0360-3016/90 $3.00 + .OO Copyright 0 1990 Pergamon Press plc
Inf. .I Rodulion Oncology Biol. Phys.. Vol. IS, pp. 1361-1375 Printed in the US A. All rights reserved.
??Original Contribution
TARGETING OF HUMAN GLIOMA XENOGRAFTS IN UTILIZING RADIOLABELED ANTIBODIES
VZVO
JEFFERY A. WILLIAMS, M.D.,’ BARRY W. WESSELS, PH.D.,* MOODY D. WHARAM, STANLEY E. ORDER, M.D., Sc.D., F.A.C.R.,’ PHILIP M. WANEK,~ J. KENNETH POGGENBURG, PH.D.~ AND JERRY L. KLEIN, PH.D.’
M.D.,’
‘Division of Radiation Oncology, The Johns Hopkins Oncology Center, 600 North Wolfe Street, Baltimore, MD 2 1205; ‘Division of Radiation Oncology and Biophysics, The George Washington University Medical Center, 901 23rd Street, N.W., Washington, D.C. 20037; and ‘Hybritech, Incorporated, San Diego, CA 92 12 1
Radiolabeled antibodies provide a potential basis for selective radiotherapy of human gliomas. We have measured tumor targeting by radiolabeled monoclonal and polyclonal antibodies directed against neuroectodermal and tumorassociated antigens in nude mice bearing human glioma xenografts. Monoclonal F’96.5, a mouse IgG2a immunoglobulin, defines an epitope of a human melanoma cell surface protein, and specifically binds the U-251 human glioma as measured by immunoperoxidase histochemistry. “‘In-radiolabeled P96.5 specifically targets the U-251 human glioma xenograft and yields 87.0 microCuries (&i) of tumor activity per gram per 100 PCi injected activity compared to 4.5 &i following administration of radiolabeled irrelevant monoclonal antibody. Calculations of targeting ratios demonstrate deposited dose to be 11.6 times greater with radiolabeled P96.5 administration compared to irrelevant monoclonal antibody. The proportion of tumor dose found in normal organs is less than lo%, further supporting specific targeting of the human glioma xenograft by this antibody. Monoclonal antibody ZME018, which defines a second melanoma-associated antigen, and polyclonal rabbit antiferritin, which defines a tumorassociated antigen, demonstraCe positive immunoperoxidase staining of the tumor, but comparatively decreased targeting. When compared to the “‘In-radiolabeled antibody, “Y-radiolabeled P96.5 demonstrates comparable tumor targeting and percentages of tumor dose found in normal organs. To test the therapeutic potential of qradiolabeled P96.5, tumors and normal sites were implanted with miniature thermoluminescent dosimeters (TLD). Seven days following administration of 100 WCig”Y-radiolabeled P96.5, average absorbed doses of 3770,980,353, and 274 cGy were observed in tumor, liver, contralateral control site, and total body, respectively. Shared cell surface antigens among neuroectodermally derived neoplasms provide a basis for exploration of human glioma radioimmunotherapy. Human glioma, Radioimmunotherapy,
Tumor localization, Thermoluminescent
dosimetry.
7, 8, 13, 14, 27, 36). The results of many studies using both monoclonal and polyclonal antibodies indicate a shared antigenicity among neuroectodermally derived tissues including glioma, melanoma, neuroblastoma, and fetal brain (5, 6, 10, 20, 33, 34, 41, 42). Radiolabeled antibodies directed against neuroectodermal antigens may provide differential delivery of radiation to gliomas and sparing of surrounding normal brain. P96.5, a murine monoclonal antibody of the IgG2a subclass, targets P97, a cell surface glycoprotein of molecular weight 97,000. Initially described in high concentrations in human melanomas (44), P97 has subsequently
INTRODUCTION Human malignant gliomas are rarely cured and conventional postoperative external beam radiotherapy only extends median survival (38). With higher radiation doses,
median survival, but not long-term survival, is improved (32). The tolerance of adjacent normal brain limits the total radiation dose which can be safely administered (26). Radiolabeled antibodies offer potential selectivity in the delivery of radiation to human neoplasms ( 12,2 1,23,28, 3 1). The role of radioimmunotherapy in the treatment of human gliomas, however, remains largely unexplored (4,
Reprint requests to: Jeffery A. Williams, M.D., Division of Neurosurgery, The University of Oklahoma Health Sciences Center, South Pavilion, Room 4SP200, Oklahoma City, OK 73190. Acknowledgements-We thank Ms. Alverta E. Fields for her assistance in the preparation of this manuscript.
Supported by Hybritech, Inc., The Preuss Foundation for Brain Tumor Research, Inc., and The Children’s Cancer Foundation, Inc. Accepted for publication 20 December 1989.
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been found in a variety of cultured cells of neuroectoderma1 origin including glioblastoma, neuroblastoma, and retinoblastoma (6, 19, 24). Similarly, monoclonal antibody ZMEO18 targets GP240, a second melanoma cell surface glycoprotein of molecular weight 240,000 (I 7). To the present, GP240 has not been assessed in human glial neoplasms. Both polyclonal rabbit antiferritin and monoclonal antibody QCIO54 target ferritin, a tumor-associated protein found in several human neoplasms (2, 11, 16, 25, 43) including neuroblastoma (17). Hence, a shared antigenic profile including P97, GP240, and ferritin may exist among neuroectodermally derived neoplasms. Radiolabeled P96.5 (30), ZME018 (37), QC1054 (S. E. Order, personal oral communication, 1988), and polyclonal rabbit antiferritin (28,29) have successfully targeted disparate human neoplasms in vivo. Despite the possible cross-reactivity with neuroectodermally derived neoplasms, the systematic analysis of these antibodies for possible human glioma immunotherapy has not been performed. Therefore, we tested histochemical binding, in vivo targeting, and therapeutic potential of these antibodies in a human glioma xenograft-nude mouse model. METHODS
AND MATERIALS
Tumor cell line Human grade 4 glioma cell line, U-25 1, was obtained from the DCT Tumor Repository, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland. Cells were cultured in Dulbecco’s modified minimal essential medium/Ham’s F- 12 nutrient mixture containing 10% fetal bovine serum* and antibiotics (penicillin and streptomycin) in 12 X 80 cm plastic culture dishes.+ Cells were grown in a humidified incubator at 37°C and gassed with a mixture of 5% carbon dioxide and 95% air. Media was changed twice weekly and cells passaged at confluence with 0.5% trypsin.* Nude mice Six-week old male athymic nude mice were injected subcutaneously in the left posterior thigh with 5 X lo6 glioma cells. Tumors were visible 1 week after injection. All experiments were performed when tumors grew to 0.3-0.4 gm. Ferritin purification and antibody production Ferritin Hodgkin’s
was purified from the spleens of patients with disease as previously described ( 12). New Zea-
land white rabbits were immunized with 100 ug of purified ferritin emulsified in complete Freund’s adjuvant injected intradermally in the thigh, followed 2 weeks later by 100 ug of purified ferritin emulsified in incomplete Freund’s adjuvant.
* GIBCO, Grand Island, NY. + Falcon Plastics, Cockeysville, MD.
June 1990, Volume 18, Number 6
Normal rabbit IgG and rabbit antiferritin IgG were purified by a combination of DEAE ion exchange chromatography and ammonium sulfate precipitation as described previously ( 12). Control IgG was isolated from normal rabbit serum taken from the same rabbits prior to immunization. Chelated monoclonal antiferritin QCI054, chelated monoclonal anti-prostatic acid phosphatase PAY276, and chelated monoclonal antibodies P96.5 and ZMEO 18 were supplied.* Immunoperoxidase staining Immunoperoxidase staining of U-25 I human glioma xenografts was performed using polyclonal and monoclonal antiferritin, monoclonal P96.5 and ZMEO 18, and nonspecific polyclonal normal rabbit IgG as well as nonspecific monoclonal anti-prostatic acid phosphatase. Subcutaneously grown U-25 1 tumors were excised and fixed sequentially in Bouin-Hollande’s solution and 70% ethanol, subsequently cut in 8 micron sections with a microtome, and mounted on glass slides. Polyclonal rabbit antiferritin and normal rabbit immunoglobulin immunoperoxidase procedure. Mounted tumor specimens were deparaffinized in one change of iodine-saturated xylene for 3 min, and sequentially hydrated through graded alcohols. Specimens were then rinsed in Tris-HCl buffered saline (TBS) 0.05 M, pH 7.6 for 5 min and incubated with 3% hydrogen peroxide for 10 min. Following rinsing in TBS for 5 min, sections were incubated with normal swine serum diluted I:20 in 0.5 M TBS in a moist incubation chamber at room temperature for 20 min. Following rinsing of sections in TBS for 5 min, sections were incubated with rabbit polyclonal antiferritin diluted 1:100 with TBS in a moist incubation chamber overnight at 4°C. Sections were then rinsed with TBS and incubated with swine anti-rabbit immunoglobulin diluted 1:20 with TBS for 30 min. Following rinsing with TBS, sections were then incubated with rabbit peroxidase-antiperoxidase diluted I : 100 with TBS for 20 min. Following rinsing with TBS, sections were incubated for 10 min in aminoethylcarbazole (AEC) solution. Following a wash in running water for 5 min, sections were counterstained with Mayer’s Hematoxylin and mounted. Monoclonal QCI antiferritin, P96.5, ZMEOl8, and PA Y276 immunoperoxidase procedure. Following deparaffinization, sections were rinsed sequentially in xylene and absolute alcohol for 2 min each. Sections were then incubated in hydrogen peroxide in methanol for 20 min (9 parts methanol to 1 part 3% H202). Following rinsing in several changes of deionized water, sections were washed in TBS for 5 min and then incubated in a moist chamber for 20 min in normal goat serum diluted 1:20
t Hybritech, Inc., San Diego, CA.
