~
Cancer ImmunolImmtmother(1992) 34:252-258
ancer
mmunology mmunotlierapy
© Springer-Verlag 1992
Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody K984 A. H. G. J. Schrijvers, M. Gerretsen, M. van Walsum, B. J. M. Braakhuis, J. J. Quak, G. B. Snow, and G. A. M. S. van Dongen TumourBiologyLaboratory,Departmentof Otolaryngology,Free UniversityHospital,R O. Box 7057, NL-1007 MB Amsterdam,The Netherlands Received 15 August 1991/Accepted22 October 1991
Summary. In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts [28, 30]. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratlos, as based on the biodistribution data, were at least ten fimes higher in case of the specific mAb K984 when compared to another non-specific, isotypematched control antibody, mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targetlng. Key words: Tumour targeting - Squamous cell carcinoma - Radioimmunoconjugate
Offprint requests to: A. H. G. J. Schrijvers
Introduction Squamous cell carcinoma (SCC) represents the vast majority of all malignant tumours of the head and neck [40]. Squamous cell carcinoma also depicts the major histological type of neoplasms arising from cervix, skin and lung. For diagnosis and therapy of SCC of the head and neck, monoclonal antibodies (mAbs) directed to tumour-associated cell-surface antigens are potentially powerful tools [7, 16]. Among the potential applications of such tumour-preferential mAbs is their use as targeting agents for the selective delivery of radionuclides [2, 8, 11, 12, 17, 20, 22, 27] or anti-tumour agents [2, 9, 10, 15, 25, 37] to primary tumours and particularly to lymph node and distant metastases. Other approaches make use of unconjugated mAbs that eliminate tumour cells by activating immunological effector mechanisms, or mAbs that block mmour-associated processes, like proliferation and metastases, by binding to cell-surface receptors directly involved in these processes [13, 33, 36, 37, 39]. Recently we described the production of a panel of mAbs with a high affinity for membrane antigens expressed at the outer cell surface of SCC cells [28, 30]. One of these antibodies is mAb K984 (isotype, IgG1), selectively reacting with a cell-surface antigen shared by the basal cells in normal epidermis and mucous membranes [28, 30]. These cells are supposed to represent the proliferative fractlon. In SCC mAb K984 is reactive with cell membranes of the microscopically poorly differentiated cells. Furthermore, the K984 antigen is homogeneously expressed by in vitro cultured SCC cells. These data suggest the involvement of the K984 antigen in growth regulatlon of squamous carcinomas. To test this hypothesis we have examined the growth kinetics of SCC cell lines cultured as monolayers in vitro in either the absence or presence of mAb K984. In this smdy a significant growth-inhibitory activity of mAb K984 in vitro will be demonstrated. Since mAb K984 is selectively reactive with the proliferating population in SCC and possesses biological activity, it may be an excellent candidate mAb
253 f o r t a r g e t i n g S C C . A s a first a p p r o a c h to d e t e r m i n e the suitability o f m A b K 9 8 4 for s e l e c t i v e t u m o u r t a r g e t i n g in v i v o w e e v a l u a t e d the a b i l i t y . o f r a d i o l a b e l l e d m A b K 9 8 4 to l o c a l i z e and v i s u a l i z e x e n o g r a f t s o f S C C o f the h e a d and n e c k in a t h y m i c n u d e m i c e . In this r e p o r t w e d e s c r i b e the biodistribution data of radiolabelled mAb K984 obtained in a t h y m i c n u d e m i c e b e a r i n g h u m a n S C C x e n o g r a f t s .
Materials and methods Immunization and hybridoma production. Immunization with SCC cells (UM-SCC-22A) was performed as previously described [28]. In short, growing hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin production, for binding with intact viable tumour cells and for lack of reactivity with erythrocytes, mAb K984 was selected after a further screening upon reactivity with frozen sections derived from a broad panel of normal tissues as well as from a panel of 59 SCC from the head and neck [30]. The isotype-matched control antibody JSB-1, directed against the P glycoprotein related to multidrug resistance and not reactive with the xenograft used in our study, has been described elsewhere [34]. Cell lines and xenograft line. Human squamous cell carcinoma cell lines of the head and heck were kindly provided by Dr. T. Carey (University of Michigan, Ann Arbor, USA); UM-SCC-14C was derived from a carcinoma of the floor of the mouth, and UM-SCC-11B from a laryngeal carcinoma, while UM-SCC-22B originated from a lymph node metastasis of the hypopharynx. A431 is a SCC of the vulva, NRK is a normal rat kidney cell line and RI is a rhabdomyosarcoma cell line. All cell lines were routinely cultured at 37°C as subconfluent monolayers in 25-cm2 plastic Nunclon flasks. Culturing medium consisted of Dulbecco's modified Eagle's medium (DMEM) buffered with 15 mM HEPES (pH 7.5) and 16 mM NaHCO3, supplemented with 10% heat-inactivated fetal calf serum (FCS), 5% CO2. The head and neck SCC xenograft line HNX-HN has been established from a SCC of the base of the tongue (T4N2) from a 54-year-old female patient. Xenografts were propagated by bilaterally implanting tumour fragments (9 mm 3) subcutaneously in the thoracic region of athymic nude mice (8-10 weeks old, female NMRI, 25-32 g; Harlan Olac, Zeist, The Netherlands) [4]. Immunohistochemistry. Expression of the K984 antigen in the xenografts (HNX-HN) was assessed on frozen tissue sections by the biotin-avidinperoxidase technique. To this end, mAb K984 was conjugated with biotin as described previously [12]. After incubation of the sections with biotinylated mAb K984, they were washed three times with phosphatebuffered saline (PBS) and incubated with a preformed avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector, Burkingham, Calif.). After subsequent washing with PBS, the peroxidase label was developed with 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, Mo., USA) plus 0.03% H202 added as substrate.
