Nttcl.Med. Biof. Vol. 18, No. 3, pp. 295-304, 1991 hr. J. Radiat. Appl. Instrum. Part B Printed in Great Britain
0883-2897/91 53.00 + 0.00 Pcrgamon Press plc
Tumor Localization in Nude Mice Bearing Human Breast Carcinoma Xenografts Using “‘In-DTPA Conjugates of Monoclonal Antibodies SHRISHAILAM YEMUL, JORGE A. LEON, DAVID W. SELDIN, MARY-JEAN LINK, PETER KRAMER, RICARDO MESA-TEJADA ALISON Departments
and
ESTABROOK*
of Surgery and Radiology, Columbia University, New York, N.Y. Johnson & Johnson, Washington Crossing, N.J. and Met-Path Inc., Teterboro, N.J., U.S.A. (Received 19 Augusi 1990)
Biodistribution of monoclonal antibody T43 and its F(ab’), i’ ‘In-DTPA conjugates were determined in nu/nu mice bearing human breast tumor and rat pituitary tumor xenografts. T43 localized in the target tumor with tumor/blood ratios of 3.9 (P < 0.01) and 4.5 (P < 0.05) at 48 and 72 h, respectively. T43 F(ab’), fragments localized with tumor/blood ratio of 14.2 (P < 0.1) at 72 h. Tumors as small as 4 mm were detected without computer subtraction technique. These studies suggest that T43 and T43 F(ab’), might be useful reagents in radioimaging.
Introduction The use of monoclonal antibodies as imaging agents and in therapy of tumors is being investigated by an increasing number of research groups. Several radionuclides have been employed in tumor imaging and therapy e.g. ‘251,13iI, “‘In, %Tc, WY, 67Ga and *‘*Bi (Goldenberg, 1988; Hnatowich et al., 1983; Koizumi et al., 1987; Kozak et al., 1986). Of these, radioiodine and “‘In are more frequently used in the preparation of antibody radioconjugates for imaging. Even though radioiodination is a much easier way to radiolabel antibody molecules, antibody inactivation by the oxidizing agents (Hayes er al., 1988) and in uivo deiodination of the radioconjugates (Stem et al., 1982) are unresolved problems associated with this technique. “‘In-labeled antibody conjugates have been shown to have longer body retention times and better imaging characteristics (Fairweather et al., 1983). Efficient localization of ‘*‘In-labeled monoclonal antibodies in mice bearing tumor xenografts has been reported (Khaw et al., 1988), and in other studies “‘In-labeled antibody conjugates were successfully employed in detecting tumors in patients (Halpem et al., 1988; Kalofonos er al., 1988).
Our interest in the radioimaging of tumors using monoclonal antibodies stems from the possibility of employing the monoclonal antibodies available to us in detecting metastatic breast cancer pre-operatively in axillary lymph nodes and in internal mammary nodes, and post-operatively at distant sites. Some studies have been reported for imaging metastatic tumors in humans using monoclonal antibodies (Rainsbury et al., 1983; Kalofonos er al., 1989). The effective localization of a radioimmunoconjugate at the tumor site depends on the specificity and the retained immunoreactivity of the antibody after derivatization and radioconjugation. We have studied the sensitivity to DTPA cyclic anhydride derivatization and rrrIn labeling of two monoclonal antibodies, T43 and CU18, specific to human breast carcinoma cell line T47D. Monoclonal antibody T43 has low specificity but high reactivity with T47D cell line as compared to CUl8. After preliminary evaluations monoclonal antibody T43 and its F(ab’), were selected for in I&O distribution studies in nude mice bearing human breast carcinoma xenografts.
Materials and Methods Mice
*Address all correspondence to: Alison Estabrook, Department of Surgery, Columbia University, 630W 168St, New York, NY 10032, U.S.A.