Targeting of human glioma xenografts 0 J. A. WILLIAMSet al.
with TBS. Sections were then washed in TBS for 5 min and incubated in a moist chamber with primary antiserum overnight at 4°C. Sections were then washed in three rinses of TBS for 5 min each and then incubated in biotinylated mouse immunoglobulin diluted 1: 100 with TBS for 20 min. Following rinsing in three changes of TBS for 5 min each, sections were incubated for 30 min in horseradish peroxidase-Avidin D diluted 1:150 with TBS. Following rinsing with TBS, sections were incubated for 10 min in aminoethylcarbazole solution. Following rinsing in running water for 5 min, sections were counterstained with Mayer’s Hematoxylin and mounted. Antibody radiolabeling Polyclonal immunoglobulins were labeled with 13’1by the lactoperoxidase glucose oxidase method” at a labeling ratio of 5 mCi of 13iI per mg of protein. The labeled immunoglobulins were separated from free iodine using a column.** Percent binding of isotope to immunoglobulin was determined by trichloroacetic acid precipitation. Monoclonal antibodies were labeled with “‘In (P96.5, ZMEO 18, QCI054, PAY276) or 9oY (P96.5) by chelation using a proprietary procedure,++ at a labeling ratio of 5 mCi per mg of protein. Percent binding of isotope to antibody was determined by thin layer chromatography. Assay of radiolabeled antibody distribution Tumor bearing nude mice were injected with 25 or 100 PCi of radiolabeled antibody by intracardiac injection and sacrificed 1,2,5, and 7 days following injection in groups of at least two animals per time point. Calibration of well counter: A known quantity of 13’1, 9oY, or ’’‘In was placed in a radioisotopic calibrator (sensitivity to 1 @i). Counts per minute were plotted from the well counter as a function of &I allowing direct conversion to activity. Expression of accumulated activity in organs and ratio calculation: The tumor and all organs were individually weighed.## Counts per minute were obtained from the well counter and then converted to PCi by a conversion factor as determined above. The counts per minute per gram of each organ and tumor were recorded. The resulting activity in the tumor and organs was then expressed as &i/gm of tissue as a function of time. A single exponential fit to the curve of activity/gram versus time was calculated and a best fit determined by the method of least squares for each organ and each experiment. The effective half-life (T,s) and maximal initial activity (Ao) of antibody in tumor or organ were calculated as previously described (3 1). The total dose deposited in tissue is equal to the sum of the penetrating and nonpenetrating radiations. The 8 Bio-Rad Laboratories, Richmond, CA. ** PD- 10 Sephadex, Pharmacia Fine Chemicals, Piscataway, NJ. ++Developed by Hybritech, Inc., San Diego, CA.
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majority of dose is deposited by the nonpenetrating component. Assuming a uniform isotropic model for the distribution of radiolabeled antibody, the total deposited dose for any organ resulting from the sum of nonpenetrating radiations (NP) accumulated over time is given by: D = 1.44 X Ao X Teff X NP(cGy)
(31)
where D = total mean dose (cGy), Ao = initial concentration of antibody in tumor or organ (&i/gm), T,R = effective half-life (hours), and NP = sum of nonpenetrating radiations (gm - cGy/&i - hr). Although the NP component is difficult to assess in small organs, expressing the results as a dose ratio of specific/nonspecific antibody cancels this term in the equation. Thus, the targeting ratio equals: Ao X Teff (specific) Ao X Teff (nonspecific) in the tumor or normal organ in question. The percentage of the tumor dose found in normal organs was determined by normalizing the product of initial activity and effective half-life of accumulated radioactivity (Ao X T,tf) in each normal organ to that in the tumor. Thermoluminescent dosimetry Paired miniature teflon-embedded CaS04 :Dy thermoluminescent dosimeters (TLD) were implanted directly into the human glioma xenografts and dorsal interscapular subcutaneous sites, while individual TLD were implanted in liver and a contralateral control subcutaneous site via a 20 gauge needle (40). Each TLD was first cross-calibrated with known activities of 9oY as previously described (39). Mice bearing 0.3-0.4 gm tumors and dosimeters were injected with 100 PCi v-labeled P96.5 antibody as described above and sacrificed 7 days following injection. TLD were removed, rinsed,@ and measured for absorbed radiation dose as follows: The TLD glow curve peak was integrated over 50 set with T, = 115°C and T2 = 275°C. Temperature ramping was 3.5”C per set with prereadout annealing cycle of 5 sec. The major integration peak occurs at 220-240°C with a minor peak (5%) at 120°C for this TLD. RESULTS
Immunoperoxidase staining Positive immunoperoxidase staining of fixed U25 1 tumors was demonstrated with monoclonal antibodies P96.5, ZME018, and QCI054, and polyclonal rabbit an** Metler balance Model H5 1AR, Metler Instrument Corp., Hightstown, NJ. @ Count-Off, New England Nuclear Research Products, Boston, MA.
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Oncology 0 Biology0 Physics
June 1990,
Table 1. Antibody activity in U-25 1 human glioma xenografts
Antibody
Immunoperoxidase staining
Initial deposited activity (Ao)*
Tee (days)
+ + + _
86.7 22.0 5.1 4.5
2.2 1.7 4.2 3.3
Monoclonal P96.5 ZME018 QC1054 PAY276 Polyclonal Polyclonal rabbit antiferritin Normal rabbit immunoglobulin
+
25.0
1.6
_
15.0
1.9
* Activity (Ao) (uCi ” ‘In monoclonal tibody/gram/ 100 uCi injected).
Volume 18, Number 6
antibody (4.5 pCi/gm) and demonstrates greater accumulation than both ZMEO 18 (22.0 &i/gm) and QC1054 (5.1 pCi/gm) monoclonal antibodies. ‘3’I-radiolabeled polyclonal rabbit antiferritin demonstrates an initial accumulation (25.0 pCi/gm) greater than control normal rabbit immunoglobulin (15.0 &i/ gm), but less than radiolabeled monoclonal P96.5 (Table 1). U25I human glioma targeting ratios The targeting ratios of specific antibody to control nonspecific antibody in the tumor and normal organs are shown in Figure 3. The targeting ratio is the quotient of absorbed dose (cGy) in tumor or normal organs following specific antibody administration, and the absorbed dose (cGy) in tumor or normal organs following nonspecific antibody administration. ’’‘In-labeled P96.5 targets the human glioma xenograft greater than radiolabeled antibodies ZMEO 18 and QC1054, whereas ‘3’I-labeled polyclonal rabbit antiferritin targets the tumor only slightly better than nonspecific rabbit immunoglobulin (Fig. 3). The dose deposited in tumor by ” 'In-radiolabeled P96.5 is I 1.6 times greater than control PAY276, 10.4 times greater than QC1054, and 5.7 times greater than ZMEO 18.
or 13’1polyclonal an-
tiferritin (Table 1). No staining was seen with irrelevant monoclonal antibody PAY276 or control normal rabbit
immunoglobulin. These results demonstrated the presence of the antigens P97, GP240, and ferritin in the U25 1 human glioma xenograft and provided a basis for radiolabeled antibody targeting studies. U251 human glioma initial accumulated activity (Ao)
U2.51 human glioma percent dose (tumor) Dose to normal organs, when calculated as a percentage of the dose to the tumor, defines the therapeutic ratio. Radiolabeled P96.5 results in a greater therapeutic ratio when compared both to monoclonal antibodies ZMEO 18 and QC1054 as well as polyclonal rabbit antiferritin (Fig. 4). With P96.5 administration, the doses to normal organs
The initial accumulated activities (Ao) of radiolabeled antibodies in tumor and normal organs are shown in Fig-
ures 1 and 2, and summarized in Table 1. The initial accumulated activity (Ao) in the glioma xenograft is 87.0 &i/gm following ’’‘In-radiolabeled monoclonal antibody P96.5 administration (Table 1). This value exceeds initial accumulated activities of control PAY276 monoclonal
80 m
F z P
70
d
60
KS.5
Ezkg
ZME018
@Z
QC!O54
LS!