resin and to sterilize the product. The percentage of protein-bound radioactivity was estimated by trichloroacetic acid precipitation.
mAb K984 in vitro binding assays. The binding characteristics of radiolabelled mAb K984 were analysed by immunoreactivity and affinity assays. The immunoreactivity assay was performed essentially as described by Lindmo et al. [21]. In short, A431 cells were fixed in 0.1% glutaraldehyde and six serial dilutions were made, ranging from 5 x 106 cells/tube to 3.1 x 105 cells/tube in PBS with 1% bovine serum albumin. Radiolabelled mAb K984 (10000 cpm) was added to the test tubes and incubated overnight at room temperature. Excess unlabelled mAb was added to the final sample to determine non-specific binding. Cells were spun down, radioactivity in the pellet and supernatant was determined in a gamma counter and the percentage of bound and free radiolabelled mAb was calculated (LKB-Wallac 1218 CompuGamma). Data were graphically analysed in a modified Lineweaver-Burke plot and the immunoreactivity was determined by linear extrapolation to conditions representing infinite antigen excess. The affinity assay was essentially as described by Badger et al. [1]. Briefly, 5 x 106 fixed A431 cells in PBS with 1% bovine serum albumin were incubated overnight at room temperature with 5000 cpm labelled K984 IgG and a serial dilution of unlabelled mAb K984 (the concentration range covering the concentration of labelled K984 IgG, as calculated from the specific activity). Cells were spun down and radioactivity in the pellet and supernatant was determined in a gamma counter. Data were graphically analysed by Scatchard analysis to determine the affinity constant and the number of antigenic sites per cell. Both the immunoreactivity assay and the affinity assay were performed in triplicate. Biodistribution experiments. The in vivo tissue distribution of radiolabelled mAb K984 was studied in nude mice bearing tumours of the SCC xenograft line HNX-HN, following i. v. administration in the retroorbital plexus of 10 gCi 13li-K984 and 10 gCi 125I-labelled control antibody in sterile saline. To this end, mice (three or four at each time point) were bled, killed and dissected under appropriate anaesthesia at 1, 2, 3 and 7 days after injection. Organs were immediately removed, rinsed in PBS and weighed. Samples were taken from blood, tumour, liver, spleen, kidney, heart, stomach, jejunum, colon, sternum, muscle, lung and tongue. All weighed tissue samples were counted in a dual-isotope gamma counter. The antibody uptake in the tumour and other tissues was calculated as a percentage of the injected dose per gram wet weight of tissue (%ID/g). The specific localization index was calculated by dividing the uptake of radiolabelled specific antibody (mAb K984) by the uptake of radiolabelled non-specific antibody in the tumour. Scintigraphic images. Mice were killed by cervical dislocation and placed under the camera. Two mice were scanned simultaneously with an Ohio gamma camera (Sigma 410S), 100000 cpm was obtained per image and data were stored in a PDP 1134 computer for further analysis and production of (colour) images. Images were taken without any subtraction procedures to correct for background activity.