Female nude mice (out-bred athymic nude) were obtained from Harlan-SpragueDawley Inc., Indi295
296
%RlSHAlLAMYEMUL
anapolis, Ind., aged 5-6 weeks and kept under controlled conventional conditions. Cell lines and cell cultures T47D clone 11 (Keydar et al., 1979), a human breast carcinoma cell line, was maintained in a culture medium of RPM1 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco Lab., Grand Island, N.Y.), L-glutamine (0.3 mg/mL), sodium pyruvate (1 mM), gentamycin (0.5 mg/mL), insulin (0.2 unit/ml) and 17-j estradiol (1 nM). GH3, a rat pituitary tumor derived cell line, was obtained from American Type Culture Collection, Rockville, Md, and cultured in a medium of RPM1 1640 supplemented with 15% horse serum, 2.5% fetal bovine serum, penicillin 200 U/mL and streptomycin 200pg/mL (Gibco Lab., Grand Island, N.Y.) (Tashjian et al., 1968). Mouse monoclonal antibodies T.43. T43 (UROlO, IgGI) antibody hybridoma was generated by immunization of BALB/c x C57Bl/6 Fl female mice with T24 cells (a human bladder cell line) and fusion of spleen cells with MOPC-21 NS/l cells. It recognizes a glycoprotein of 85,000 Da (Fradet et al., 1984, 1986). In an indirect immunofluorescence assay T43 bound to 100% T47D clone 11 cells; GH3 cells were negative. CU18, A hybridoma secreting monoclonal antibody CU18 (IgGl) was produced by fusion of NS/l cells with spleen cells from BALB/c mice immunized with the 1.16-1.19 g/mL sucrose density gradient isolate from T47D cells. U 18 has been shown to bind to a high molecular weight glycoprotein (BCA-225) on T47D cells and in culture supernatant (MesaTejada ef al., 1988). URO4. Antibody UR04 (S-27, IgGl) was raised against cell surface antigen of renal cancer cell line SK-RC-7. It recognizes a glycoprotein of 120,000 Da (Ueda et al., 1981) and it does not bind to either T47D cells or GH3 cells. All mouse monoclonal antibodies were purified from ascites by protein A affinity chromatography using Afhgel protein A MAPS II kit (Bio-Rad, Richmond, Calif.). Monoclonal antibodies T43 and UR04 were supplied by Dr T. Parish, Cambridge Research Laboratories, Cambridge, Mass. and CU18 was made at Columbia University. Preparation of F(ab’)z fragments
F(ab’), fragments were prepared according to the method of Parham by pepsin digestion (Parham, 1983). F(ab’), fragments were obtained in 60-80% yield and migrated as a single band at about 100,000 Da in 7.5% SDS_PAGE/7M urea. Derivatization of antibodies and their F(ab’), ments with DTPA cyclic anhydride
frag-
Purified IgGs were treated in 0.1 M NaHCO,, pH 8.2, with DTPA cyclic anhydride (1 mg/mL in
et ai.
anhydrous DMSO, Aldrich Chem. Co., Milwaukee, Wis.) at room temperature in a protein/DTPA ratio of 1:5, l:lO, 1:20, 1:40 and 1:lOO (mol/mol) for 1 h (Paik et al., 1983). Excess DTPA was then removed by dialysis. The purity of the conjugates was analyzed by 7.5% SDS-PAGE/7 M urea. The extent of derivatization was determined by TLC method (Paik et al., 1985) and InCl,/“‘In dialysis method (Hnatowich et al., 1982). l-2 DTPA groups/IgG were usually introduced with a protein/DTPA ratio of 1:20. T43 F(ab’), DTPA conjugate was prepared similarly using a protein/DTPA ratio of 1:20. “‘In labeling of the DTPA conjugates
DTPA conjugates (1 mg/mL) in 0.1 M NaOAc, pH 5.0, were reacted with “‘InCl, (NEN, Boston, Mass.) in a ratio of l-2 mCi/mg protein at room temperature for 1 h and then fractionated on a column of Sephadex G-50 using PBS pH 7.4 as eluant. The specific activities of l-2 mCi/mg were usually obtained and radiochemical purities were greater than 90% (by TLC). Immunoreactivity determination of the conjugates by ELBA
T47D cells (25,000 cells/well) were cultured in a 96 well round bottom plate (Costar) and fixed with 0.1% glutaraldehyde for 1 h at 4°C. The wells were blocked with 2% BSA in phosphate buffered saline (PBS) for 1 h at 37°C. The conjugates and underivatized antibody samples were then added by serial 1:2 dilution to the wells ranging from 25 to 0.78 pg/mL in PBS pH 7.6/l% BSA/O.OS% Tween 20 and incubated for 2 h at 37°C. After washing, biotinylated anti-mouse IgG made in horse (1 pg/mL, Vector Lab., Burlingame, Calif.) was added and incubated for 1 h at 37°C. The wells were washed and incubated with avidin-biotinylated horseradish peroxidase Complex (Vector Lab., Burlingame, Calif.) at 1: 800 dilution in PBS for 0.5 h at 37°C. Finally, after washing the plates, color was developed using o-phenylene diamine (OPD) as chromogen in McIlvan’s buffer pH 6.0. The wells were read after 0.5 h at 492 nm in an ELISA Plate reader (Titertek Multiscan, Flow Lab., McLean, Va). Determination conjugates
of immunoreactive fraction
of the
True immunoreactive fractions of “‘In-labeled antibodies or F(ab’), fragments were determined by the method of Lindmo and Bunn (1986). Establishment of tumors in mice
T47D clone 11 and GH3 cells (> 95% viable) were injected subdermally into the flanks of the mice using a 25 gage needle and a tuberculin syringe (10’ T47D cells in 0.6 mL left flank, IO6 GH3 cells in 0.4 mL right flank), A pellet of 0.5 mg of estradiol (Innovative Research of America, Toledo, Ohio) was placed subdennally in the neck area through a 1 cm
Imaging breast tumor xenografts in mice and closed with silk sutures. The tumors were allowed to grow for about 3 weeks, when the T47D tumors were approx. 4-8 mm in diameter. incision,
Injection of “‘In -labeled IgG and F(ab ‘)2 fragments into nude mice bearing tumor xenografts
“‘In-labeled IgG and F(ab’), fragments were sterilized by filtration through 0.45 pm filter (Acrodisc, Gelman, Ann Arbor, Mich.). 15-30 flCi of “‘In/15 pg of IgG or F(ab’)* in 100~1 PBS were injected per mouse into tail vein. Groups of 3-6 mice were used for each antibody in each experiment.
