibody based tumor imaging, the T:N ratio of localization of the antibody is regarded as the single most important determinant of scan sensitivity followed by the absolute amount of radioactivity localized at the tumor site [19]. Thus, even though the intact K45 IgG showed higher tumor localization than its F(ab)z moiety, the latter produced images of better quality at 24 h because of its higher T:N ratios of localization. Furthermore, the longer biological tr,z of K45 IgG was associated with a relatively high whole body background activity. Consistent with our own previously reported observations [20] and those of others [19,21], F(ab)z moieties cleared faster from the serum of other tissues compared to their respective parent IgGs. The faster clearance of F(ab)z-linked radioactivity is likely to be associated with the smaller size of the protein (and hence ease of transcapillary
42
passage and renal clearance) as well as the decrease in K,. The decrease in the K, of the F(ab), moieties compared to their respective parent IgGs may be due to steric changes in the antigen-binding sites and/or increased susceptibility of the fragments to partial denaturation during radioiodination [ZZ] . Both K45 F(ab), and K29 F(ab)z showed much faster clearance from the tumor xenografts than the parent IgGs. However, both of our tumor-specific antibodies and their F(ab)s moieties were retained longer in the target tumors compared to the isotype matched nonspecific IgG or its F(ab)* moiety. Such longer retention of antitumor antibodies or their immunoreactive fragments in target tumor tissues has been observed with other, (but not all) antitumor MABs [26]. The rapid decrease in the absolute amount of tumor-associated radioactivity observed at 48 h in our mice given ‘311-labeled F(ab)s moieties of K45 or K29 adversely affected the quality of imaging even though significantly more radioactivity remained in the tumors compared to the normal tissues in mice given 1311-labeled K45 F(ab),. Following the observation of Pressman and Keighley [23] that radiolabeled antibodies did localize in the target tissue in vivo, a number of laboratories, including ours [ 13,24 - 261, have demonstrated that it is indeed possible to use radiolabeled antitumor antibodies for tumor diagnosis and therapy [21,27] both in experimental models and in patients. However, results from clinics have generally been less impressive than those obtained in experimental models [21,27,28]. We have a long standing interest in developing RCC specific antibodies [29] mainly because there is no tumor marker like CEA, AFP, etc. that can be of help in the diagnosis and monitoring of RCC. Using polyclonal anti-RCC antibodies [30] we could successfully image 13 out of 15 primary lesions and metastases in 7 out of 8 RCC patients [24]. Due to well recognized limitations of most polyclonal antibodies [25] (e.g. low IRF, the presence of antibodies that are directed against tissues other than the target tumor, batch to batch variations in immunological pro-
per-ties, etc.), we produced several MABs against RCC associated antigens [4]. It is very encouraging that PAL K45 which has the most tumor-restricted specificity has also optimal qualities for localizing in tumors either as such or as the F(ab)s derivative and therefore has the potential for targeting radionuclides and chemotherapeutic agents in cancer patients. Of the rather limited number of reported MABs against human RCC associated antigens, only a few have been evaluated for tumor localization in vivo [31]. Acknowledgment This investigation was supported by the Medical Research Council of Canada (Grant No. MT 10964), Cancer Research Society, Inc., Montreal and the Kidney Foundation of Canada. We are grateful to Ms. H. Maxner for the preparation of this manuscript. References Ghose,
T. and Blair,
agent-antibody
A.H.
conjugates.
(1987) CRC
Design of cytotoxic
Crit.
Rev.
Ther.
Drug
Carrier Syst., 3, 263 - 361. Ghose, T., Blair, A.H.
and Kulkarni, P.N. (1983)
tion of antibody-linked zymol.,
agents.
Bracken,
R.B.
(1987)
Semin.
Luner,
Ghose,
S.J..
Belitsky, P. (1986) tumor-associated
Renal carcinoma: Roentgenol., T.,
Chatterjee,
Monoclonal
clinical aspects
S., Cruz,
and
surface antigens of human renal cell car-
(1991)
Inhibition
of human
monoclonal-anti-body-linked 331-
H.N.
antibodies to kidney and
Cancer Res., 46, 5816 - 5820.
P.
tumor
En-
22, 241-247.
Singh. M., Ghose, R., Kralovec. E., Blair, A.H. sky,
Prepara-
Methods
93. 280 - 333.
and therapy.
cinoma.
cytotoxic
model.
Cancer
renal
methotrexate Immunol.
and Belitcancer
by
in an ascites
Immunother.,
32,
334.
Singh, M., Kralovec,
J., Mezei, M. and Ghose, T. (1989)
Inhibition of human renal cancer by methotrexate a monoclonal
antibody.
Singh,
M.,
Ghose,
Mezei,
M.
(1989)
liposomes
with
renal cancer.
T.,
J. Ural., Faulkner,
Targeting
linked to
141, 428-431. G.,
Kralovec,
J. and
of methotrexate-containing
a monoclonal
antibody
against
human
Cancer Res., 49, 3965 - 3984.