PAY276
$j E E
=O
g
40
g 8 0”
30
i TUMOR
BRAIN
LIVER
SPLEEN
KIDNEY
LUNG
FEMUR
HEART
MUSCLE
Organ Fig. 1. Comparison of maximum activity (Ao) (rCi/gm) deposited in the tumor and normal organs by “‘Inradiolabeled monoclonal antibodies following injection of 100 cLCiof radiolabeled antibody: [Percent injected dose (% I.D.) per gram equals Ao (&i/gm)/(injected dose (PCi)) X 100.1. P96.5, ZMEO18, QCIO54, PAY276.
1371
Targeting of human glioma xenografts 0 J. A. WILLIAMSet d. 26 I
PolyclonalRXF
2 0 TUMOR
BRAIN
LUNG
LIVER
SPLEEN
KIDNEY TH MUSCLE FEMUR
Organ Fig. 2. Comparison of maximum activity (Ao) (&i/gm) deposited in the tumor and normal organs by ‘3’I-radiolabeled polyclonal antibody following injection of 100 &I of radiolabeled antibody: RXF = polyclonal rabbit antiferritin, NML IgG = normal rabbit immunoglobulin.
P96.5 versus “‘In P96.5 (Fig. 5) provided a basis for comparative tumor absorbed dose measurements in tumorbearing animals using thermoluminescent dosimetry.
are 10% or less of that in tumor. In contrast, doses to spleen and all remaining normal organs are a higher percentage of tumor dose following ZMEO 18, QCI054, and polyclonal rabbit antiferritin administration. The superior maximal initial activity in tumor (Ao) and tumor targeting ratio of “‘In-radiolabeled P96.5, and the low percentages of absorbed dose in normal organs, suggested measurement of the therapeutic potential of 9oYradiolabeled P96.5. The comparable biodistribution of 9oY
Thermoluminescent dosimetry Direct measurement of absorbed radiation dose by “rradiolabeled P96.5 in the human glioma xenograft by microthermoluminescent dosimetry revealed average absorbed doses (cGy) of 3770. 980, 353, and 275 in tumor,
12 11
m
lJ96.5
9
KS3
ZME018
8
B
Qc054
7
fSS
Polyclonal RXF
10
6 5 4 3 2 1 0 TUMOR
BRAIN
SPLEEN
LIVER
FEMUR
MUSCLE
LUNG
KIDNEY
Organ Fig. 3. Dose deposition in tumor and normal tissues expressed as a targeting ratio of ”'In-radiolabeled specific monoclonal antibodies (P96.5, ZMEOl8, and QCIO54) to irrelevant monoclonal antibody (PAY276), and 13’1polyclonal rabbit antifenitin to normal rabbit immunoglobulin: P96.5, ZME018, QC1054. RXF = polyclonal rabbit antifenitin.
I. J. Radiation Oncology 0 Biology0 Physics
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s
June 1990, Volume 18, Number 6
P96.5 K?zG ZMEOl8 Kz QclO54
90
6
c
a0
F x
70
.E
40
m
Polyckmal RXF
% 0” 30 E $ 20 $ a 10 0 TUMOR
BRAIN
SPLEEN
LIVER
KDNEY
LUNG
FEMUR
MUSCLE
Organ Fig. 4. Dose deposition in tumor and normal tissues by ’’‘In-radiolabeled monoclonal antibodies P96.5, ZMEO18, QC1054, and ‘3’I-polyclonal rabbit antiferritin expressed as a percentage of the tumor dose: P96.5, ZMEO18, QCI054, RXF = polyclonal rabbit antiferritin.
liver, contralateral control site, and total body (40) respectively, during the 7 days following injection of 100 j&i seY-radiolabeled P96.5 (Table 2).
and ZME018 antibodies were raised against antigens found in human melanoma. These results show that neuroectodermally derived antigens of one tumor may be expressed by a second neuroectodermally derived tumor and demonstrated by immunoperoxidase methods. Positive immunoperoxidase staining indicates synthesis of these same antigens by the U25 1 human glioma. Immunoperoxidase staining was positive for polyclonal rabbit antiferritin, and was negative for normal rabbit immunoglobulin. The extent of positive staining was greater for polyclonal rabbit antiferritin than for monoclonal
DISCUSSION Immunoperoxidase
staining
Immunoperoxidase staining of the U25 1 human xenograft was positive for monoclonal antibodies ZME018, and QCI054, but was negative for monoclonal antibody PAY276. Both monoclonal
glioma P96.5, control P96.5
100
7
=
QO-YP96.5
m
Ill-hPQ6.5
0 TUMOR
BRAIN
SPLEEN
LIVER
LUNG
KMJEY
!+ART
MUSCLE FEMUR
Organ Fig. 5. Comparison of maximum activity (Ao) (r.&i/gm) deposited in tumor and normal organs by “‘In- versus 9aY-radiolabeled P96.5 following injection of 100 &i radiolabeled antibody: “‘In-radiolabeled P96.5, y-radiolabeled P96.5.
Targeting of human glioma xenografis ??J. A. WILLIAMS et al. Table 2. U25 1 human glioma absorbed dose following 9oYlabeled P96.5 administration (cGy/lOO uCi administered)
Site Tumor Liver Contralateral Total body
Dose f S.E.M.
control
3770 980 353 275
f 445 + 127 f 41 f 14
QCI054. This result may indicate both the expression of a wide variety of ferritin epitopes within this tumor and the limitations of using a single monoclonal antibody recognizing a single epitope. Radiolabeled antibody targeting The results indicate superior targeting of the U25 1 human glioma by radiolabeled P96.5, an antibody specific for P97, a cell membrane protein originally purified from a human melanoma ( 18). Numerous authors have demonstrated cross-reactivity of anti-glioma antibodies with melanoma cells in vitro. Sikora et al. (35) and Schnegg et al. (33) noted binding of hybridoma anti-glioma antibody to six different cultured melanoma cell lines. Bourdon et al. (3) noted cross-reactivity of anti-8 lC6 antibody with one of seven cultured human melanoma cell lines by cell surface radioimmunoassay. By peroxidase-antiperoxidase staining, however, four of four melanoma cell lines were positive. Yoshida et al. (45) noted binding of hybridoma anti-glioma antibody to two of two cultured melanoma cell lines determined by indirect immunofluorescence. The cross-reactivity of anti-melanoma antibodies with cultured glioma cells has been noted as well. Herlyn et al. ( 19) noted specific immunoreactivity of monoclonal antimelanoma antibodies to cultured glioma cell lines. One hybridoma antibody bound to 13 of 17 melanoma cell lines and 6 of 7 astrocytoma lines by cell surface radioimmunoassay. Liao et al. (24) studied two monoclonal antibodies from a mouse immunized with a melanoma cell line and found cross-reactivity with different melanoma cell lines. Additional testing revealed a broad cross-reactivity among melanomas, neuroblastomas, retinoblastomas, and glioblastomas. DeMuralt et al. (9) studied the reactivity spectrum of three monoclonal antibodies reactive with a human glioma and five monoclonal antibodies reactive with a melanoma by cell surface radioimmunoassay. One of three anti-glioma monoclonal antibodies reacted with melanomas, whereas the five antimelanoma monoclonal antibodies reacted with gliomas, neuroblastomas, and medulloblastomas. Carrel et al. (6) studied the reactivity spectrum of five different monoclonal anti-melanoma antibodies which cross-reacted with gliomas and neuroblastomas and one monoclonal anti-glioma antibody which cross-reacted with a melanoma cell line. Comparison of binding activity of these monoclonal antibodies for 11 melanoma, 7