297
linked products with the increasing DTPA concentrations (data not shown). The immunoreactivities of T43 DTPA-conjugates before and after “‘In labeling were unchanged in ELISA assay showing no further reduction in immunoreactivity after “‘In labeling. However, CU18 showed more sensitivity to both DTPA conjugation and “‘In labeling. Figure l(A) and (B) shows the immunoreactivities of T43 and CU18 conjugates. The immunoreactivities of T43
A
(T431DTPA)hlll
Scanning and dissection of injected mice
Mice were scanned at intervals ranging from 48 to 144 h following radioconjugate injection. The mice were anesthetized with ketamine and xylazine for the duration of the scan. All images were obtained using a small-field-of-view 37 photomultiplier tube Anger camera with a pinhole collimator (Searle Pho-Gam HP). The single pulse height analyzer was set to the upper principle photopeak of “‘In (247 keV) with a 20% window width. The data were acquired by a dedicated nuclear medicine minicomputer system (MDS A*, Ann Arbor, Mich.) in a 128 x 128 word mode image matrix. Acquisition times were either 5 or 10 min per image depending on the count rate. Data analysis included qualitative visual assessment of the video display of contrast enhanced antibody images by a consensus of two or three observers. The mouse body outlines were clearly evident, and blood pool subtraction images were not necessary. Quantitative comparisons of target and non-target count ratios were obtained from manually drawn regionsof-interest on the images. After scanning the animals were sacrificed. The blood was collected by transecting the jugular vein. The entire T47D breast and GH3 pituitary xenografts were excised. Portions of other organs, including heart, lung, liver, spleen, kidney, intestine, muscle and bone were excised. The tissues were weighed and the radioactivity was counted gamma counter (Packard Multi-Prias 1).
1
10
100
ug/mL
I
.”
(CU18IDTPA)ln-111 ___ Q___
B
l:o
in a
Results Derivatization of monoclonal antibodies with DTPA cyclic anhydride using d$erent protein/DTPA ratios
To determine the optima1 derivatization of monoclonal antibodies with DTPA cyclic anhydride, antibodies were derivatized using protein/DTPA ratios of 1:5, l:lO, 1:20, 1:40 and 1:lOO. The purity and immunoreactivities were assessed by SDS-PAGE and Elisa before and after “‘In labeling. The SDS-PAGE analysis of the conjugates prepared with a protein/DTPA ratio of 1: 5 to 1: 20 revealed a single band corresponding to monomeric form and no high molecular weight aggregates or cross-linked polymeric products were seen. However, the conjugates prepared using a protein/DTPA ratio of 1: 40 and 1: 100 showed formation of high molecular weight cross-
1
10
10 0
uglmL
Fig. 1. ELISA assays of monoclonal antibodies (A) T43 and (B) CU 18 and their DTPA conjugates prepared with different protein/DTPA ratios and “‘In labeling are shown (see Materials and Methods). Underivatized antibodies were used as a control in each assay. Lower immunoreactivities were seen with increasing DTPA conjugation of CU18 as compared to control antibody. T43 showed less damage at lower protein/DTPA ratios.
298
ai.