Singh, M., Ghose, T.. Mezei, M. and Belitsky, P. (1991) Inhibition of human renal cancer by monoclonal targeted
methotrexate-containing
tumor model.
Cancer Lett.,
Fogh. J. and Trempe.
antibody
liposomes in an ascites
56, 97 - 102.
G. (1975)
New human tumor cell
43
154.
21
Editor: J. Fogh, Plenum, New York. McKinney, M.M. and Parkinson, A. (1987) A simple, non chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J. Immunol. Methods, 96,
22
lines. In: Human 10
11
12
13
14
15
Tumor
Cells in Vitro,
271- 278. Kulkarni, P.N., Blair, A.H., Ghose. (1985) Conjugation of methotrexate their F(ab’)n fragments and the
pp.
115-
T. and Mammen, M. to IgG antibodies and effect of conjugated
methotrexate on tumor growth in viva. Cancer Immunol. Immunother., 16, 127 - 129. Lowry, O.H. 1Rosebrough, N. J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol
Immunol. Methods, 72, 77 - 89. Lindmo, T. and Bunn, P.A., Jr. (1986) Determination of the true immunoreactive fraction of monoclonal antibodies after radiolabeling. Methods Enzymol., 121, 678 - 691.
19
Munson, P.J. and Rodbard, D. (1980) Ligand: a versatile computerized approach for characterization of ligandbinding systems. Anal. Biochem.. 107. 220- 239. MacPherson, G.A. (1985) Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. J. Pharmacol. Methods, 14, 213-228. Ghose, T.J., Kulkami, N., Ferrone. S., Giacomini, P Norvell, S.T., Kulkami. P. and Blair, A.H. (1983) Imaging human tumors in nude mice. In: Radioimmunoimaging, pp. 255-263, Editors: S. Burchiel and B.A. Rhodes, Elvsevier, New York. Bradwell, A.R., Fairweather, D.S.. Dykes, P.W., Keeling,
20
A., Vaughan, A. and Taylor, J. (1985) Limiting factors in the localization of tumours with radiolabelled antibodies. Immumol. Today, 6, 163- 170. Ghose. T., Blair, A.H.. Martin, R.H., Novell, S.T.,
16
17
18
Taube, R.A., Adelstin, S.J. and Kassis, A.1. (1991) Antigen-binding site protection during radiolabeling leads to a higher immunoreactive fraction. J. Nucl. Med., 32, 116 - 122. 23
Pressman, D. and Keighley, G. (1948) The zone of activity of antibodies as determined by the use of radioactive tracers: the zone of activity of nephrotoxic antikidney serum. J. Immunol.. 59. 141- 146.
24
Ghose, Guclu, labeled mouse
reagent. J. Biol. Chem., 193, 265- 275. Ghose, T. and Guclu. A. (1974) Cure of a mouse lymphoma with radioiodinated antibody. Eur. J. Cancer, 10, 787 - 792. Lindmo, T., Boven, E., Cutitta, F., Fedorko, J. and Bunn, P.A. Jr. (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J.
Ramakrishnan, S., Belitsky, P. and Ferrone, S. (1982) Tumor imaging by antitumor antibody linked radionuclides. In: Tumor Imaging, pp. 167 - 178, Editor: S. Burchiel. Masson, New York.
Goldenberg, D.M. (1988) Targeting of cancer with radiolabeled antibodies. Prospects for imaging and therapy. Arch. Pathol. Lab. Med.. 112. 580-587. Van den Abbeele, A.D., Aaronson, R.A., Daher, S.,
25
26
27
28
29
30
31
T., Norvell. S.T., Aquino. J., Belitsky, P.. Tai, J., A. and Blair A.H. (1980) Localization of 1311antibodies in human renal cell carcinomas and in a hepatoma and correlation with tumor detection by
photoscanning. Cancer Res., 40, 3018-3031. Ghose. T., Ferrone. S., Imai, K.. Norvell, S.T.. Luner, S.J., Martin, R.H. and Blair, A.H. (1981) Imaging of human melanoma xenografts in nude mice with a radiolabeled monoclonal antibody. J Natl. Cancer Inst., 69. 823 - 826. Ghose, T., Tai, J. and Aquino. J. (1975) Tumor localization of ‘311-labeled antibodies by radionuclide imaging. Radiology, 116. 445 - 448. Kramer, E.L. and Larson, S.M. (1991) Tumor targeting with radiolabeled antibody for diagnosis and therapy, Immunol. Allerg. Clin. North Am., 11, 301-339. Begent, R.H.J. and Pedley. R.B. (1990) Antibody targeted therapy in cancer: comparison of murine and clinical studies. Cancer Treat. Rev., 17. 373 - 378. Nairn. R.C.. Philip, J., Ghose. T., Porteous, I.B. and Fothergill, J.E. (1963) Production of a precepitin against renal cancer. Br. Med. J., 1, 1702- 1704. Ghose. T.. Belitsky. P., Tai. J. and Janigan. D.T. (1979) Production and characterization of xenogeneic antisera to a human renal cell carcinoma associated antigen. J. Natl. Cancer Inst., 63, 301- 308. Vessella, R.L., Alvarez. V . Chiou. R-K., Rodwell. J., Elson, M., Plame. D., Shafter, R. and Lange, P. (1987) Radioimmunoscintigraphy and radioimmunotherapy of renal cell carcinoma xenografts. Natl. Cancer Inst. Monogr.. 3, 159- 167.