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ghoma, and 3 neuroblastoma cell lines showed that each of these clones had a different pattern of cross-reactivity. The results indicated that the antigenic determinants detected by these antibodies were not associated with the same antigen, and thus suggested the existence of at least six different antigens common to melanomas, gliomas, and neuroblastomas. Our results confirm the observed sharing of antigens in melanoma and glioma and now extend the observation to significant blood-borne targeting of a human glioma by a radiolabeled antibody in vivo. The results show a clear distinction between accumulated activity in tumor compared to normal organs and blood. The dose (expressed as a targeting ratio compared to irrelevant monoclonal antibody) to the human glioma xenograft from P96.5 exceeds 11.6. Both antibodies P96.5 and ZME018 are synthesized using human melanoma cell-surface antigens (P97 and GP240, respectively), but only P96.5 targets the human glioma xenograft significantly. Therefore, although antigens are likely shared between this glioma and human melanomas determined by peroxidase-immunoperoxidase studies, the concentration or availability of P97 for binding by antibody exceeds that of GP240 in the U251 human glioma xenograft in vivo. Both polyclonal antiferritin and monoclonal QCI054 antiferritin bind the U25 1 human glioma by peroxidaseantiperoxidase staining, and both demonstrate a moderate targeting ratio when compared with irrelevant immunoglobulin. These results may indicate that synthesized ferritin is less available for binding by antibody in this tumor. When compared to irrelevant immunoglobulin, the targeting ratios both of polyclonal rabbit antiferritin and monoclonal QC1054 antiferritin are greater than unity, indicating targeting. However, the magnitude of this ratio is much less for antiferritin antibodies compared with P96.5 when each is compared to its irrelevant counterpart. This difference indicates increased surface antigen concentration, increased availability for binding by antibody, or increased affinity of antibody for antigen when monoclonal antibody P96.5 is compared to polyclonal antiferritin. Direct thermoluminescent dosimetry of absorbed radiation dose in tumor following “Y-labeled P96.5 administration indicates disparate absorbed radiation dose in the tumor compared to the contralateral control site (Table 2) and corroborates in vivo biodistribution calculations discussed above. Absorbed dose in liver is moderate, a finding supported both by the current mouse biodistribution data and by human biodistribution studies of radiolabeled P96.5 (30). Thermoluminescent dosimetry demonstrates delivery of 3770 cGy to the tumor in 7 days following administration of 100 &i 9oY-P96.5 (3770 cGy/lOO PCi). These values for tumor absorbed dose, duration of study, and administered activity compare favorably to that seen in other studies (1, 15, 22, 46). Lee et al. (22) reported de-
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livery of 97 19 cGy over 11 days to human glioma xenografts using 1,000 PCi of 13’I-labeled 8 lC6 antibody (972 cGy/ 100 &I). Badger et al. ( 1) reported absorbed dose of 3300 cGy in a subcutaneous AKR T-cell lymphoma following administration of 1,000 &i ‘3’I-labeled anti-Thy 1.1 (330 cGy/ 100 &I), whereas Zalcberg et al. (46) calculated absorbed dose of 700 cGy to a human colon carcinema xenograft in athymic mice using 1,000 PC1 ‘j’Ilabeled 250-30.6 (70 cGy/lOO &I). Goldenberg et al. (15) calculated an absorbed dose of approximately 1500 cGy
June 1990, Volume 18, Number 6
to a human colon carcinoma xenograft using 1,000 &i of 13’1-labeled anti-CEA ( 150 cGy/ 100 &i). “Y-labeled P96.5 effectively targets human glioma xenografts in vivo and yields substantial tumor absorbed radiation doses as measured directly by thermoluminescent dosimetry. In addition, low to modest uptake in various normal organs implies a very favorable therapeutic ratio. The shared antigenicity among neuroectodermally derived tumors provides a basis for exploration of radioimmunotherapy of human gliomas.
REFERENCES 1. Badger, C.; Krohn, K. A.; Peterson, A. V.; Shulman. H.; Bernstein, I. D. Experimental radiotherapy of murine lymphoma with 13 l-Iodine-labeled anti-Thy 1.1 monoclonal antibody. Cancer Res. 45: 1536- 1544; 1985. 2. Beck, G.; Bullock, C. Synthesis of ferritin in cultured hepatoma cells. Fed. Exp. Biol. Sot. 47:3 14-3 17; 1974. 3. Bourdon, M. A.; Wikstrand, C. J.; Furthmayr, H.; Matthews. T. J.; Bigner, D. D. Human ghoma-mesenchymal extracellular matrix antigen defined by monoclonal antibody. Cancer Res. 43:2796-2805; 1983. 4. Bullard, D. E.; Gillespie, G. Y.; Mahaley, M. S.: Bigner, D. D. Immunobiology of human gliomas. Sem. Oncol. 13(1):96-109; 1986. 5. Cairncross, J. G.; Mattes, M. J.; Beresford, H. R.; Albino, A. P.; Houghton, A. N.; Lloyd, K. 0.; Old, L. J. Cell surface antigens of human astrocytoma defined by mouse monoclonal antibodies: Identification of astrocytoma subsets. Proc. Natl. Acad. Sci. USA 79:5641-5645; 1982. 6. Carrel, S.; Tribolet, N.; Mach, J. P. Expression of neuroectodermal antigens common to melanomas, ghomas, and neuroblastomas. Acta Neuropathol. (Berl.) 57: 158-164: 1982. 7. Coakham, H. B.; Garson, J. A.: Brownell, B. Diagnosis of cerebral neoplasms using monoclonal antibodies. Prog. Exp. Tumor Res. 29:57-77; 1985. 8. Day, E. D.; Lassiter, S.; Woodhall, B.; Mahaley, M. S. Localization of radioantibodies in human brain tumors. 1. Preliminary exploration. Cancer Res. 25:773-778; 1965. 9. DeMuralt, B.; deTribolet, N.; Diserens, A. C.; Stavrou. D.; Mach, J. P.; Carrel, S. Phenotyping of 60 cultured human gliomas and 34 other neuroectodermal tumors by means of monoclonal antibodies against glioma, melanoma, and HLA-DR antigens. Eur. J. Cancer Clin. Oncol. 21(2):207216; 1985. 10. deTribolet, N.; Carrell, S.; Mach, J. P. Brain tumor associated antigens. Prog. Exp. Tumor Res. 27: 1 I8- 13 1; 1984. Il. Eshhar, Z.; Order, S. E.; Katz, D. H. Fenitin, a Hodgkin’s disease associated antigen. Proc. Natl. Acad. Sci. USA 7 1: 3956-3969; 1974. 12. Ettinger, D. S.; Order, S. E.; Wharam, M. D.; Parker, M. K.; Klein, J. L.; Leichner, P. K. Phase I-II study of isotopic immunoglobulin therapy for primary liver cancer. Cancer Treat. Rep. 66:289-297; 1982. 13. Fischer, D. K.; Chen, T. L.; Raj, N. K. Immunological and biochemical strategies for the identification of brain tumor associated antigens. J. Neurosurg. 68: 165-l 80; 1988. 14. Garson, J. A.; Coakham, H. B.; Kemshead, J. T.; Brownell, B.; Harper, E. I.; Allan, P.; Boume, S. The role of monoclonal antibodies in brain tumor diagnosis and cerebrospinal fluid cytology. J. Neurosurg. 3: 165- 17 1; 1985. 15. Goldenberg, D. M.; Gaffar, S. A.; Bennett, S. J.; Beach,
J. L. Experimental radioimmunotherapy of xenografted human colonic tumor (GW-39) producing carcinoembryonic antigen. Cancer Res. 41:4354-4360; 198 1. 16. Gropp, C.; Havemann, K.: Lehmann, E. G. Carcinoembryonic antigen and ferritin in patients with lung cancer before and during therapy. Cancer 42:2802-2808; 1978. 17. Hann, H. L.; Levy, H. M.; Evans, A. E. Serum ferritin and neuroblastoma. Proc. Am. Sot. Chn. Oncol. 20: 176; 1979. 18. Hellstrom, I.; Garrigues, J.; Cabasco, T.; Mosely, G. H.: Brown, J. P.; Hellstrom, K. E. Studies of a high molecular weight human melanoma-associated antigen. J. Immun. 130(3):1467-1472; 1983. 19. Herlyn, M.; Clark, W. H.; Mastrangelo, M. J.; Guerry, D. P.; Elder, D. E.; LaRossa, D.; Hamilton, R.; Bondi, E.; Tuthill, R.; Steplewski, Z.; Koprowski, H. Specific immunoreactivity of hybridoma-secreted monoclonal anti-melanoma antibodies to cultured cells and freshly derived human cells. Cancer Res. 40:3602-3609; 1980. 20. Kemshead, J. T.; Goldman, A.; Jones, D.; Pritchard, J.; Malpas, J. S.; Gordon, I.; Malone, J. F.; Hurley, G. D.; Breatnach, F. Therapeutic application of radiolabeled monoclonal antibody UJ 13A in children with disseminated neuroblastoma-a phase I study. Prog. Clin. Biol. Res. 175: 533-544; 1985. 2 1. Larson, S. M. Radiolabeled monoclonal anti-tumor antibodies in diagnosis and therapy. J. Nucl. Med. 26(5):538545; 1985. 22. Lee, Y. S.; Bullard, D. E.; Zalutsky, M. R.; Coleman, R. E.; Wikstrand, C. J.; Friedman, H. S.; Calapinto, E. V.; Bigner, D. D. Therapeutic efficacy of antiglioma mesenchymal extracellular matrix 13 1-Iodine-radiolabeled murine monoclonal antibody in a human glioma xenograft model. Cancer Res. 48:559-566; 1988. 23. Lenhard, R. E.; Order, S. E.; Spunberg, J. J. Isotopic immunoglobulin: a new systemic therapy for advanced Hodgkin’s disease. J. Clin. Oncol. 3( 10): 1296-1300; 1985. 24. Liao, S. K.; Clarke, B. J.; Kwong, P. C.; Brickenden, A.; Gallic, B. L.; Dent, P. B. Common neuroectodermal antigens on human melanoma, neuroblastoma, retinoblastoma, ghoblastoma and fetal brain revealed by hybridoma antibodies raised against melanoma cells. Eur. J. Immunol. 11: 450-454; 198 1. 25. Marcus, D. M.; Zinberg, N. Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoadsorbents. Arch. Biochem. Biophys. 162:493-50 1; 1974. 26. Marks, J. E.; Baglan, R. J.; Prassad, S. C.; Blank, W. F. Cerebral radionecrosis: incidence and risk in relation to dose, time, fractionation, and volume. Int. J. Radiat. Oncol. Biol. Phys. 7~243-252; 198 1. 27. Marrack, D.; Kubala, M.; Carey, P.; Leavens, M.; Howze,