Results Growth inhibition assay. Suspensions of single cells were plated in 2 ml DMEM plus 10% FCS. After 24 h the medium was exchanged for fresh medium with or without the addition of mAb K984. Further exchanges were performed every 3 days. The number of cells was counted using a haematocytometer at various times after seeding. Radioiodination. Iodination of mAb K984 was essentially as described by Häisma et al. [14]. In short, 250 gg mAb in phosphate buffer pH 7.4 was mixed with 0.5 mCi 1311 (for mAb K984) or 125I (for the control antibody) in a vial previously coated with iodogen. After 10 min of incubation at room temperature, a sample was removed to determine the amount of incorporated iodine. Subsequently, 1 ml AG1-X-8 resin (BioRad, Veenendaal, The Netherlands), previously mixed with PBS+1% bovine serum albumin, was added to the vial to adsorb unbound iodine. The reaction mixture was filtered through a 0.22-gm filter to remove the
mAb K98"4 as functional probe W e h a v e p r e v i o u s l y s h o w n that m A b K 9 8 4 r e c o g n i z e s an ( 9 0 - 9 5 k D a ) a n t i g e n s e l e c t i v e l y e x p r e s s e d o n the o u t e r c e l l s u r f a c e o f the p r o l i f e r a t i n g p o p u l a t i o n in n o r m a l e p i d e r m i s and S C C [28, 30]. m A b K 9 8 4 w a s also f o u n d to b e e x p r e s s e d o n S C C c e l l lines c u l t u r e d in vitro. T h i s s t a i n i n g p a t t e r n o f m A b K 9 8 4 r a i s e d the i d e a that the K 9 8 4 a n t i g e n m a y b e i n v o l v e d in the g r o w t h r e g u l a t i o n o f S C C . T o test this h y p o t h e s i s w e h a v e e x a m i n e d the g r o w t h kinetics o f S C C c e l l lines c u l t u r e d as m o n o l a y e r s in v i t r o in the p r e s e n c e o f m A b K 9 8 4 . I n d e e d a s i g n i f i c a n t g r o w t h inhibi-
254
a 106 Number of cells
1 05
104 0
~
' 2
' 4
' 6
' 8
' 10
Fig. 2. Cryo-section of xenograft HNX-HN, labelled with biotinylated mAb K984 by the indirect immunoperoxidase method. Note reactivity of mAb K984 with the outer rim of the tumour nests representing the proliferating cell compartment
Days
b 100
qumber of oells (% of control)
80 60
__õ
40 20 I
I
I
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tion of the head and neck SCC cell line UM-SCC-14C was observed when it was cultured in presence of mAb K984 (Fig. 1 a). The lag phase was extended while the doubling time during logarithmic growth significantly increased from 1.9 to 3.1 days. The growth-inhibitory effect was found to be dose-dependent for concentrations up to 10 ~g/ml (Fig. 1 b). Higher dosages had no further effect. Comparable inhibition of growth by mAb K984 was also observed in other antigen-positive cell lines; UM-SCC11B, UM-SCC-22B and A431 (Fig. 1 c). No inhibition effects were seen in the antigen-negative normal rat kidney (NRK) or rhabdomyosarcoma (R~) cell lines (Fig. lc). Under the same conditions, there was no reduction of growth when an isotype-matched control antibody mAb K931 [29] was used, which also binds to the outer cell surface to a large extent (data not shown).
2O
Immunohistochemistry 0 0
L
i
i
i
i
5
10
15
20
25
MAb K984
(IJg/ml)
Fig. I. a Effect of mAh K984 on growth of UM-SCC-14C cell line. Cells were seeded on 8-cmz plastic dishes (3 x 104 cells/dish) in 2 ml Dulbecco's modified Eagle's medium +10% fetal calf serum, and 24 h later (arrow) the medium was exchanged for 2 ml medium with (10 ~tg/ml, O) or without mAb K984 ( 0 ) . The number of cells was counted at various times after seeding. Points represent means of triplicate determinations with SEM. b Dose/response curve for growth inhibition of UMSCC-14C cell line induced by mAb K984. Procedure as in a, cells were counted 4 days after seeding. Data expressed as means with SEM. c Dose/response curves for mAb K984-induced growth inhibition of antigen-positive head and neck squamous cell carcinoma (SCC) cell lines UM-SCC-11B (O), UM-SCC-14C (&), UM-SCC-22B (D), and the vulva SCC cell line A431 ( V ). As control, the antigen-negative normal rat kidney (NRK; O) and rhabdomyosarcoma (R1; A) cell lines were included. For further explanation see a
Since mAb K984 is selectively reactive with the proliferating population in SCC and possesses growth-inhibitory activity in vitro, it may be an excellent candidate for targeting SCC, especially in a therapeutic setting. Therefore we evaluated whether mAb K984 is capable of selective tumour targeting in vivo. To this end we have attempted to localize and visualize SCC xenografts in athymic nude mice with radiolabelled mAb K984. A panel of eight SCC xenografts was immunohistochemically evaluated by incubating cryo-sections with protein-A-purified biotinylated mAb K984 or control antibody. The staining pattern of the xenograft line HNX-HN, selected for the biodistribution and imaging studies described here (Fig. 2), was exemplary for the xenografts tested and for the 59 human tumours evaluated previously [30]. It is demonstrated that mAb K984 preferentially binds to membranes of the cells in the outer rim of the tumour cell nests. No labelling was observed using the control antibody (data not shown).