SHRIS~AILAMYEMUL~~
conjugates prepared with protein/DTPA ratios of 1: 5, 1: 10 and I:20 were similar to underivatized antibody, but CU18 conjugates prepared with the same protein/DTPA ratios had lower immunoreactivities. The conjugates prepared with protein/DTPA ratios of 1: 40 and 1: 100 showed even more reduction in immunoreactivities for both antibodies. The true immunoreactive fractions for these conjugates were determined and compared. T43. Figure 2 shows plots of total applied antibody concentration/specific bound antibody concentration versus inverse cell concentration (mL x lo+) of “‘In-labeled DTPA conjugates of T43. The conjugate prepared using a protein/DTPA ratio of 1: 5 had an immunoreactive fraction of -9O%, which was similar to the immunoreactivity observed in ELBA assay; however, the conjugates prepared using ratios of l:lO, 1:20, 1:40 and 1:lOO contained 31, 25, 22 and 21% immunoreactive fractions, respectively. CU18. The “‘In-labeled DTPA conjugates of CU18 showed negligible binding to T47D cells at the concentration (40-800 ng/mL) employed in this assay and the immunoreactive fractions of less than 10% were obtained. The immunoreactivities of both of these conjugates determined by ELBA were higher than the true immunoreactive fractions determined at infinite antigen excess by linear extrapolation. The higher immunoreactivities observed in ELISA might be due to the presence of aggregates, and some underivatized antibody. In these preliminary evaluations, antibody CU18 conjugates showed reasonable reactivity in ELISA, but the immunoreactive fractions determined at antigenie excess were less than 10% and for this reason CU18 was withdrawn from further studies. A proteinjDTPA ratio of 1: 20 was used for the preparation
30
-)
143
W
I1
----o---
T43 F(ab32/DTPA/lnl UR04 F(ab’)P
F(ab.12
_
UR04
I 1I
F(ab’)P/DTPA/ln
I .o 0.0 0.6 N
s d d
0.4 0.2 0.0 I
I
o0.0
q
T43
0.1 inverse
2
111
F(ab’)P/DTPA/ln-
0.2 of
ug/mL
cell
0.3
0.4
concentration
0.5
I
0.6 (mL/
7
10’6)
Fig. 3. (A) ELISA assay of T43 F(ab’), and UR04 F(ab’), and their DTPA conjugates prepared with a protein/DTPA of 1: 20 and “‘In labeling. (B) Immunoreactivity plot of “‘In-labeled T43 F(ab’), DTPA conjugate. Immunoreactivity fraction = 42%.
of T43 antibody-DTPA conjugates. 1-2 DTPA groups/antibody molecules were normally introduced at this ratio and specific activities of l-2 mCi/mg were obtained. T43 F(ab’)* conjugate prepared similarly with this ratio of protein/DTPA and subsequent “‘In labeling showed an immunoreactive fraction of 42% (Fig. 3). Serum stability
0 0 Invww
7 of cell
2
3
4
eoncentmllon (mUlO%)
Fig. 2. Immunoreactive fractions of “‘In-labeled T43 DTPA conjugates prepared with different protein/DTPA ratios were determined at infinite antigen excess by linear extrapolation to the ordinate. Immunoreactive fractions of 90, 31, 25, 22 and 21% were obtained for the conjugates with protein/DTPA ratios of 1: 5 to 1:100, respectively.
SDS-PAGE and autoradiography analyses of the radioconjugates incubated in mouse sera showed that all the “‘In radioactivity was associated with the protein bands corresponding to IgG or F(ab’),. Thus, the l”In-labeled immunoconjugates were stable in mouse sera at 37°C for at least 48 h. Biodistribution The radioactivity distribution in various tissues in tumor bearing mice at 48 and 72 h after injecting “‘In-labeled DTPA conjugates of T43, and UR04
Imaging breast tumor xenografts in mice
299
L”
n q
18
Br. Tumor Pit Tumor
16 -
16 14
T
._
•j
Blood
•3
Liver
n q
18 -
14 12 -
Br. Tumor Pit. Tumor
•j
Blood
a
Liver
10 -%
IO
8 6 4 2 n UR04 (n=3)
T43 (n=lS)
Blodlrtrlbution
at 48h
T43
UR04 (n=3)
(n=ll)
Blodlstributlon
at 72h
Fig. 4. Tissue distributions of “‘In-labeled T43 and UR04 conjugates in nude mice bearing human breast tumor as targeted and rat pituitary tumor as co’ntrol tumor xenografts. Higher accumulation of T43 was seen in breast tumor at 48 h. n = number of mice.
antibodies is presented in Fig. 4. Antibody T43 readioconjugate showed higher accumulation in breast tumor than other tissues at 48 h. The radioactivity associated with the breast tumor was 11.l -+_6.0% of the injected dose per g tissue (ID/g tissue); however, the control antibody UR04 showed 4.0 +_3.1% ID/g tissue. The radioactivity in pituitary tumor was three times less than breast tumor. Liver and kidneys were the relatively high radioactivity accumulation sites. There were about equal amounts of radioactivity (7.3-7.9% ID/g tissue) in these organs. Moderate accumulation of radioactivity was
‘OY
l
4 J
seen in spleen (about 4% ID/g tissue). Lung and bone also showed -4% uptake. The sites of least radioactivity localization were heart, muscle and intestine (- 1.5% ID/g). After 72 h there was considerable clearance of T43 radioconjugate from the circulation which resulted in a better tumor/blood ratio. There was a gradual decrease in radioactivity in other organs except liver and kidneys, where the radioactivity persisted for longer time. Figure 5 shows the tissue distribution of T43 F(ab’), and UR04 F(ab’)z in tumor bearing animals at 48 and 72 h. Compared to the control antibody
Er. Tumor
Tq q
T43 Flob’)2 Biodistribution
(n-81
Liver
Kidney
UR04 F(ob’)2 (n-3)
of F (ob’)2
ot
48h
T43 F(ab’)P (n=6)’ Biodistribution
UR04 Fob’l2(n=21 of F(ab’)2
at 72h
Fig. 5. Tissue distributions of “‘In-labeled T43 F(ab’), and UR04 F(ab’), conjugates in nude mice bearing human breast tumor as targeted and rat pituitary tumor as control tumor xenografts. Higher % ID/g tissue in breast tumor was seen with T43 F(ab’), than UR04 F(ab’),. Kidneys were the site of clearance for these radioconjugates. n = number of mice.