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J. Localization of intracranial tumors: comparative study with “‘I-labeled antibody to human fibrinogen and neohydrin 203-Hg. Cancer 20:75 l-755; 1967. Order, S. E.; Stillwagon, G. B.; Klein, J. L.; Leichner, P. K.; Singelman, S. S.; Fishman, E. K.; Ettinger, D. S.; Haulk, T.; Kopher, K.; Finney, K. Iodine- 13 1 antiferritin, a new treatment modality in hepatoma: a Radiation Therapy Oncology Group study. J. Clin. Oncol. 3(12):1573-1582; 1985. Order, S. E.; Klein, J. L.; Leichner, P. K.; Frincke, J.; Lollo, C.; Carlo, D. J. 90-Yttrium antiferritin-a new therapeutic radiolabeled antibody. Int. J. Radiat. Oncol. Biol. Phys. 12: 277-28 1; 1986. Rosenblum, M. G.; Murray, J. L.; Haynie, T. P.; Glenn, H. J.; Jahns, M. F.; Benjamin, R. S.; Frincke, J. M.; Carlo, D. J.; Hersh, E. M. Pharmacokinetics of 11 l-In-labeled antiP97 monoclonal antibody in patients with metastatic melanoma. Cancer Res. 45:2382-2386; 1985. Restock, R. A.; Klein, J. L.; Leichner, P. K.; Kopher, K. A.; Order, S. E. Selective tumor localization in experimental hepatoma by radiolabeled antiferritin. Int. J. Radiat. Oncol. Biol. Phys. 9:1345-1350; 1983. Salazar, 0. M.; Rubin, P.; Fieldstein, M. L.; Pizzutiello, R. High dose radiation therapy in the treatment of malignant gliomas: final report. Int. J. Radiat. Oncol. Biol. Phys. 5: 1733-1748; 1979. Schnegg, J. F.; Diserens, A. C.; Carrel, S. Human gliomaassociated antigens detected by monoclonal antibodies. Cancer Res. 41:1209-1213; 1981. Seeger, R. C.; Zeltezer, P. M.; Rayner, S. A. Onto-neural antigen: a new neural differentiation antigen expressed by neuroblastoma, oat cell carcinoma, Wilms’ tumor, and sarcoma cells. J. Immunol. 122:1548-1555; 1979. Sikora, K.; Alderson, T.; Phillips, J.; Watson, J. V. Human hybridomas from malignant gliomas. Lancet 1:1 1- 14; 1982. Takahashi, H.; Herlyn, D.; Atkinson, B.; Alavi, A.; Powe, J.; Rodeck, U.; Bruce, D. A.; Koprowski, H. Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor. Cancer Res. 47:3847-3850; 1987. Taylor, A.; Milton, W.; Eyre, H.; Christian, P.; Wu, F.; Ha-
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gan, P.; Alazaraki, N.; Datz, F. L.; Unger, M. Radioimmunodetection of human melanoma with ’’‘In-labeled monoclonal antibody. J. Nucl. Med. 29:329-337; 1988. Walker, M. D.; Alexander, E.; Hunt, W. E.; MacCarty, S.; Mahaley, M.; Mealey, J.; Norrell, H. A.; Owens, G.; Ransohoff, J.; Wilson, C. B.; Gehan, E. A.; Strike, T. A. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. J. Neurosurg. 49:333-343; 1978. Wessels, B. W.; Griffith, M. H. Miniature thermoluminescent dosimetry absorbed dose measurements in tumor phantom models. J. Nucl. Med. 27: 1308- 13 14; 1986. Wessels, B. W.; Griffith, M. H.; Bradley, E. W. Dosimetric measurements and radiobiological consequences of radioimmunotherapy in tumor-bearing mice. Fourth International Radiopharmaceutical Dosimetry Symposium, CONF-85 1113, Oak Ridge, 1985:446-457. Wikstrand, C. J.; Bigner, D. D. Expression of human fetal brain antigens by human tumors of neuroectodermal origin as defined by monoclonal antibodies. Cancer Res. 42:267275; 1982. Wikstrand, C. J.; Mclendon, R. E.; Carrel, S.; Kemshead, J. T.; Mach, J. P.; Coakham, H. B.; deTribolet, N.; Bullard, D. E.; Zalutsky, M. R.; Bigner, D. D. Comparative localization of glioma-reactive monoclonal antibodies in an athymic nude mouse human glioma xenograft model. J. Neuroimmunol. 15:37-56; 1987. White, G. P.; Worwood, M.; Parry, D. H. Ferritin synthesis in normal and leukemic leukocytes. Nature 250:584-585, 1974. Woodbury, R. G.; Brown, J. P.; Yeh, M. Y.; Hellstrom, I.; Hellstrom, K. E. Identification of a cell surface protein, P97, in human melanomas and certain other neoplasms. Proc. Natl. Acad. Sci. USA 77(4):2183-2187; 1980. Yoshida, J.; Wakabayashi, T.; Kito, A. Clinical application of monoclonal antibodies against glioma-associated antigens. Prog. Exp. Tumor. Res. 30:44-56; 1987. Zalcberg, J. R.; Thompson, C. H.; Lichtenstein, M.; McKenzie, I. F. Tumor immunotherapy in the mouse with the use of 13 1-Iodine-labeled monoclonal antibodies. J. Natl. Cancer Inst. 72:697-704; 1984.
Inf. .I Rodulion Oncology Biol. Phys.. Vol. IS, pp. 1361-1375 Printed in the US A. All rights reserved.
??Original Contribution
TARGETING OF HUMAN GLIOMA XENOGRAFTS IN UTILIZING RADIOLABELED ANTIBODIES
VZVO
JEFFERY A. WILLIAMS, M.D.,’ BARRY W. WESSELS, PH.D.,* MOODY D. WHARAM, STANLEY E. ORDER, M.D., Sc.D., F.A.C.R.,’ PHILIP M. WANEK,~ J. KENNETH POGGENBURG, PH.D.~ AND JERRY L. KLEIN, PH.D.’
M.D.,’
‘Division of Radiation Oncology, The Johns Hopkins Oncology Center, 600 North Wolfe Street, Baltimore, MD 2 1205; ‘Division of Radiation Oncology and Biophysics, The George Washington University Medical Center, 901 23rd Street, N.W., Washington, D.C. 20037; and ‘Hybritech, Incorporated, San Diego, CA 92 12 1
Radiolabeled antibodies provide a potential basis for selective radiotherapy of human gliomas. We have measured tumor targeting by radiolabeled monoclonal and polyclonal antibodies directed against neuroectodermal and tumorassociated antigens in nude mice bearing human glioma xenografts. Monoclonal F’96.5, a mouse IgG2a immunoglobulin, defines an epitope of a human melanoma cell surface protein, and specifically binds the U-251 human glioma as measured by immunoperoxidase histochemistry. “‘In-radiolabeled P96.5 specifically targets the U-251 human glioma xenograft and yields 87.0 microCuries (&i) of tumor activity per gram per 100 PCi injected activity compared to 4.5 &i following administration of radiolabeled irrelevant monoclonal antibody. Calculations of targeting ratios demonstrate deposited dose to be 11.6 times greater with radiolabeled P96.5 administration compared to irrelevant monoclonal antibody. The proportion of tumor dose found in normal organs is less than lo%, further supporting specific targeting of the human glioma xenograft by this antibody. Monoclonal antibody ZME018, which defines a second melanoma-associated antigen, and polyclonal rabbit antiferritin, which defines a tumorassociated antigen, demonstraCe positive immunoperoxidase staining of the tumor, but comparatively decreased targeting. When compared to the “‘In-radiolabeled antibody, “Y-radiolabeled P96.5 demonstrates comparable tumor targeting and percentages of tumor dose found in normal organs. To test the therapeutic potential of qradiolabeled P96.5, tumors and normal sites were implanted with miniature thermoluminescent dosimeters (TLD). Seven days following administration of 100 WCig”Y-radiolabeled P96.5, average absorbed doses of 3770,980,353, and 274 cGy were observed in tumor, liver, contralateral control site, and total body, respectively. Shared cell surface antigens among neuroectodermally derived neoplasms provide a basis for exploration of human glioma radioimmunotherapy. Human glioma, Radioimmunotherapy,
Tumor localization, Thermoluminescent
dosimetry.