35 Ltd.
Tumor localization of monoclonal antibodies renal carcinoma in a xenograft model A.K. Guha”, T. Ghose”, M. Mammena Departments
of “Pathology,
M. Singh”,
bNuclear
Medicine
J. Aquinob,
against human
A.H. Blair’, S.J. Lunera and
and ‘Biochemistry,
Dalhousie
University.
Halifax.
N.S.
B3H
4H7
[Canada) (Received
2 July 1991)
(Accepted
9 September
1991)
Summary
ingly used to target chemotherapeutic agents or radionuclides selectively towards cancer cells [1,2]. Metastatic renal cell carcinoma (RCC) is a relentless, progressive disease that is hardly affected by any form of current therapy including chemotherapy [3]. We have produced several monoclonal antibodies against human RCC associated antigens [4]. We have also demonstrated that when these MABs are linked to the antimetabolite methotrexate (MTX) [5,6], or to MTX containing small unilamellar vesicles, the conjugated antibodies bind to human RCC cell lines such as Caki-1 and are more effective inhibitors of Caki-1 cells in vitro [7] or Caki-1 cells grown as ascites tumors in nude mice [8] compared to equimolar amounts of the drug alone or the drug linked to a non-specific immunoglobulin. However, to be clinically useful, tumorselective localization of these MABs in vivo is essential. Using intrarenal xenografts of Caki- 1 cells in nude mice, we demonstrate that the two of our MABs tested i.e. DAL K29 and DAL K45 and their respective F(ab)* fragments indeed localize in the tumor xenografts.
We investigated the localization of intravenously injected DAL K45 and DAL K29, two monoclonal antibodies (MABs) against human renal cell carcinoma (RCC), and their F(abj2 fragments in nude mice bearing intratenal transplants of the RCC line Caki-I. More of the MABs or their F(ab)g specifically localized in the tumor than in any normal tissue with the exception of blood. Compared to parent MABs, F(ab)+ were cleared faster from all tissues. In serum, the MABs and F(ab)+ showed a single radioactive peak reimmunoreactiuiry. taining partial DAL K45-F(ab)* showed the highest tumor:normal tissue localization ratios and the most distinct gamma-camera image at 24 h. Keywords: monoclonal antibodies; tumor localization; tumor imaging; human renal carcinoma Introduction Monoclonal antibodies tumor associated antigens
(MAB) to human are being increas-
Correspondence to: T. Ghose, Department Dalhousie University, Sir Charles Tupper Halifax,
N.S. B3H 4H7.
0304-3835/91/$03.50 Published .znd Printed
Materials and Methods Tumor and monoclonal antibodies The Caki-1 cell line, originally derived from a skin metastasis of renal cell carcinoma from
of Pathology, Medical Bldg.,
Canada.
0 1991 Elsevier Scientific Publishers in Ireland
Ireland
Ltd
36
an adult Caucasian male [9] and the nonspecific mouse IgGi-producing myeloma line ATCC TIB9 were obtained from the Human Tumor Cell Bank through the American Type Culture Collection (Rockwell, MD). A subline of Caki-1 cells was developed by serial transplantation of Caki-1 cells subcapsularly into the kidney of adult female, athymic (nu/nu) BALB/c mice (Life Science Inc., St. Petersburg, FL.) so that intrarenal inoculation of 2 x lo6 Caki-1 cells produced progressive tumors in 100% of inoculated mice. For intrarenal inoculation, a longitudinal incision, approximately 1 cm long, was made in the skin of the mice under anesthesia, the kidney was exposed after dissection of the perirenal fat and tumor cells were inoculated subcapsularly using a 25 gauge needle. Tumors could be palpated in the left kidney approximately 15 - 20 days after tumor inoculation and weighed about 500 mg at the conclusion of the imaging studies. MAB DAL K29 reacted with 8/9 renal carcinomas in our laboratory and precipitated molecules with molecular masses of 118 kDa and 150 kDa from extracts of surface-labeled Caki-1 cells. MAB DAL K45 reacted with 4/6 RCCs tested and precipitated 177-kDa and 150-kDa antigens from surface-labeled Caki-1 cells. The details of the specificity and reactivity of these two MABs have been reported [4]. The non-specific murine monoclonal IgGi and the MABs were isolated by precipitation with caprylic acid [lo] from the ascites fluid of BALB/c mice (Life Science Inc.) intraperitoneally inoculated with lo7 myeloma or appropriate hybridoma cells. Polyacrylamide gel electrophoresis (PAGE) of the purified preparations revealed a homogeneous IgG band and a very small amount of contaminating albumin. F(ab)z fragments of the different IgGi preparations were obtained by digestion with pepsin (Sigma, St. Louis, MO) at 37OC, (pH 4.2) at a pepsin protein ratio of 1:33. The optimal duration of digestion for each of the IgGi preparations was determined by prior studies. F(ab)z fragments were isolated using chromatography with a pre-