255 day 1 T:B= 1.4
biodistribution day 2 ~ day 3 T:B=n.d. T:B=3,1
~
Tumor to non.tumor ratlos d•y 2 SLI-5,4
day I SLI'4.1
[-----I clay • T:B=3.8
~ß~/J day 3 SLI-5.1
["---1 day 7 SLI=4.3
3O
20
a 0
25
15
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15
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O
blood 20
sternum .
.
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liver .
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0
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sternum
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colon
% 30
b 15
0 T--
25
2o 0
5
2 5
bIood
sternurn
Lturnor
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spleen
kidney
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Fig. 3. Biodistribution data for (a) l0 gCi 13tI-labelledmAb K984 and (b) a25I-labelledJSB 1 IgG1 in athymic nude mice bearing tumours of the SCC xenograft line HNX-HN. Tissues were dissected 1, 2, 3 and 7 days after injection. Radioactivity(means _+ SEM) is expressed as a percentage of the injected dose per gram of wet weight of tissue (%ID/gram). Each day three or four mice were dissected
Radiolabelling of mAb K984 Radiolabelling of 250 gg K984 IgG with 0.5 mCi 131I resulted in a specific activity of 929.3+85.8 gCi mg-1 (mean___ SD, n = 3). Trichloroacetic acid precipitation revealed that 86.7 + 1.2% (n = 3) of the iodine was bound. Labelling of 500 gg control IgG with 1 mCi 125I resulted in a specific activity of 810 gCi mg -1, while more than 98% of the iodine was bound.
mAb K984 in vitro binding assays As determined by the modified Lineweaver-Burke plot, the immunoreactive fraction of radiolabelled m A b K984 was 9 0 . 8 + 0 . 5 % (mean_+SD of three triplicate determinations), mAb K984 appeared to be a high-affinity antibody, the affinity constant of the radiolabelled K984 was 3 x 1010 M -1, as determined by Scatchard plot. The A431 cells used expressed 1.25 x 104 antigenic sites/cell.
Pharmacokinetics At days 1, 2, 3 and 7, serum samples were collected to determine free iodine. Less than 10% free iodine was pre-
blood
Fig. 4. Tumour to non-tumour ratios of (a) a31I-labelledmAb K984 and (b) 125I-labelledJSB 1 IgG1 in athymic nude mice bearing tumours of the SCC xenograft line HNX-HN. Antibody uptake is measured as %ID/g and expressed as means + SD, the specific localization index (SL1)is calculated by dividing the uptake (%ID/g) of specific mAb K984 in the tumour by that of control antibody. Each day three or four mice were dissected sent as revealed by trichloroacetic acid precipitation. On day 1 afer injection of 10 gCi 131I-K984 IgG, 9.23 + 2 . 9 1 % of the injected dose/g was present in the blood, as compared to 12.21 _+ 1.01% for the radiolabelled control antibody.
Biodistribution in HNX-HN-bearing mice The tumour uptake ofK984 IgG. The amount of 131I-K984 IgG in the xenografts and various organs, expressed as the average percentage of radioactivity of the injected dose per gram wet weight of tissue (%ID/g), is shown in Fig. 3 a, b. As the amount of radiolabelled K984 IgG in the blood diminishes within the first few days after injection, the level in the tumour remains relatively constant. Mean tumour uptake (as %ID/g___ SD) of 13q-K984 IgG1 on days 1, 2, 3 and 7 was 13.2+_5.4%, 1 4 . 0 + 2 . 9 % , 1 1 . 7 + 5 . 2 % and 10.7 + 1.2% respectively. Specificity of the accumulation of radiolabelled m A b K984 is demonstrated by the absence of accumulation of the non-specific, isotype-matched control antibody mAb JSB-1 (Fig. 3b). Mean tumour uptake (as % I D / g + S D ) of the control antibody was 3.2 + 0.5%, 2.6 + 0.4%, 2 . 3 + 0 . 4 % and 2.5_+0.7% on days 1, 2, 3 and 7 respectively.