7, 8, 13, 14, 27, 36). The results of many studies using both monoclonal and polyclonal antibodies indicate a shared antigenicity among neuroectodermally derived tissues including glioma, melanoma, neuroblastoma, and fetal brain (5, 6, 10, 20, 33, 34, 41, 42). Radiolabeled antibodies directed against neuroectodermal antigens may provide differential delivery of radiation to gliomas and sparing of surrounding normal brain. P96.5, a murine monoclonal antibody of the IgG2a subclass, targets P97, a cell surface glycoprotein of molecular weight 97,000. Initially described in high concentrations in human melanomas (44), P97 has subsequently
INTRODUCTION Human malignant gliomas are rarely cured and conventional postoperative external beam radiotherapy only extends median survival (38). With higher radiation doses,
median survival, but not long-term survival, is improved (32). The tolerance of adjacent normal brain limits the total radiation dose which can be safely administered (26). Radiolabeled antibodies offer potential selectivity in the delivery of radiation to human neoplasms ( 12,2 1,23,28, 3 1). The role of radioimmunotherapy in the treatment of human gliomas, however, remains largely unexplored (4,
Reprint requests to: Jeffery A. Williams, M.D., Division of Neurosurgery, The University of Oklahoma Health Sciences Center, South Pavilion, Room 4SP200, Oklahoma City, OK 73190. Acknowledgements-We thank Ms. Alverta E. Fields for her assistance in the preparation of this manuscript.
Supported by Hybritech, Inc., The Preuss Foundation for Brain Tumor Research, Inc., and The Children’s Cancer Foundation, Inc. Accepted for publication 20 December 1989.
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been found in a variety of cultured cells of neuroectoderma1 origin including glioblastoma, neuroblastoma, and retinoblastoma (6, 19, 24). Similarly, monoclonal antibody ZMEO18 targets GP240, a second melanoma cell surface glycoprotein of molecular weight 240,000 (I 7). To the present, GP240 has not been assessed in human glial neoplasms. Both polyclonal rabbit antiferritin and monoclonal antibody QCIO54 target ferritin, a tumor-associated protein found in several human neoplasms (2, 11, 16, 25, 43) including neuroblastoma (17). Hence, a shared antigenic profile including P97, GP240, and ferritin may exist among neuroectodermally derived neoplasms. Radiolabeled P96.5 (30), ZME018 (37), QC1054 (S. E. Order, personal oral communication, 1988), and polyclonal rabbit antiferritin (28,29) have successfully targeted disparate human neoplasms in vivo. Despite the possible cross-reactivity with neuroectodermally derived neoplasms, the systematic analysis of these antibodies for possible human glioma immunotherapy has not been performed. Therefore, we tested histochemical binding, in vivo targeting, and therapeutic potential of these antibodies in a human glioma xenograft-nude mouse model. METHODS
AND MATERIALS
Tumor cell line Human grade 4 glioma cell line, U-25 1, was obtained from the DCT Tumor Repository, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland. Cells were cultured in Dulbecco’s modified minimal essential medium/Ham’s F- 12 nutrient mixture containing 10% fetal bovine serum* and antibiotics (penicillin and streptomycin) in 12 X 80 cm plastic culture dishes.+ Cells were grown in a humidified incubator at 37°C and gassed with a mixture of 5% carbon dioxide and 95% air. Media was changed twice weekly and cells passaged at confluence with 0.5% trypsin.* Nude mice Six-week old male athymic nude mice were injected subcutaneously in the left posterior thigh with 5 X lo6 glioma cells. Tumors were visible 1 week after injection. All experiments were performed when tumors grew to 0.3-0.4 gm. Ferritin purification and antibody production Ferritin Hodgkin’s
was purified from the spleens of patients with disease as previously described ( 12). New Zea-
land white rabbits were immunized with 100 ug of purified ferritin emulsified in complete Freund’s adjuvant injected intradermally in the thigh, followed 2 weeks later by 100 ug of purified ferritin emulsified in incomplete Freund’s adjuvant.
* GIBCO, Grand Island, NY. + Falcon Plastics, Cockeysville, MD.
June 1990, Volume 18, Number 6
Normal rabbit IgG and rabbit antiferritin IgG were purified by a combination of DEAE ion exchange chromatography and ammonium sulfate precipitation as described previously ( 12). Control IgG was isolated from normal rabbit serum taken from the same rabbits prior to immunization. Chelated monoclonal antiferritin QCI054, chelated monoclonal anti-prostatic acid phosphatase PAY276, and chelated monoclonal antibodies P96.5 and ZMEO 18 were supplied.* Immunoperoxidase staining Immunoperoxidase staining of U-25 I human glioma xenografts was performed using polyclonal and monoclonal antiferritin, monoclonal P96.5 and ZMEO 18, and nonspecific polyclonal normal rabbit IgG as well as nonspecific monoclonal anti-prostatic acid phosphatase. Subcutaneously grown U-25 1 tumors were excised and fixed sequentially in Bouin-Hollande’s solution and 70% ethanol, subsequently cut in 8 micron sections with a microtome, and mounted on glass slides. Polyclonal rabbit antiferritin and normal rabbit immunoglobulin immunoperoxidase procedure. Mounted tumor specimens were deparaffinized in one change of iodine-saturated xylene for 3 min, and sequentially hydrated through graded alcohols. Specimens were then rinsed in Tris-HCl buffered saline (TBS) 0.05 M, pH 7.6 for 5 min and incubated with 3% hydrogen peroxide for 10 min. Following rinsing in TBS for 5 min, sections were incubated with normal swine serum diluted I:20 in 0.5 M TBS in a moist incubation chamber at room temperature for 20 min. Following rinsing of sections in TBS for 5 min, sections were incubated with rabbit polyclonal antiferritin diluted 1:100 with TBS in a moist incubation chamber overnight at 4°C. Sections were then rinsed with TBS and incubated with swine anti-rabbit immunoglobulin diluted 1:20 with TBS for 30 min. Following rinsing with TBS, sections were then incubated with rabbit peroxidase-antiperoxidase diluted I : 100 with TBS for 20 min. Following rinsing with TBS, sections were incubated for 10 min in aminoethylcarbazole (AEC) solution. Following a wash in running water for 5 min, sections were counterstained with Mayer’s Hematoxylin and mounted. Monoclonal QCI antiferritin, P96.5, ZMEOl8, and PA Y276 immunoperoxidase procedure. Following deparaffinization, sections were rinsed sequentially in xylene and absolute alcohol for 2 min each. Sections were then incubated in hydrogen peroxide in methanol for 20 min (9 parts methanol to 1 part 3% H202). Following rinsing in several changes of deionized water, sections were washed in TBS for 5 min and then incubated in a moist chamber for 20 min in normal goat serum diluted 1:20
t Hybritech, Inc., San Diego, CA.
Targeting of human glioma xenografts 0 J. A. WILLIAMSet al.
with TBS. Sections were then washed in TBS for 5 min and incubated in a moist chamber with primary antiserum overnight at 4°C. Sections were then washed in three rinses of TBS for 5 min each and then incubated in biotinylated mouse immunoglobulin diluted 1: 100 with TBS for 20 min. Following rinsing in three changes of TBS for 5 min each, sections were incubated for 30 min in horseradish peroxidase-Avidin D diluted 1:150 with TBS. Following rinsing with TBS, sections were incubated for 10 min in aminoethylcarbazole solution. Following rinsing in running water for 5 min, sections were counterstained with Mayer’s Hematoxylin and mounted. Antibody radiolabeling Polyclonal immunoglobulins were labeled with 13’1by the lactoperoxidase glucose oxidase method” at a labeling ratio of 5 mCi of 13iI per mg of protein. The labeled immunoglobulins were separated from free iodine using a column.** Percent binding of isotope to immunoglobulin was determined by trichloroacetic acid precipitation. Monoclonal antibodies were labeled with “‘In (P96.5, ZMEO 18, QCI054, PAY276) or 9oY (P96.5) by chelation using a proprietary procedure,++ at a labeling ratio of 5 mCi per mg of protein. Percent binding of isotope to antibody was determined by thin layer chromatography. Assay of radiolabeled antibody distribution Tumor bearing nude mice were injected with 25 or 100 PCi of radiolabeled antibody by intracardiac injection and sacrificed 1,2,5, and 7 days following injection in groups of at least two animals per time point. Calibration of well counter: A known quantity of 13’1, 9oY, or ’’‘In was placed in a radioisotopic calibrator (sensitivity to 1 @i). Counts per minute were plotted from the well counter as a function of &I allowing direct conversion to activity. Expression of accumulated activity in organs and ratio calculation: The tumor and all organs were individually weighed.## Counts per minute were obtained from the well counter and then converted to PCi by a conversion factor as determined above. The counts per minute per gram of each organ and tumor were recorded. The resulting activity in the tumor and organs was then expressed as &i/gm of tissue as a function of time. A single exponential fit to the curve of activity/gram versus time was calculated and a best fit determined by the method of least squares for each organ and each experiment. The effective half-life (T,s) and maximal initial activity (Ao) of antibody in tumor or organ were calculated as previously described (3 1). The total dose deposited in tissue is equal to the sum of the penetrating and nonpenetrating radiations. The 8 Bio-Rad Laboratories, Richmond, CA. ** PD- 10 Sephadex, Pharmacia Fine Chemicals, Piscataway, NJ. ++Developed by Hybritech, Inc., San Diego, CA.