equilibrated Sephacryl S200 column [ 111. PAGE of the different F(ab)s revealed prominent F(ab), bands with minor smaller molecular weight contaminants. Protein was determined by the Lowry method [12]. Radioiodination Carrier free lz51 or i3iI (DuPont New England Nuclear) were incorporated into appropriate IgGr or F(ab)s preparations using chloramine T (Sigma, St. Louis, MO) as described before [13]. Free iodine was separated from the protein bound iodine using PD-10 Sephadex G25 columns. 1311and ‘*‘I activities were counted using appropriate windows of a gamma emission counter (Nuclear Chicago, IL). Determination of the immunoreactive fraction (IRF), the number of binding sites on Caki-1 cells and the affinity of binding Determination of the IRF according to the method of Lindmo and co-workers 114,151 was carried out by incubating varying numbers of cells (between lo5 and 107) with a constant amount of labeled antibody (typically 0.3 pg). Determination of the number of binding sites for the MABs and the apparent I&,,,, was carried out by incubating a constant number of cells (usually 1 x 10’) with varying amounts of labeled antibody in a total volume of 100 ~1 in glass tubes (12 x 77 mm pre-coated with a 1% BSA solution in phosphate buffered saline (PBS)). After 2 h at 4OC, the unbound radioactivity was removed by washing four times with 2.0 ml of 0.1% BSA plus 0.02% sodium azide in PBS at 4OC. Washing was carried out in the same tubes with centrifugation for 3 min in a Beckman serafuge. Radioactivity associated with the cell pellet was determined. Control incubations, either of cells with a labeled non-specific IgG of the same subclass or of cells with labeled specific MAB plus an excess of unlabeled specific MAB showed that less than 5% of the bound radioactivity was non-specifically bound even at the highest levels of MAB in incubation mixtures. Appropriate corrections were introduced in the curve fitting of the binding data. The Ligand pro-
37
gram of Munson and Rodbard [16] as adapted for a PC by MacPherson [17] was used to calculate the number of binding sites per cell and the apparent K,,,,. Values for the total amount of MAB radioactivity added (dpm) were multiplied by the immunoreactive fraction for that radioiodinated MAB preparation to correct for inactive MAB in the preparation. Gamma camera imaging and determination of distribution of radioiodine-labeled MA& in Caki-1 xenografted nude mice Tumor-bearing mice in groups of 3 were each given intravenous injections of approximately 50 PC1 of 1311-labeled MABs or their F(ab)* fragments and 2 PC1 of 1251 labeled MMG or its F(ab)* fragment. Mice were given Lugol’s iodine in drinking water starting 3 days before administration of radiolabeled agents. Animals under pentobarital anesthesia (35 mg/kg, intraperitoneally) were scanned every 24 h with a large field-of-view gamma scintillation camera interfaced to a PDP 11/34 computer. For assay of radioactivity in tissues, groups were killed at 24, 48 and 96 h after administration of the radiolabeled agents. A 0.5 ml sample of blood was obtained by cardiac puncture from each anesthetized mouse. The axillary vessels were then severed and blood was flushed out of the organs by injection of warm PBS into the heart. Aliquots of tissues were washed in PBS, blotted dry, weighed and individually digested in concentrated sulfuric acid. Radioactivity was determined in duplicate aliquots using at least two different dilutions of each tissue digest. In the analysis of tissues from the mice given both 1311and 1251 labeled preparations, appropriate windows were used for separate assays of 1311and 1251 activites, corrections being made for the counts contributed by 1311to the rz51 window. The total body count of radioactivity was measured using a Mediac Dose Calibrator (Nuclear, Chicago, IL) at 8-h intervals up to a maximum of 96 h to obtain the biological halflife. Radioactivity measurements on the animals were corrected for decay of radioactivity
HPLC
assay of MABs and mouse sera HPLC assays were performed using a BlO-SILO column (BioRad, CA) on 50 ~1 samples of serum obtained from nude mice at 24 and 48 h post injection. Samples of sera were diluted 1:l with PBS. The eluting buffer contained 0.05 M sodium sulfate plus 0.02 M sodium dihydrogen phosphate (pH 6.8). One milliliter fractions were collected at a rate of 1 fraction per min and counted for radioactivity associated with each of the iodine isotopes. The radioactivity data were plotted versus fraction number and the recovery of radioactivity calculated. A sample of the original serum was also counted so the recovery of radioactivity from HPLC could be related to the original serum. For determination of antitumor antibody activity in mouse serum, sera from antibody injected mice were incubated with a large number of Caki-1 cells (i.e. lo7 cells) to provide an excess of antigen-binding sites. After incubation at 37OC for 1 h, the cells were washed and the amount of cell associated radioactivity was determined. Statistical evaluation The significance of the differences amount of the MABs and their fragments that localized in the tumor grafts and various normal tissues was mined by Student’s t-test.