256
Fig. 5 a - d . Whole-bodyscintigraphicimages of pairs of athymicnude
mice bearing two subcutaneousHNX-HNxenograftsgiven an injection of 10 ktCi13q-labelledK984IgG.Imagesweretakenwithoutsubtraction
There was also a striking difference in the tumour to blood (T: B) ratios between the radiolabelled non-specific antibody (T:B ~
Cancer ImmunolImmtmother(1992) 34:252-258
ancer
mmunology mmunotlierapy
© Springer-Verlag 1992
Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody K984 A. H. G. J. Schrijvers, M. Gerretsen, M. van Walsum, B. J. M. Braakhuis, J. J. Quak, G. B. Snow, and G. A. M. S. van Dongen TumourBiologyLaboratory,Departmentof Otolaryngology,Free UniversityHospital,R O. Box 7057, NL-1007 MB Amsterdam,The Netherlands Received 15 August 1991/Accepted22 October 1991
Summary. In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts [28, 30]. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratlos, as based on the biodistribution data, were at least ten fimes higher in case of the specific mAb K984 when compared to another non-specific, isotypematched control antibody, mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targetlng. Key words: Tumour targeting - Squamous cell carcinoma - Radioimmunoconjugate
Offprint requests to: A. H. G. J. Schrijvers
Introduction Squamous cell carcinoma (SCC) represents the vast majority of all malignant tumours of the head and neck [40]. Squamous cell carcinoma also depicts the major histological type of neoplasms arising from cervix, skin and lung. For diagnosis and therapy of SCC of the head and neck, monoclonal antibodies (mAbs) directed to tumour-associated cell-surface antigens are potentially powerful tools [7, 16]. Among the potential applications of such tumour-preferential mAbs is their use as targeting agents for the selective delivery of radionuclides [2, 8, 11, 12, 17, 20, 22, 27] or anti-tumour agents [2, 9, 10, 15, 25, 37] to primary tumours and particularly to lymph node and distant metastases. Other approaches make use of unconjugated mAbs that eliminate tumour cells by activating immunological effector mechanisms, or mAbs that block mmour-associated processes, like proliferation and metastases, by binding to cell-surface receptors directly involved in these processes [13, 33, 36, 37, 39]. Recently we described the production of a panel of mAbs with a high affinity for membrane antigens expressed at the outer cell surface of SCC cells [28, 30]. One of these antibodies is mAb K984 (isotype, IgG1), selectively reacting with a cell-surface antigen shared by the basal cells in normal epidermis and mucous membranes [28, 30]. These cells are supposed to represent the proliferative fractlon. In SCC mAb K984 is reactive with cell membranes of the microscopically poorly differentiated cells. Furthermore, the K984 antigen is homogeneously expressed by in vitro cultured SCC cells. These data suggest the involvement of the K984 antigen in growth regulatlon of squamous carcinomas. To test this hypothesis we have examined the growth kinetics of SCC cell lines cultured as monolayers in vitro in either the absence or presence of mAb K984. In this smdy a significant growth-inhibitory activity of mAb K984 in vitro will be demonstrated. Since mAb K984 is selectively reactive with the proliferating population in SCC and possesses biological activity, it may be an excellent candidate mAb
253 f o r t a r g e t i n g S C C . A s a first a p p r o a c h to d e t e r m i n e the suitability o f m A b K 9 8 4 for s e l e c t i v e t u m o u r t a r g e t i n g in v i v o w e e v a l u a t e d the a b i l i t y . o f r a d i o l a b e l l e d m A b K 9 8 4 to l o c a l i z e and v i s u a l i z e x e n o g r a f t s o f S C C o f the h e a d and n e c k in a t h y m i c n u d e m i c e . In this r e p o r t w e d e s c r i b e the biodistribution data of radiolabelled mAb K984 obtained in a t h y m i c n u d e m i c e b e a r i n g h u m a n S C C x e n o g r a f t s .
Materials and methods Immunization and hybridoma production. Immunization with SCC cells (UM-SCC-22A) was performed as previously described [28]. In short, growing hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin production, for binding with intact viable tumour cells and for lack of reactivity with erythrocytes, mAb K984 was selected after a further screening upon reactivity with frozen sections derived from a broad panel of normal tissues as well as from a panel of 59 SCC from the head and neck [30]. The isotype-matched control antibody JSB-1, directed against the P glycoprotein related to multidrug resistance and not reactive with the xenograft used in our study, has been described elsewhere [34]. Cell lines and xenograft line. Human squamous cell carcinoma cell lines of the head and heck were kindly provided by Dr. T. Carey (University of Michigan, Ann Arbor, USA); UM-SCC-14C was derived from a carcinoma of the floor of the mouth, and UM-SCC-11B from a laryngeal carcinoma, while UM-SCC-22B originated from a lymph node metastasis of the hypopharynx. A431 is a SCC of the vulva, NRK is a normal rat kidney cell line and RI is a rhabdomyosarcoma cell line. All cell lines were routinely cultured at 37°C as subconfluent monolayers in 25-cm2 plastic Nunclon flasks. Culturing medium consisted of Dulbecco's modified Eagle's medium (DMEM) buffered with 15 mM HEPES (pH 7.5) and 16 mM NaHCO3, supplemented with 10% heat-inactivated fetal calf serum (FCS), 5% CO2. The head and neck SCC xenograft line HNX-HN has been established from a SCC of the base of the tongue (T4N2) from a 54-year-old female patient. Xenografts were propagated by bilaterally implanting tumour fragments (9 mm 3) subcutaneously in the thoracic region of athymic nude mice (8-10 weeks old, female NMRI, 25-32 g; Harlan Olac, Zeist, The Netherlands) [4]. Immunohistochemistry. Expression of the K984 antigen in the xenografts (HNX-HN) was assessed on frozen tissue sections by the biotin-avidinperoxidase technique. To this end, mAb K984 was conjugated with biotin as described previously [12]. After incubation of the sections with biotinylated mAb K984, they were washed three times with phosphatebuffered saline (PBS) and incubated with a preformed avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector, Burkingham, Calif.). After subsequent washing with PBS, the peroxidase label was developed with 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, Mo., USA) plus 0.03% H202 added as substrate.