1369
majority of dose is deposited by the nonpenetrating component. Assuming a uniform isotropic model for the distribution of radiolabeled antibody, the total deposited dose for any organ resulting from the sum of nonpenetrating radiations (NP) accumulated over time is given by: D = 1.44 X Ao X Teff X NP(cGy)
(31)
where D = total mean dose (cGy), Ao = initial concentration of antibody in tumor or organ (&i/gm), T,R = effective half-life (hours), and NP = sum of nonpenetrating radiations (gm - cGy/&i - hr). Although the NP component is difficult to assess in small organs, expressing the results as a dose ratio of specific/nonspecific antibody cancels this term in the equation. Thus, the targeting ratio equals: Ao X Teff (specific) Ao X Teff (nonspecific) in the tumor or normal organ in question. The percentage of the tumor dose found in normal organs was determined by normalizing the product of initial activity and effective half-life of accumulated radioactivity (Ao X T,tf) in each normal organ to that in the tumor. Thermoluminescent dosimetry Paired miniature teflon-embedded CaS04 :Dy thermoluminescent dosimeters (TLD) were implanted directly into the human glioma xenografts and dorsal interscapular subcutaneous sites, while individual TLD were implanted in liver and a contralateral control subcutaneous site via a 20 gauge needle (40). Each TLD was first cross-calibrated with known activities of 9oY as previously described (39). Mice bearing 0.3-0.4 gm tumors and dosimeters were injected with 100 PCi v-labeled P96.5 antibody as described above and sacrificed 7 days following injection. TLD were removed, rinsed,@ and measured for absorbed radiation dose as follows: The TLD glow curve peak was integrated over 50 set with T, = 115°C and T2 = 275°C. Temperature ramping was 3.5”C per set with prereadout annealing cycle of 5 sec. The major integration peak occurs at 220-240°C with a minor peak (5%) at 120°C for this TLD. RESULTS
Immunoperoxidase staining Positive immunoperoxidase staining of fixed U25 1 tumors was demonstrated with monoclonal antibodies P96.5, ZME018, and QCI054, and polyclonal rabbit an** Metler balance Model H5 1AR, Metler Instrument Corp., Hightstown, NJ. @ Count-Off, New England Nuclear Research Products, Boston, MA.
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June 1990,
Table 1. Antibody activity in U-25 1 human glioma xenografts
Antibody
Immunoperoxidase staining
Initial deposited activity (Ao)*
Tee (days)
+ + + _
86.7 22.0 5.1 4.5
2.2 1.7 4.2 3.3
Monoclonal P96.5 ZME018 QC1054 PAY276 Polyclonal Polyclonal rabbit antiferritin Normal rabbit immunoglobulin
+
25.0
1.6
_
15.0
1.9
* Activity (Ao) (uCi ” ‘In monoclonal tibody/gram/ 100 uCi injected).
Volume 18, Number 6
antibody (4.5 pCi/gm) and demonstrates greater accumulation than both ZMEO 18 (22.0 &i/gm) and QC1054 (5.1 pCi/gm) monoclonal antibodies. ‘3’I-radiolabeled polyclonal rabbit antiferritin demonstrates an initial accumulation (25.0 pCi/gm) greater than control normal rabbit immunoglobulin (15.0 &i/ gm), but less than radiolabeled monoclonal P96.5 (Table 1). U25I human glioma targeting ratios The targeting ratios of specific antibody to control nonspecific antibody in the tumor and normal organs are shown in Figure 3. The targeting ratio is the quotient of absorbed dose (cGy) in tumor or normal organs following specific antibody administration, and the absorbed dose (cGy) in tumor or normal organs following nonspecific antibody administration. ’’‘In-labeled P96.5 targets the human glioma xenograft greater than radiolabeled antibodies ZMEO 18 and QC1054, whereas ‘3’I-labeled polyclonal rabbit antiferritin targets the tumor only slightly better than nonspecific rabbit immunoglobulin (Fig. 3). The dose deposited in tumor by ” 'In-radiolabeled P96.5 is I 1.6 times greater than control PAY276, 10.4 times greater than QC1054, and 5.7 times greater than ZMEO 18.
or 13’1polyclonal an-
tiferritin (Table 1). No staining was seen with irrelevant monoclonal antibody PAY276 or control normal rabbit
immunoglobulin. These results demonstrated the presence of the antigens P97, GP240, and ferritin in the U25 1 human glioma xenograft and provided a basis for radiolabeled antibody targeting studies. U251 human glioma initial accumulated activity (Ao)
U2.51 human glioma percent dose (tumor) Dose to normal organs, when calculated as a percentage of the dose to the tumor, defines the therapeutic ratio. Radiolabeled P96.5 results in a greater therapeutic ratio when compared both to monoclonal antibodies ZMEO 18 and QC1054 as well as polyclonal rabbit antiferritin (Fig. 4). With P96.5 administration, the doses to normal organs
The initial accumulated activities (Ao) of radiolabeled antibodies in tumor and normal organs are shown in Fig-
ures 1 and 2, and summarized in Table 1. The initial accumulated activity (Ao) in the glioma xenograft is 87.0 &i/gm following ’’‘In-radiolabeled monoclonal antibody P96.5 administration (Table 1). This value exceeds initial accumulated activities of control PAY276 monoclonal
80 m
F z P
70
d
60
KS.5
Ezkg
ZME018
@Z
QC!O54
LS!
PAY276
$j E E
=O
g
40
g 8 0”
30
i TUMOR
BRAIN
LIVER
SPLEEN
KIDNEY
LUNG
FEMUR
HEART
MUSCLE
Organ Fig. 1. Comparison of maximum activity (Ao) (rCi/gm) deposited in the tumor and normal organs by “‘Inradiolabeled monoclonal antibodies following injection of 100 cLCiof radiolabeled antibody: [Percent injected dose (% I.D.) per gram equals Ao (&i/gm)/(injected dose (PCi)) X 100.1. P96.5, ZMEO18, QCIO54, PAY276.
1371
Targeting of human glioma xenografts 0 J. A. WILLIAMSet d. 26 I
PolyclonalRXF
2 0 TUMOR
BRAIN
LUNG
LIVER
SPLEEN
KIDNEY TH MUSCLE FEMUR
Organ Fig. 2. Comparison of maximum activity (Ao) (&i/gm) deposited in the tumor and normal organs by ‘3’I-radiolabeled polyclonal antibody following injection of 100 &I of radiolabeled antibody: RXF = polyclonal rabbit antiferritin, NML IgG = normal rabbit immunoglobulin.
P96.5 versus “‘In P96.5 (Fig. 5) provided a basis for comparative tumor absorbed dose measurements in tumorbearing animals using thermoluminescent dosimetry.
are 10% or less of that in tumor. In contrast, doses to spleen and all remaining normal organs are a higher percentage of tumor dose following ZMEO 18, QCI054, and polyclonal rabbit antiferritin administration. The superior maximal initial activity in tumor (Ao) and tumor targeting ratio of “‘In-radiolabeled P96.5, and the low percentages of absorbed dose in normal organs, suggested measurement of the therapeutic potential of 9oYradiolabeled P96.5. The comparable biodistribution of 9oY
Thermoluminescent dosimetry Direct measurement of absorbed radiation dose by “rradiolabeled P96.5 in the human glioma xenograft by microthermoluminescent dosimetry revealed average absorbed doses (cGy) of 3770. 980, 353, and 275 in tumor,
12 11
m
lJ96.5
9
KS3
ZME018
8
B
Qc054
7
fSS
Polyclonal RXF
10
6 5 4 3 2 1 0 TUMOR
BRAIN
SPLEEN
LIVER
FEMUR
MUSCLE
LUNG
KIDNEY
Organ Fig. 3. Dose deposition in tumor and normal tissues expressed as a targeting ratio of ”'In-radiolabeled specific monoclonal antibodies (P96.5, ZMEOl8, and QCIO54) to irrelevant monoclonal antibody (PAY276), and 13’1polyclonal rabbit antifenitin to normal rabbit immunoglobulin: P96.5, ZME018, QC1054. RXF = polyclonal rabbit antifenitin.