in the F(ab), xenodeter-
Results Characterization of DAL K45, DAL K29 and their F(ab), moieties with respect to binding to Caki- 1 cells Table I shows the number
of sites on Caki-1 cells available for binding to DAL K45, K29 and their F(ab)* moieties. The immunoreactive fraction (IRF) of all DAL K29 and its F(ab), preparations was 270.75; those of DAL K45 and DAL K45 F(ab)z were ~0.50. The fraction of the MAB that localized in different tissues (Figs. la-d) show the fractions of injected radioactivity that localized in different tissues. The highest tumor localization
38 Table 1. MAB or F(ab),
No. of binding sites per Caki- 1 cells (x 105)
K,,,, (M” x 109)
K45 K45 F(ab), K29 K29 F(ab),
1.2 1.2 10.4 10.7
27 2.4 0.2 0.15
Binding of MABs DAL K29, DAL K45 and their F(ab), fragments to human renal cell carcinoma Caki-1 cells. Values for the total number of binding sites per cell and the K,,, were determined as described in Materials and Methods.
FRACTION MEAN
VALUES
OF K29 THAT OF TISSUES
FROM
LOCALIZED 3 NUDE
MICE
FRACTION PEACENTAGE
% OF K29 F(ab)2 MEAN
VALUES
THAT
OF TISSUES
FROM
LOCALIZED 3 NUDE
MICE
PERCENTAGE
PERCENTAGE
MEAN
was found after injection of MAB DAL K45, i.e. 2.5% of the injected dose which remained virtually unchanged during the 96 h period of observation. In contrast, only 0.9% of the injected DAL K29 localized in the tumor. This was observed at 48 h after injection and declined to approximately 0.5% of the injected dose at 96 h. As regards F(ab), fragments, approximately 1.3% of the injected dose of K45 F(ab)s localized in the tumor at 24 h and this declined to 0.9% at 48 h. The corresponding figures for K29 F(ab), were, respectively, 1.5 and 0.4% of the injected dose. Irrespective of whether labeled IgG or F(ab)s moieties were administered, the highest tumor localization was achieved at 24 h after injection. How-
VALUES
OF K45 OF TISSUES
THAT FROM
LOCALIZED 3 NUDE
MICE
% OF K45 MEAN
VALUES
F(ab)2
THAT
OF TISSUES
FROM
LOCALIZED 3 NUDE
MICE
PEACENTAGE
Fig. 1. Fractions of injected radioactivity that localized in different tissues at various times after intravenous of 1311-labeled MAB DAL K29, DAL K45 or their F(ab), fragments.
injection
39
ever, at 48 h the amounts of intact K29 and K45 IgG in the tumor xenograft remained virtually unchanged, but the amounts of the corresponding F(ab)z moieties were reduced to l/3 at 48 h. Both K29 and K29 F(ab)zshowed the highest hepatic and splenic localization at 48 h and the lowest at 96 h. There was no difference between K29 IgG and K29 F(ab)z with respect to the proportion of injected dose that localized in liver and spleen. Compared to K29 IgG, a higher proportion of K45 IgG showed hepatic and splenic localizaton that remained virtually unchanged during the 96 h period of observation. The proportion of K45 F(ab)z that localized in the liver and spleen was approximately half that of the parent IgG. Compared to all the normal
TUMOR
MEAN
TISSUE
RATIOS
K29
TUMOR
! NORMAL
/ NORMAL
TISSUE
RATIOS
K45
TUMOR
/ NORMAL
MEANVALUESOF 9 7
Tumor to normal tissue ratios of localization Excluding whole blood and serum, both DAL K29 and K45 showed tumor to normal tissue (T:N) ratio above 1.0 at all the times of assay (Fig. 2a,c). The T:N ratios were generally higher after injection of DAL K45 than after the injection of DAL K29. This includes the T:N kidney ratios of localization. Compared to the intact DAL K29 IgG, higher T:N ratios were obtained with the F(ab)z moieties at 24 h, but the ratios went down considerably at 48
I NORMAL
VALUES OF TISSUES FROM
TUMOR
tissues (except blood or serum), there was significantly more tumor localization of K45 IgG at 96 h (P < O.Ol), K45 F(ab)z at 48 h (P < 0.005) and K29 F(ab)z at 24 h (P < 0.05).
TISSUES FROM
3 NUDEMICE
MEAN
MEANVALUESOF
3 NUDEMICE
RATIOS
10 9 8 7 6 5 4 3 2 1 0
6 5 4 3 2 , 0
TlME;DAyS)
Fig. 2.