%I~ISHAILAM YEMUL
300
UR04 F(ab’),, the specific antibody T43 F(ab’), showed 2-fold higher localization in breast tumor at 48 h. There was 8.2 + 2.4% ID/g breast tumor tissue accumulation of T43 F(ab’), , whereas UR04 F(ab’), accumulated up to 4.2 k 1.2% ID/g tissue. Much less radioactivity was found in other organs except liver, spleen and kidneys. For both the fragments, kidneys were the site of highest radioactivity accumulation (up to 42.1% ID/g). At 72 h most of the antibody fragment radioconjugates had cleared from the circulation. The concentration of the immunoconjugate localized in the breast tumor remained unchanged. Thus at 72 h higher breast tumor/blood counts were obtained. Kidneys continued to be the site of radioactivity retention with 27.0 + 15.4% ID/g tissue. Breast tumor/blood ratios of the radioactivity of the antibodies studied are shown in Table 1. Antibody T43 showed a greater tumor/blood ratio than UR04. With increasing time the tumor/blood ratio of T43 had increased as the radioconjugate had cleared from the circulation. The F(ab’), fragment showed a higher tumor/blood ratio than the whole antibodies. The ratio as high as 14.3 was obtained at 72 h. Even higher tumor/blood ratios were obtained at 120 and 144h. The scintigrams of the mice injected with radioconjugates of T43 and F(ab’), are shown in Fig. 6. At 48 h the breast tumor in the left flank was discernible in the mice injected with the whole antibody radioconjugate despite the high background of the liver. The nontarget pituitary tumor in the tight flank was relatively larger and well vascularized than the breast tumor but it was not detected in these images. The fragments produced less background compared to the whole antibody and the breast tumor was clearly visible at 48 h. The images at 120 h showed reduction of counts in background as well as in tumor site, but there were sufficient counts to visualize the tumor.
Discussion In this paper we have described the effect of DTPA derivatization on immunoreactivities of a monoclonal antibody, T43, directed to T47D cell membrane antigen, and a second monoclonal antibody, CU18, directed to an antigen (BCA-225) expressed on T47D cell membrane as well as in the cytoplasm (Mesa-Tejada et al., 1988). A nonspecific monoclonal antibody, UR04, that did not bind to T47D and GH3 cells was used as a control.
el al.
The immunoreactivities of the conjugates of the antibodies prepared by DTPA cyclic anhydride coupling method were affected by the extent of the derivatization of lysine amino groups in the antigen binding region. Other investigators (Kulmann and Steinstrasser, 1988) have reported similar effects on immunoreactivities of antibodies with DTPA derivatization. The loss of immunoreactivity was thought to be due to cross linking and the steric hindrance caused by the DTPA groups introduced in the antigen binding sites. The damaging effect of increased derivatization on immunoreactivity was much more pronounced in CU18, which suggested the presence of highly exposed lysines in antigen binding sites. T43 and CU18 showed different degrees of sensitivity to DTPA derivatization. Such variation in deleterious effect of conjugations from antibody to antibody was also observed by other workers (Paik et al., 1983). When we compared the immunoreactivities determined by ELISA with the true immunoreactive fraction determined by the methods of Lindmo et al. (1986), higher immunoreactivities were observed in ELISA. This may be attributed to the presence of some underivatized antibody molecules in the conjugate preparations. The DTPA cyclic anhydride derivatization produces a range of conjugation products with different degrees of substitution in addition to the presence of some underivatized antibody molecules. Since the reaction is thought to obey Poisson statistics, after the introduction of an average of l-2 groups per protein molecule there will be 1535% unmodified protein molecules in the conjugate prep aration (Petrella et al., 1987). The purification of the DTPA conjugates by dialysis and size exclusion chromatography does not remove undervatized antibody molecules, and ion exchange chromatography or affinity chromatography might be required for further purification. In our in vivo model the nude mice were transplanted with T47D (human breast carcinoma cell line) and GH3 (rat pituitary tumor cell line) xenografts. Monoclonal antibody T43 localized in the tumor site better than the control antibody UR04. At 48 h a tumor/blood ratio of 3.9 was obtained for T43, whereas UR04 showed a ratio of 1.3 (P < 0.01). Liver, spleen and kidney were the organs that sequestered radioconjugates very efficiently. Such sequestration by a reticuloendothelial system has been reported for most IgG radioconjugates and major uptake by liver seems to be a disadvantage with the use of radioimmunoconjugates. We have observed liver uptake between 3-15%, whereas others
Table 1. Breast tumor/blood ratios Antibody
1 2 3 4
T43 UR04 T43 F(ab’), UR04 F(ab’),
*ND = not determined.