resin and to sterilize the product. The percentage of protein-bound radioactivity was estimated by trichloroacetic acid precipitation.
mAb K984 in vitro binding assays. The binding characteristics of radiolabelled mAb K984 were analysed by immunoreactivity and affinity assays. The immunoreactivity assay was performed essentially as described by Lindmo et al. [21]. In short, A431 cells were fixed in 0.1% glutaraldehyde and six serial dilutions were made, ranging from 5 x 106 cells/tube to 3.1 x 105 cells/tube in PBS with 1% bovine serum albumin. Radiolabelled mAb K984 (10000 cpm) was added to the test tubes and incubated overnight at room temperature. Excess unlabelled mAb was added to the final sample to determine non-specific binding. Cells were spun down, radioactivity in the pellet and supernatant was determined in a gamma counter and the percentage of bound and free radiolabelled mAb was calculated (LKB-Wallac 1218 CompuGamma). Data were graphically analysed in a modified Lineweaver-Burke plot and the immunoreactivity was determined by linear extrapolation to conditions representing infinite antigen excess. The affinity assay was essentially as described by Badger et al. [1]. Briefly, 5 x 106 fixed A431 cells in PBS with 1% bovine serum albumin were incubated overnight at room temperature with 5000 cpm labelled K984 IgG and a serial dilution of unlabelled mAb K984 (the concentration range covering the concentration of labelled K984 IgG, as calculated from the specific activity). Cells were spun down and radioactivity in the pellet and supernatant was determined in a gamma counter. Data were graphically analysed by Scatchard analysis to determine the affinity constant and the number of antigenic sites per cell. Both the immunoreactivity assay and the affinity assay were performed in triplicate. Biodistribution experiments. The in vivo tissue distribution of radiolabelled mAb K984 was studied in nude mice bearing tumours of the SCC xenograft line HNX-HN, following i. v. administration in the retroorbital plexus of 10 gCi 13li-K984 and 10 gCi 125I-labelled control antibody in sterile saline. To this end, mice (three or four at each time point) were bled, killed and dissected under appropriate anaesthesia at 1, 2, 3 and 7 days after injection. Organs were immediately removed, rinsed in PBS and weighed. Samples were taken from blood, tumour, liver, spleen, kidney, heart, stomach, jejunum, colon, sternum, muscle, lung and tongue. All weighed tissue samples were counted in a dual-isotope gamma counter. The antibody uptake in the tumour and other tissues was calculated as a percentage of the injected dose per gram wet weight of tissue (%ID/g). The specific localization index was calculated by dividing the uptake of radiolabelled specific antibody (mAb K984) by the uptake of radiolabelled non-specific antibody in the tumour. Scintigraphic images. Mice were killed by cervical dislocation and placed under the camera. Two mice were scanned simultaneously with an Ohio gamma camera (Sigma 410S), 100000 cpm was obtained per image and data were stored in a PDP 1134 computer for further analysis and production of (colour) images. Images were taken without any subtraction procedures to correct for background activity.