RATIOS
K29
TISSUE
RATIOS
K45 Flab)2
TISSUES FROM
F(ab)Z
3 NUDEMICE
3 NUDE MICE
RATIOS
i-
i
TISSUE
VALUES OF TISSUES FROM
Tumor to normal tissue ratios of localization of radioactivity labeled DAL K29, DAL K45 or their F(ab)z fragments.
i
1 TIME [DAYS)
at various times after intravenous
injection of 13*1-
40
and 96 h (Fig. 2b). In contrast, high T:N values were maintained by K45 F(ab)s for at least 48 h (Fig. 2d).
Specificity index of localization The specificity index of localization (SPIL i.e. tumor:normal tissue ratio of the specific antibody/tumor:normaI tissue ratio of an isotype matched non-specific IgG) was > 1 with respect to most tissues (Fig. 3) indicating that the localization of the MABs and their F(ab), moieties was not due to non-specific factors such as increased permeability of tumor vasculature. The SPIL for K45 and K45 F(ab), were higher compared to K29 and K29 F(ab),. SPECIFICITY MEAN
VALUES
RATIOS
OF TISSUES
FROM
Clearance of radioactiuity The t1,2 of whole body radioactivity corresponded closely with the tlj2 of radioactivity in whole blood or serum. The t1,2 of i311labeled K45, K29, K45 F(ab)s and K29 F(ab)2 were respectively 42, 12, 11 and 8 h. Both the MABs and their F(ab)s fragments cleared slower from the tumor xenograft than from any normal tissue. Gamma camera imaging Of the agents tested, the most distinct tumor images were produced by K45 F(ab), at 24 h (Fig. 4), followed in order by K45 at 96 and 48 h, K29 at 96 h and then K29 F(ab), at 48 h. HPLC assay of mouse sera On HPLC assay, the two MABs and their
OF K29 3 NUDE
SPECIFICITY
MICE
RATIOS
MEAN
100
VALUES
RATIOS
OF TISSUES
OF K29 F(ab)2
FROM
3 NUDE
MICE
RATIOS
,_______________.............................. m ..__...____...._.--.-----_................., 10 _ _ __
._ _
1
I
SPECIFICITY MEAN
VALUES
RATIOS
OF TISSUES
FROM
OF K45 3 NUDE
SPECIFICITY
MICE
MEAN
AATl”S
100
VALUES
2
4
RATIOS OF K45 OF TISSUES
FROM
3 NUDE
F(ab)2 MICE
RATIOS
Fig. 3. Specificity indices of localization (i.e. T:N ratio of localization of the specific MAB or F(ab),/T:N ratio of localization of an isotype matched non-specific Ig or its F(ab), fragment) of DAL K29, DAL K45 and their F(ab), fragments.
41
Fig. 4.
A large field-of-view gamma camera image of an intrarenal human xenograft bearing nude mouse 24 h after intravenous injection of 50 j&i of i3’I bound to the F(ab)s moiety of MAB DAL K45. The scan of *311 distribution was obtained at 364 KeV with a 20% window, stored in a PDP 11/34 computer and then displayed in a linear color scale from dark blue (lowest activity) to yellow (highest activity), Localization of radioactivity in excess of the normal tissue background can be seen in the renal cancer xenograft in the right kidney (arrow).
F(ab)z fragments, showed a single radioactive protein peak that corresponded exactly with control radiolabeled MABs or their F(ab), fragments. When sera from the mice obtained at 24 or 48 h after intravenous administration of the labeled MAB or their F(ab)z fragments were incubated with Caki-1 cells, approximately 50% of the radioactivity could be absorbed out by Caki-1 cells indicating that 50% of the radiolabeled preparations in circulation had retained antibody activity. Discussion
There was no difference MAB IgGs and their
between the parent respective F(ab)z
fragments as regards the number of sites on Caki-1 cells to which they could bind. K45 intact IgG showed the highest tumor localization even though fewer binding sites were available to it compared to K29. Our preliminary experiments had demonstrated that the amounts of the two MABs and their F(ab)z fragments injected per mouse were below the saturation dose for the tumor. Therefore the higher tumor localization of tl,z K45 IgG and the prolonged maintenance of this high level of tumor localization may be due to its higher affinity and/or its longer biological tt,z compared to K29 IgG and the two F(ab)* moieties. Consistent with our previously reported observation on radioiodine-labeled antimelanoma MABs [US], the results of HPLC assay of the sera from the mice given radioiodine labeled IgG or F(ab)z demonstrate that, after intravenous administration of radiolabeled MABs or their little dissociated free F(ab)z fragments, radioiodine or radioiodine-containing small catabolic fragments are detectable in serum during the 96 h period of observation even though there is partial loss of antibody activity in the circulating labeled immunoglobulins and their F(ab)z fragments. Among factors that determine the effectiveness of antibody based tumor imaging, the T:N ratio of localization of the antibody is regarded as the single most important determinant of scan sensitivity followed by the absolute amount of radioactivity localized at the tumor site [19]. Thus, even though the intact K45 IgG showed higher tumor localization than its F(ab)z moiety, the latter produced images of better quality at 24 h because of its higher T:N ratios of localization. Furthermore, the longer biological tr,z of K45 IgG was associated with a relatively high whole body background activity. Consistent with our own previously reported observations [20] and those of others [19,21], F(ab)z moieties cleared faster from the serum of other tissues compared to their respective parent IgGs. The faster clearance of F(ab)z-linked radioactivity is likely to be associated with the smaller size of the protein (and hence ease of transcapillary