48 b 3.92 1.3 a.79 8.54
+ f f +
1.57 1.22 5.43 3.75
72 b 4.57 1.9 14.26 9.44
f * f +
2.16 0.87 4.13 0.35
120b
144h
9.35 + 0.29 ND* 15.4 * 5.5 ND
16.75 _+ 1.24 ND 53.48 + 6.3 ND
Fig. 6. Examples of gamma camera images of nude mice with human beast tumor xenograft (T47D) in the left flank and rat pituitary tumor xenograft (GH3) in the right flank, after injecting with whole antibody or F[ab’jz fragment are shown. Breast tumors were visualized with T43 and T43 F(ab’)2 at 48 h. Pituitary tumors were not seen at 48 and 120 h in these scintigrams.
301
Imaging breast tumor xenografts in mice
have reported bodies
higher than 20% for different anti(Kalofonos et al., 1988). The mechanism for
liver uptake is believed to be multifactorial, e.g. transchelation of “‘In to transferrin and/or interaction of Fc part of IgG with hepatocytes receptors (Halpem el al., 1983; Hopf et al., 1976). Although the detection of lymph node and distal metastates may not be affected by such high liver background, the abdomenal observations and liver metastates might be obscured. However, recently single photon emission computed tomography (SPECT) technique has been employed to detect liver metastates using ‘*‘Inlabeled antibodies with considerable success (Halpern et al., 1988; Abdel-Nabi et al., 1988). Improved localization of F(ab’), compared to whole IgG has been reported by several workers (Moseley et al., 1988; Andrew et al., 1988). Similarly, in our studies T43 F(ab’), showed better contrast than T43. Due to much rapid clearance of F(ab’), from the circulation tumor/blood ratios as high as 8.79 were obtained at 48 h. Although the control antibody UR04 F(ab’), showed similar tumor/blood ratios at 48 h, the % ID/g in the tumor was 2-fold lower than T43 F(ab’), and for this reason the target tumors were visible only with T43 F(ab’),. Even higher ratios were seen for T43 F(ab’), at later time points (see Table 1). The rapid clearance of F(ab’), from the circulation is thought to be due to the absence of the Fc portion of the IgG molecules and/or the concurrent reduction in molecular size (Holten et al., 1987). The fragments were captured by liver, spleen and kidneys. The site of highest radioactivity accumulation was the kidneys. The role of kidneys in the excretion and catabolism of IgG and IgG fragments was studied previously (Wochner et al., 1967). The kidneys were shown to be the site of glomerular filtration and catabolism of the IgG fragments but not the intact IgG. A recent comparative study of tumor localization using “‘I and “‘Inlabeled anti-CEA antibody Cl98 fragments showed similar higher uptake in the kidneys of nude mice (Andrew et al., 1988). Although radioiodine from ‘3’I-labeled antibody fragments is removed much more rapidly from the kidneys, radioactivity of “‘Inlabeled antibody fragments is retained for a prolonged time. The reasons for such preferential retention of “‘In in the kidney are not clear. These studies demonstrated that a 4mm human breast tumor xenograft could be visualized at 48 h with tumor/blood ratios of 3.92 & 1.57 for the whole antibody, T43, and 8.79 + 5.43 for its F(ab’)z fragments. With increasing time even higher tumor/blood ratios were observed, and so 48-72 h appears to be suitable time for imaging. We now plan to extend the applicability of T43 F(ab’), radioconjugates to humans. Since CU18 has very high specificity to breast carcinoma, we are investigating derivatizations using very selective heterofunctional chelating agents and use of these conjugates in different tumor xenografts/nude mice models.
303
Acknowledgements-We
thank DC Tom Parish for supplying the antibodies T43 and UR04, Irene Ho and Eileen Kelley for technical assistance. This research was supported in part by Johnson & Johnson Special Biological Projects, Irving A. Hansen Memorial Foundation, Florence and Herbert Irving Foundation and NIH Grant 5ROl CA 32984-07.
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Fradet Y., Cordon-Cardo C., Whitmore W. F. Jr ef al. (1986) Cell surface antigens of human bladder tumors: definition of tumor subs& by monoclonal antibodies and correlation with growth characteristics. Cancer Res. 46, 5183-5188. Goldenberg D. M. (1988) Targeting of cancer with radiolabeled antibodies. Prospects for imaging and therapy. Archiv. Pathol. Lab. Med. 112, 580-587. Halpern S. E., Hagan P. L., Garver P. R. et al. (1983) Stability, characterization and kinetics of In-labeled monoclonal antitumor antibodies in normal and nude mouse-tumor models. Cancer Res. 43, 5347-5355. Halpern S. E., Haindl W., Beauregard J. et al. (1988) Scintigraphy with In-l 1l-labeled monoclonal antitumor antibodies: kinetics, biodistribution, and tumor detection. Radiology 168, 529-536. Hayes D. F., Noska M. A., Kufe D. W. et al. (1988) Effect of radioiodiodination on the binding of monoclonal antibody DF3 to breast carcinoma cells. Nucf. Med. Biol. 15, 235-241, Hnatowich D. J., Layne W. W. and Childs R. L. (1982) The preparation and labeling of DTPA-coupled albumin. In/. J. Appl. Radiut. Isot. 33, 327-332.