Results Growth inhibition assay. Suspensions of single cells were plated in 2 ml DMEM plus 10% FCS. After 24 h the medium was exchanged for fresh medium with or without the addition of mAb K984. Further exchanges were performed every 3 days. The number of cells was counted using a haematocytometer at various times after seeding. Radioiodination. Iodination of mAb K984 was essentially as described by Häisma et al. [14]. In short, 250 gg mAb in phosphate buffer pH 7.4 was mixed with 0.5 mCi 1311 (for mAb K984) or 125I (for the control antibody) in a vial previously coated with iodogen. After 10 min of incubation at room temperature, a sample was removed to determine the amount of incorporated iodine. Subsequently, 1 ml AG1-X-8 resin (BioRad, Veenendaal, The Netherlands), previously mixed with PBS+1% bovine serum albumin, was added to the vial to adsorb unbound iodine. The reaction mixture was filtered through a 0.22-gm filter to remove the
mAb K98"4 as functional probe W e h a v e p r e v i o u s l y s h o w n that m A b K 9 8 4 r e c o g n i z e s an ( 9 0 - 9 5 k D a ) a n t i g e n s e l e c t i v e l y e x p r e s s e d o n the o u t e r c e l l s u r f a c e o f the p r o l i f e r a t i n g p o p u l a t i o n in n o r m a l e p i d e r m i s and S C C [28, 30]. m A b K 9 8 4 w a s also f o u n d to b e e x p r e s s e d o n S C C c e l l lines c u l t u r e d in vitro. T h i s s t a i n i n g p a t t e r n o f m A b K 9 8 4 r a i s e d the i d e a that the K 9 8 4 a n t i g e n m a y b e i n v o l v e d in the g r o w t h r e g u l a t i o n o f S C C . T o test this h y p o t h e s i s w e h a v e e x a m i n e d the g r o w t h kinetics o f S C C c e l l lines c u l t u r e d as m o n o l a y e r s in v i t r o in the p r e s e n c e o f m A b K 9 8 4 . I n d e e d a s i g n i f i c a n t g r o w t h inhibi-
254
a 106 Number of cells
1 05
104 0
~
' 2
' 4
' 6
' 8
' 10
Fig. 2. Cryo-section of xenograft HNX-HN, labelled with biotinylated mAb K984 by the indirect immunoperoxidase method. Note reactivity of mAb K984 with the outer rim of the tumour nests representing the proliferating cell compartment
Days
b 100
qumber of oells (% of control)
80 60
__õ
40 20 I
I
I
10
20
30
0
I
MAb K984
C 100
~lumber of ~ells
I
40
50
(ug/ml)
(% of control)
8O 60 40
tion of the head and neck SCC cell line UM-SCC-14C was observed when it was cultured in presence of mAb K984 (Fig. 1 a). The lag phase was extended while the doubling time during logarithmic growth significantly increased from 1.9 to 3.1 days. The growth-inhibitory effect was found to be dose-dependent for concentrations up to 10 ~g/ml (Fig. 1 b). Higher dosages had no further effect. Comparable inhibition of growth by mAb K984 was also observed in other antigen-positive cell lines; UM-SCC11B, UM-SCC-22B and A431 (Fig. 1 c). No inhibition effects were seen in the antigen-negative normal rat kidney (NRK) or rhabdomyosarcoma (R~) cell lines (Fig. lc). Under the same conditions, there was no reduction of growth when an isotype-matched control antibody mAb K931 [29] was used, which also binds to the outer cell surface to a large extent (data not shown).
2O
Immunohistochemistry 0 0
L
i
i
i
i
5
10
15
20
25
MAb K984
(IJg/ml)
Fig. I. a Effect of mAh K984 on growth of UM-SCC-14C cell line. Cells were seeded on 8-cmz plastic dishes (3 x 104 cells/dish) in 2 ml Dulbecco's modified Eagle's medium +10% fetal calf serum, and 24 h later (arrow) the medium was exchanged for 2 ml medium with (10 ~tg/ml, O) or without mAb K984 ( 0 ) . The number of cells was counted at various times after seeding. Points represent means of triplicate determinations with SEM. b Dose/response curve for growth inhibition of UMSCC-14C cell line induced by mAb K984. Procedure as in a, cells were counted 4 days after seeding. Data expressed as means with SEM. c Dose/response curves for mAb K984-induced growth inhibition of antigen-positive head and neck squamous cell carcinoma (SCC) cell lines UM-SCC-11B (O), UM-SCC-14C (&), UM-SCC-22B (D), and the vulva SCC cell line A431 ( V ). As control, the antigen-negative normal rat kidney (NRK; O) and rhabdomyosarcoma (R1; A) cell lines were included. For further explanation see a
Since mAb K984 is selectively reactive with the proliferating population in SCC and possesses growth-inhibitory activity in vitro, it may be an excellent candidate for targeting SCC, especially in a therapeutic setting. Therefore we evaluated whether mAb K984 is capable of selective tumour targeting in vivo. To this end we have attempted to localize and visualize SCC xenografts in athymic nude mice with radiolabelled mAb K984. A panel of eight SCC xenografts was immunohistochemically evaluated by incubating cryo-sections with protein-A-purified biotinylated mAb K984 or control antibody. The staining pattern of the xenograft line HNX-HN, selected for the biodistribution and imaging studies described here (Fig. 2), was exemplary for the xenografts tested and for the 59 human tumours evaluated previously [30]. It is demonstrated that mAb K984 preferentially binds to membranes of the cells in the outer rim of the tumour cell nests. No labelling was observed using the control antibody (data not shown).