42
passage and renal clearance) as well as the decrease in K,. The decrease in the K, of the F(ab), moieties compared to their respective parent IgGs may be due to steric changes in the antigen-binding sites and/or increased susceptibility of the fragments to partial denaturation during radioiodination [ZZ] . Both K45 F(ab), and K29 F(ab)z showed much faster clearance from the tumor xenografts than the parent IgGs. However, both of our tumor-specific antibodies and their F(ab)s moieties were retained longer in the target tumors compared to the isotype matched nonspecific IgG or its F(ab)* moiety. Such longer retention of antitumor antibodies or their immunoreactive fragments in target tumor tissues has been observed with other, (but not all) antitumor MABs [26]. The rapid decrease in the absolute amount of tumor-associated radioactivity observed at 48 h in our mice given ‘311-labeled F(ab)s moieties of K45 or K29 adversely affected the quality of imaging even though significantly more radioactivity remained in the tumors compared to the normal tissues in mice given 1311-labeled K45 F(ab),. Following the observation of Pressman and Keighley [23] that radiolabeled antibodies did localize in the target tissue in vivo, a number of laboratories, including ours [ 13,24 - 261, have demonstrated that it is indeed possible to use radiolabeled antitumor antibodies for tumor diagnosis and therapy [21,27] both in experimental models and in patients. However, results from clinics have generally been less impressive than those obtained in experimental models [21,27,28]. We have a long standing interest in developing RCC specific antibodies [29] mainly because there is no tumor marker like CEA, AFP, etc. that can be of help in the diagnosis and monitoring of RCC. Using polyclonal anti-RCC antibodies [30] we could successfully image 13 out of 15 primary lesions and metastases in 7 out of 8 RCC patients [24]. Due to well recognized limitations of most polyclonal antibodies [25] (e.g. low IRF, the presence of antibodies that are directed against tissues other than the target tumor, batch to batch variations in immunological pro-
per-ties, etc.), we produced several MABs against RCC associated antigens [4]. It is very encouraging that PAL K45 which has the most tumor-restricted specificity has also optimal qualities for localizing in tumors either as such or as the F(ab)s derivative and therefore has the potential for targeting radionuclides and chemotherapeutic agents in cancer patients. Of the rather limited number of reported MABs against human RCC associated antigens, only a few have been evaluated for tumor localization in vivo [31]. Acknowledgment This investigation was supported by the Medical Research Council of Canada (Grant No. MT 10964), Cancer Research Society, Inc., Montreal and the Kidney Foundation of Canada. We are grateful to Ms. H. Maxner for the preparation of this manuscript. References Ghose,
T. and Blair,
agent-antibody
A.H.
conjugates.
(1987) CRC
Design of cytotoxic
Crit.
Rev.
Ther.
Drug
Carrier Syst., 3, 263 - 361. Ghose, T., Blair, A.H.
and Kulkarni, P.N. (1983)
tion of antibody-linked zymol.,
agents.
Bracken,
R.B.
(1987)
Semin.
Luner,
Ghose,
S.J..
Belitsky, P. (1986) tumor-associated
Renal carcinoma: Roentgenol., T.,
Chatterjee,
Monoclonal
clinical aspects
S., Cruz,
and
surface antigens of human renal cell car-
(1991)
Inhibition
of human
monoclonal-anti-body-linked 331-
H.N.
antibodies to kidney and
Cancer Res., 46, 5816 - 5820.
P.
tumor
En-
22, 241-247.
Singh. M., Ghose, R., Kralovec. E., Blair, A.H. sky,
Prepara-
Methods
93. 280 - 333.
and therapy.
cinoma.
cytotoxic
model.
Cancer
renal
methotrexate Immunol.
and Belitcancer
by
in an ascites
Immunother.,
32,
334.
Singh, M., Kralovec,
J., Mezei, M. and Ghose, T. (1989)
Inhibition of human renal cancer by methotrexate a monoclonal
antibody.
Singh,
M.,
Ghose,
Mezei,
M.
(1989)
liposomes
with
renal cancer.
T.,
J. Ural., Faulkner,
Targeting
linked to
141, 428-431. G.,
Kralovec,
J. and
of methotrexate-containing
a monoclonal
antibody
against
human
Cancer Res., 49, 3965 - 3984.
Singh, M., Ghose, T.. Mezei, M. and Belitsky, P. (1991) Inhibition of human renal cancer by monoclonal targeted
methotrexate-containing
tumor model.
Cancer Lett.,
Fogh. J. and Trempe.
antibody
liposomes in an ascites
56, 97 - 102.
G. (1975)
New human tumor cell
43
154.