Hnatowich D. J., Layne W. W. and Childs R. L. et al. (1983) Radioactive labeling of antibody: a simple and efficient method. Science 220, 613-615. Holten 0. D. III, Black C. D. V., Parker R. J. et al. (1987) Biodistribution of monoclonal IgG 1, F(ab’), , and Fab’ in mice after intravenous injection: comparison between anti-B [anti-LyB8.21 and irrelevant (MOPC-211 antibodies. J. Immunol. 139, 304-3049. Hopf V., Meyer Z. U., Bushenfeld K. H. er al. (1976) Demonstration of binding sites of IgG Fc and the third complement component (C) on isolated hepatocytes. J. Immunol. 117, 639-645. Kalofonos H. P., Sivolapenko G. B., Courtenay-Luck N. S. et al. (1988) Antibody guided targeting of non-small cell lung cancer using “‘In-labeled HMFGl F(ab’), fragments. Cancer Res.-48, 1977-1984. Kalofonos H. P.. Sackier J. M.. Hatzistvlianou M. cr al. (1989) Kinetics, quantitative anal& and radioimmunolocalisation using indium-I 1I-HMFGI monoclonal antibody in patients with breast cancer. Br. J. Cancer 59, 939-942.
Khaw B. A., Bailes J. S., Schneider S. L. et al. (1988) Human breast tumor imaging using “‘In labeled monoclonal antibody: athymic mouse model. Eur. J. Nucl. Med. 14, 362-366.
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%lUIiAILAM
Keydar I., Chen L., Karby S. et al. (1979) Establishment and characterization of a cell line of human breast carcinoma origin, Eur. J. Nucl. Med. 15, 659-670. Koizumi M., Endo K., Kunimatsu M. et al. (1987) Preparation of 67Ga-labeled antibodies using deferoximine as a bifunctional chelate. An improved method. J. Immunol. Methouk 104, 93-102. Kozak R. W., Atcher R. W., Gansow 0. A. et al. (1986) Bismuth-212 labeled anti-Tat monoclonal antibody: alpha-particle emitting radionuclides as modalities for radioimmunotherapy. Proc. Nat/. Acad. Sci. U.S.A. 83, 474-478.
Kulmann L. and Steinstrasser A. (1988) Effect of DTPA to antibody ratio on chemical, immunological and biological properties of the l’lIn-labelled F(ab’), fragment of the monoclonal antibody 431/31. Nucl. Med. Biol. 15, 617-627.
Lindmo T. and Bunn P. A. Jr (1986) Determination of the true immunoreactive fraction of monoclonal antibodies after radiolabeling. MethorLF Enzymol. 121, 678-691. Mesa-Tejada R., Palakodety R. B., Leon J. A. et al. (1988) Immunocytochemical distribution of breast carcinoma associated glycoprotein identified by monoclonal antibodies. Am. J. Pathol. 130, 305-314. K. R., A., Knapp C. er (1988) Localization radiolabeled F(ab’), of monoantibodies in mice bearing growing human cancer xenografts. J. Cancer
C. H., reactivity of conjugates
M. A. al. (1985) immunoreactive antibody-DTPA and nonimmunoreactive toward 11. J. Nucl. Med. 26, J. J.,
482-487.
Parham P. (1983) On the fragmentation of monoclonal IeGl. IaG2a. and IaG2b from BALB/c mice. J. Immunol. 131, i8k2do2.
w
Petrella E. C., Wilke S. D., Smith C. A. et al. (1987) Antibody conjugates with cobra venom factor. Synthesis and biochemical characterization. J. Immunol. Methodr 104, 159-172. Rainsbury R. M., Westwood J. H., Coombes R. C. et al. (1983) Location of metastatic breast carcinoma by a monoclonal antibody chelate labelled with Indium- 111. Lancer ii, 934-938.
Stem P., Hagan P., Halpem N. et al. (1982) The effect of the radiolabel on the kinetics of monoclonal anti-CEA in a nude mouse-human colon tumor model. In Hybridomas in Cancer Diaanosis and Management (Edited bv Michell M. S. and Getigen H. F.), pp. 245-253.‘Raven Pie.%, New York. Tashjian A. H. Jr, Yasumura Y., Levine L. et al. (1968) Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82, 342-352. Ueda R., Ogata S. I., Morrissey D. M. et al. (1981) Cell surface antigens of human renal cancer defined by mouse monoclonal antibodies: identification of tissue specific kidney glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 78, 5122-5126.
368-372.