255 day 1 T:B= 1.4
biodistribution day 2 ~ day 3 T:B=n.d. T:B=3,1
~
Tumor to non.tumor ratlos d•y 2 SLI-5,4
day I SLI'4.1
[-----I clay • T:B=3.8
~ß~/J day 3 SLI-5.1
["---1 day 7 SLI=4.3
3O
20
a 0
25
15
2O 0 ~E
15
0
10 5
O
blood 20
sternum .
.
I.tu:qor .
r.tumor .
liver .
S!01een kidney .
0
colon
blood
$ternum
liver
spleen
kidney
colon
sternum
liver
spleen
kMney
colon
% 30
b 15
0 T--
25
2o 0
5
2 5
bIood
sternurn
Lturnor
r,turnor
liver
spleen
kidney
colon
Fig. 3. Biodistribution data for (a) l0 gCi 13tI-labelledmAb K984 and (b) a25I-labelledJSB 1 IgG1 in athymic nude mice bearing tumours of the SCC xenograft line HNX-HN. Tissues were dissected 1, 2, 3 and 7 days after injection. Radioactivity(means _+ SEM) is expressed as a percentage of the injected dose per gram of wet weight of tissue (%ID/gram). Each day three or four mice were dissected
Radiolabelling of mAb K984 Radiolabelling of 250 gg K984 IgG with 0.5 mCi 131I resulted in a specific activity of 929.3+85.8 gCi mg-1 (mean___ SD, n = 3). Trichloroacetic acid precipitation revealed that 86.7 + 1.2% (n = 3) of the iodine was bound. Labelling of 500 gg control IgG with 1 mCi 125I resulted in a specific activity of 810 gCi mg -1, while more than 98% of the iodine was bound.
mAb K984 in vitro binding assays As determined by the modified Lineweaver-Burke plot, the immunoreactive fraction of radiolabelled m A b K984 was 9 0 . 8 + 0 . 5 % (mean_+SD of three triplicate determinations), mAb K984 appeared to be a high-affinity antibody, the affinity constant of the radiolabelled K984 was 3 x 1010 M -1, as determined by Scatchard plot. The A431 cells used expressed 1.25 x 104 antigenic sites/cell.
Pharmacokinetics At days 1, 2, 3 and 7, serum samples were collected to determine free iodine. Less than 10% free iodine was pre-
blood
Fig. 4. Tumour to non-tumour ratios of (a) a31I-labelledmAb K984 and (b) 125I-labelledJSB 1 IgG1 in athymic nude mice bearing tumours of the SCC xenograft line HNX-HN. Antibody uptake is measured as %ID/g and expressed as means + SD, the specific localization index (SL1)is calculated by dividing the uptake (%ID/g) of specific mAb K984 in the tumour by that of control antibody. Each day three or four mice were dissected sent as revealed by trichloroacetic acid precipitation. On day 1 afer injection of 10 gCi 131I-K984 IgG, 9.23 + 2 . 9 1 % of the injected dose/g was present in the blood, as compared to 12.21 _+ 1.01% for the radiolabelled control antibody.
Biodistribution in HNX-HN-bearing mice The tumour uptake ofK984 IgG. The amount of 131I-K984 IgG in the xenografts and various organs, expressed as the average percentage of radioactivity of the injected dose per gram wet weight of tissue (%ID/g), is shown in Fig. 3 a, b. As the amount of radiolabelled K984 IgG in the blood diminishes within the first few days after injection, the level in the tumour remains relatively constant. Mean tumour uptake (as %ID/g___ SD) of 13q-K984 IgG1 on days 1, 2, 3 and 7 was 13.2+_5.4%, 1 4 . 0 + 2 . 9 % , 1 1 . 7 + 5 . 2 % and 10.7 + 1.2% respectively. Specificity of the accumulation of radiolabelled m A b K984 is demonstrated by the absence of accumulation of the non-specific, isotype-matched control antibody mAb JSB-1 (Fig. 3b). Mean tumour uptake (as % I D / g + S D ) of the control antibody was 3.2 + 0.5%, 2.6 + 0.4%, 2 . 3 + 0 . 4 % and 2.5_+0.7% on days 1, 2, 3 and 7 respectively.
256
Fig. 5 a - d . Whole-bodyscintigraphicimages of pairs of athymicnude
mice bearing two subcutaneousHNX-HNxenograftsgiven an injection of 10 ktCi13q-labelledK984IgG.Imagesweretakenwithoutsubtraction
There was also a striking difference in the tumour to blood (T: B) ratios between the radiolabelled non-specific antibody (T:B ~