21
Editor: J. Fogh, Plenum, New York. McKinney, M.M. and Parkinson, A. (1987) A simple, non chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J. Immunol. Methods, 96,
22
lines. In: Human 10
11
12
13
14
15
Tumor
Cells in Vitro,
271- 278. Kulkarni, P.N., Blair, A.H., Ghose. (1985) Conjugation of methotrexate their F(ab’)n fragments and the
pp.
115-
T. and Mammen, M. to IgG antibodies and effect of conjugated
methotrexate on tumor growth in viva. Cancer Immunol. Immunother., 16, 127 - 129. Lowry, O.H. 1Rosebrough, N. J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol
Immunol. Methods, 72, 77 - 89. Lindmo, T. and Bunn, P.A., Jr. (1986) Determination of the true immunoreactive fraction of monoclonal antibodies after radiolabeling. Methods Enzymol., 121, 678 - 691.
19
Munson, P.J. and Rodbard, D. (1980) Ligand: a versatile computerized approach for characterization of ligandbinding systems. Anal. Biochem.. 107. 220- 239. MacPherson, G.A. (1985) Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. J. Pharmacol. Methods, 14, 213-228. Ghose, T.J., Kulkami, N., Ferrone. S., Giacomini, P Norvell, S.T., Kulkami. P. and Blair, A.H. (1983) Imaging human tumors in nude mice. In: Radioimmunoimaging, pp. 255-263, Editors: S. Burchiel and B.A. Rhodes, Elvsevier, New York. Bradwell, A.R., Fairweather, D.S.. Dykes, P.W., Keeling,
20
A., Vaughan, A. and Taylor, J. (1985) Limiting factors in the localization of tumours with radiolabelled antibodies. Immumol. Today, 6, 163- 170. Ghose. T., Blair, A.H.. Martin, R.H., Novell, S.T.,
16
17
18
Taube, R.A., Adelstin, S.J. and Kassis, A.1. (1991) Antigen-binding site protection during radiolabeling leads to a higher immunoreactive fraction. J. Nucl. Med., 32, 116 - 122. 23
Pressman, D. and Keighley, G. (1948) The zone of activity of antibodies as determined by the use of radioactive tracers: the zone of activity of nephrotoxic antikidney serum. J. Immunol.. 59. 141- 146.
24
Ghose, Guclu, labeled mouse
reagent. J. Biol. Chem., 193, 265- 275. Ghose, T. and Guclu. A. (1974) Cure of a mouse lymphoma with radioiodinated antibody. Eur. J. Cancer, 10, 787 - 792. Lindmo, T., Boven, E., Cutitta, F., Fedorko, J. and Bunn, P.A. Jr. (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J.
Ramakrishnan, S., Belitsky, P. and Ferrone, S. (1982) Tumor imaging by antitumor antibody linked radionuclides. In: Tumor Imaging, pp. 167 - 178, Editor: S. Burchiel. Masson, New York.
Goldenberg, D.M. (1988) Targeting of cancer with radiolabeled antibodies. Prospects for imaging and therapy. Arch. Pathol. Lab. Med.. 112. 580-587. Van den Abbeele, A.D., Aaronson, R.A., Daher, S.,
25
26
27
28
29
30
31
T., Norvell. S.T., Aquino. J., Belitsky, P.. Tai, J., A. and Blair A.H. (1980) Localization of 1311antibodies in human renal cell carcinomas and in a hepatoma and correlation with tumor detection by
photoscanning. Cancer Res., 40, 3018-3031. Ghose. T., Ferrone. S., Imai, K.. Norvell, S.T.. Luner, S.J., Martin, R.H. and Blair, A.H. (1981) Imaging of human melanoma xenografts in nude mice with a radiolabeled monoclonal antibody. J Natl. Cancer Inst., 69. 823 - 826. Ghose, T., Tai, J. and Aquino. J. (1975) Tumor localization of ‘311-labeled antibodies by radionuclide imaging. Radiology, 116. 445 - 448. Kramer, E.L. and Larson, S.M. (1991) Tumor targeting with radiolabeled antibody for diagnosis and therapy, Immunol. Allerg. Clin. North Am., 11, 301-339. Begent, R.H.J. and Pedley. R.B. (1990) Antibody targeted therapy in cancer: comparison of murine and clinical studies. Cancer Treat. Rev., 17. 373 - 378. Nairn. R.C.. Philip, J., Ghose. T., Porteous, I.B. and Fothergill, J.E. (1963) Production of a precepitin against renal cancer. Br. Med. J., 1, 1702- 1704. Ghose. T.. Belitsky. P., Tai. J. and Janigan. D.T. (1979) Production and characterization of xenogeneic antisera to a human renal cell carcinoma associated antigen. J. Natl. Cancer Inst., 63, 301- 308. Vessella, R.L., Alvarez. V . Chiou. R-K., Rodwell. J., Elson, M., Plame. D., Shafter, R. and Lange, P. (1987) Radioimmunoscintigraphy and radioimmunotherapy of renal cell carcinoma xenografts. Natl. Cancer Inst. Monogr.. 3, 159- 167.