I. J. Radiation Oncology 0 Biology0 Physics
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s
June 1990, Volume 18, Number 6
P96.5 K?zG ZMEOl8 Kz QclO54
90
6
c
a0
F x
70
.E
40
m
Polyckmal RXF
% 0” 30 E $ 20 $ a 10 0 TUMOR
BRAIN
SPLEEN
LIVER
KDNEY
LUNG
FEMUR
MUSCLE
Organ Fig. 4. Dose deposition in tumor and normal tissues by ’’‘In-radiolabeled monoclonal antibodies P96.5, ZMEO18, QC1054, and ‘3’I-polyclonal rabbit antiferritin expressed as a percentage of the tumor dose: P96.5, ZMEO18, QCI054, RXF = polyclonal rabbit antiferritin.
liver, contralateral control site, and total body (40) respectively, during the 7 days following injection of 100 j&i seY-radiolabeled P96.5 (Table 2).
and ZME018 antibodies were raised against antigens found in human melanoma. These results show that neuroectodermally derived antigens of one tumor may be expressed by a second neuroectodermally derived tumor and demonstrated by immunoperoxidase methods. Positive immunoperoxidase staining indicates synthesis of these same antigens by the U25 1 human glioma. Immunoperoxidase staining was positive for polyclonal rabbit antiferritin, and was negative for normal rabbit immunoglobulin. The extent of positive staining was greater for polyclonal rabbit antiferritin than for monoclonal
DISCUSSION Immunoperoxidase
staining
Immunoperoxidase staining of the U25 1 human xenograft was positive for monoclonal antibodies ZME018, and QCI054, but was negative for monoclonal antibody PAY276. Both monoclonal
glioma P96.5, control P96.5
100
7
=
QO-YP96.5
m
Ill-hPQ6.5
0 TUMOR
BRAIN
SPLEEN
LIVER
LUNG
KMJEY
!+ART
MUSCLE FEMUR
Organ Fig. 5. Comparison of maximum activity (Ao) (r.&i/gm) deposited in tumor and normal organs by “‘In- versus 9aY-radiolabeled P96.5 following injection of 100 &i radiolabeled antibody: “‘In-radiolabeled P96.5, y-radiolabeled P96.5.
Targeting of human glioma xenografis ??J. A. WILLIAMS et al. Table 2. U25 1 human glioma absorbed dose following 9oYlabeled P96.5 administration (cGy/lOO uCi administered)
Site Tumor Liver Contralateral Total body
Dose f S.E.M.
control
3770 980 353 275
f 445 + 127 f 41 f 14
QCI054. This result may indicate both the expression of a wide variety of ferritin epitopes within this tumor and the limitations of using a single monoclonal antibody recognizing a single epitope. Radiolabeled antibody targeting The results indicate superior targeting of the U25 1 human glioma by radiolabeled P96.5, an antibody specific for P97, a cell membrane protein originally purified from a human melanoma ( 18). Numerous authors have demonstrated cross-reactivity of anti-glioma antibodies with melanoma cells in vitro. Sikora et al. (35) and Schnegg et al. (33) noted binding of hybridoma anti-glioma antibody to six different cultured melanoma cell lines. Bourdon et al. (3) noted cross-reactivity of anti-8 lC6 antibody with one of seven cultured human melanoma cell lines by cell surface radioimmunoassay. By peroxidase-antiperoxidase staining, however, four of four melanoma cell lines were positive. Yoshida et al. (45) noted binding of hybridoma anti-glioma antibody to two of two cultured melanoma cell lines determined by indirect immunofluorescence. The cross-reactivity of anti-melanoma antibodies with cultured glioma cells has been noted as well. Herlyn et al. ( 19) noted specific immunoreactivity of monoclonal antimelanoma antibodies to cultured glioma cell lines. One hybridoma antibody bound to 13 of 17 melanoma cell lines and 6 of 7 astrocytoma lines by cell surface radioimmunoassay. Liao et al. (24) studied two monoclonal antibodies from a mouse immunized with a melanoma cell line and found cross-reactivity with different melanoma cell lines. Additional testing revealed a broad cross-reactivity among melanomas, neuroblastomas, retinoblastomas, and glioblastomas. DeMuralt et al. (9) studied the reactivity spectrum of three monoclonal antibodies reactive with a human glioma and five monoclonal antibodies reactive with a melanoma by cell surface radioimmunoassay. One of three anti-glioma monoclonal antibodies reacted with melanomas, whereas the five antimelanoma monoclonal antibodies reacted with gliomas, neuroblastomas, and medulloblastomas. Carrel et al. (6) studied the reactivity spectrum of five different monoclonal anti-melanoma antibodies which cross-reacted with gliomas and neuroblastomas and one monoclonal anti-glioma antibody which cross-reacted with a melanoma cell line. Comparison of binding activity of these monoclonal antibodies for 11 melanoma, 7
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ghoma, and 3 neuroblastoma cell lines showed that each of these clones had a different pattern of cross-reactivity. The results indicated that the antigenic determinants detected by these antibodies were not associated with the same antigen, and thus suggested the existence of at least six different antigens common to melanomas, gliomas, and neuroblastomas. Our results confirm the observed sharing of antigens in melanoma and glioma and now extend the observation to significant blood-borne targeting of a human glioma by a radiolabeled antibody in vivo. The results show a clear distinction between accumulated activity in tumor compared to normal organs and blood. The dose (expressed as a targeting ratio compared to irrelevant monoclonal antibody) to the human glioma xenograft from P96.5 exceeds 11.6. Both antibodies P96.5 and ZME018 are synthesized using human melanoma cell-surface antigens (P97 and GP240, respectively), but only P96.5 targets the human glioma xenograft significantly. Therefore, although antigens are likely shared between this glioma and human melanomas determined by peroxidase-immunoperoxidase studies, the concentration or availability of P97 for binding by antibody exceeds that of GP240 in the U251 human glioma xenograft in vivo. Both polyclonal antiferritin and monoclonal QCI054 antiferritin bind the U25 1 human glioma by peroxidaseantiperoxidase staining, and both demonstrate a moderate targeting ratio when compared with irrelevant immunoglobulin. These results may indicate that synthesized ferritin is less available for binding by antibody in this tumor. When compared to irrelevant immunoglobulin, the targeting ratios both of polyclonal rabbit antiferritin and monoclonal QC1054 antiferritin are greater than unity, indicating targeting. However, the magnitude of this ratio is much less for antiferritin antibodies compared with P96.5 when each is compared to its irrelevant counterpart. This difference indicates increased surface antigen concentration, increased availability for binding by antibody, or increased affinity of antibody for antigen when monoclonal antibody P96.5 is compared to polyclonal antiferritin. Direct thermoluminescent dosimetry of absorbed radiation dose in tumor following “Y-labeled P96.5 administration indicates disparate absorbed radiation dose in the tumor compared to the contralateral control site (Table 2) and corroborates in vivo biodistribution calculations discussed above. Absorbed dose in liver is moderate, a finding supported both by the current mouse biodistribution data and by human biodistribution studies of radiolabeled P96.5 (30). Thermoluminescent dosimetry demonstrates delivery of 3770 cGy to the tumor in 7 days following administration of 100 &i 9oY-P96.5 (3770 cGy/lOO PCi). These values for tumor absorbed dose, duration of study, and administered activity compare favorably to that seen in other studies (1, 15, 22, 46). Lee et al. (22) reported de-
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livery of 97 19 cGy over 11 days to human glioma xenografts using 1,000 PCi of 13’I-labeled 8 lC6 antibody (972 cGy/ 100 &I). Badger et al. ( 1) reported absorbed dose of 3300 cGy in a subcutaneous AKR T-cell lymphoma following administration of 1,000 &i ‘3’I-labeled anti-Thy 1.1 (330 cGy/ 100 &I), whereas Zalcberg et al. (46) calculated absorbed dose of 700 cGy to a human colon carcinema xenograft in athymic mice using 1,000 PC1 ‘j’Ilabeled 250-30.6 (70 cGy/lOO &I). Goldenberg et al. (15) calculated an absorbed dose of approximately 1500 cGy
June 1990, Volume 18, Number 6
to a human colon carcinoma xenograft using 1,000 &i of 13’1-labeled anti-CEA ( 150 cGy/ 100 &i). “Y-labeled P96.5 effectively targets human glioma xenografts in vivo and yields substantial tumor absorbed radiation doses as measured directly by thermoluminescent dosimetry. In addition, low to modest uptake in various normal organs implies a very favorable therapeutic ratio. The shared antigenicity among neuroectodermally derived tumors provides a basis for exploration of radioimmunotherapy of human gliomas.
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