Paik H., Ebbert A., Murphy R. et Factors DTPA conjugation bodies cyclic DTPA J. Nucl. 1158-1163.
YEhfuLet al.
(1983) anti24,
Wochner R. D., Strober W. and Waldmann T. A. (1967) The role of the kidney in the catabolism of Bence Jones proteins and immunoglobulin fragments. J. Exp. Med. 126, 207-221.
0883-2897/91 53.00 + 0.00 Pcrgamon Press plc
Tumor Localization in Nude Mice Bearing Human Breast Carcinoma Xenografts Using “‘In-DTPA Conjugates of Monoclonal Antibodies SHRISHAILAM YEMUL, JORGE A. LEON, DAVID W. SELDIN, MARY-JEAN LINK, PETER KRAMER, RICARDO MESA-TEJADA ALISON Departments
and
ESTABROOK*
of Surgery and Radiology, Columbia University, New York, N.Y. Johnson & Johnson, Washington Crossing, N.J. and Met-Path Inc., Teterboro, N.J., U.S.A. (Received 19 Augusi 1990)
Biodistribution of monoclonal antibody T43 and its F(ab’), i’ ‘In-DTPA conjugates were determined in nu/nu mice bearing human breast tumor and rat pituitary tumor xenografts. T43 localized in the target tumor with tumor/blood ratios of 3.9 (P < 0.01) and 4.5 (P < 0.05) at 48 and 72 h, respectively. T43 F(ab’), fragments localized with tumor/blood ratio of 14.2 (P < 0.1) at 72 h. Tumors as small as 4 mm were detected without computer subtraction technique. These studies suggest that T43 and T43 F(ab’), might be useful reagents in radioimaging.
Introduction The use of monoclonal antibodies as imaging agents and in therapy of tumors is being investigated by an increasing number of research groups. Several radionuclides have been employed in tumor imaging and therapy e.g. ‘251,13iI, “‘In, %Tc, WY, 67Ga and *‘*Bi (Goldenberg, 1988; Hnatowich et al., 1983; Koizumi et al., 1987; Kozak et al., 1986). Of these, radioiodine and “‘In are more frequently used in the preparation of antibody radioconjugates for imaging. Even though radioiodination is a much easier way to radiolabel antibody molecules, antibody inactivation by the oxidizing agents (Hayes er al., 1988) and in uivo deiodination of the radioconjugates (Stem et al., 1982) are unresolved problems associated with this technique. “‘In-labeled antibody conjugates have been shown to have longer body retention times and better imaging characteristics (Fairweather et al., 1983). Efficient localization of ‘*‘In-labeled monoclonal antibodies in mice bearing tumor xenografts has been reported (Khaw et al., 1988), and in other studies “‘In-labeled antibody conjugates were successfully employed in detecting tumors in patients (Halpem et al., 1988; Kalofonos er al., 1988).
Our interest in the radioimaging of tumors using monoclonal antibodies stems from the possibility of employing the monoclonal antibodies available to us in detecting metastatic breast cancer pre-operatively in axillary lymph nodes and in internal mammary nodes, and post-operatively at distant sites. Some studies have been reported for imaging metastatic tumors in humans using monoclonal antibodies (Rainsbury et al., 1983; Kalofonos er al., 1989). The effective localization of a radioimmunoconjugate at the tumor site depends on the specificity and the retained immunoreactivity of the antibody after derivatization and radioconjugation. We have studied the sensitivity to DTPA cyclic anhydride derivatization and rrrIn labeling of two monoclonal antibodies, T43 and CU18, specific to human breast carcinoma cell line T47D. Monoclonal antibody T43 has low specificity but high reactivity with T47D cell line as compared to CUl8. After preliminary evaluations monoclonal antibody T43 and its F(ab’), were selected for in I&O distribution studies in nude mice bearing human breast carcinoma xenografts.
Materials and Methods Mice
*Address all correspondence to: Alison Estabrook, Department of Surgery, Columbia University, 630W 168St, New York, NY 10032, U.S.A.
Female nude mice (out-bred athymic nude) were obtained from Harlan-SpragueDawley Inc., Indi295
296
%RlSHAlLAMYEMUL
anapolis, Ind., aged 5-6 weeks and kept under controlled conventional conditions. Cell lines and cell cultures T47D clone 11 (Keydar et al., 1979), a human breast carcinoma cell line, was maintained in a culture medium of RPM1 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco Lab., Grand Island, N.Y.), L-glutamine (0.3 mg/mL), sodium pyruvate (1 mM), gentamycin (0.5 mg/mL), insulin (0.2 unit/ml) and 17-j estradiol (1 nM). GH3, a rat pituitary tumor derived cell line, was obtained from American Type Culture Collection, Rockville, Md, and cultured in a medium of RPM1 1640 supplemented with 15% horse serum, 2.5% fetal bovine serum, penicillin 200 U/mL and streptomycin 200pg/mL (Gibco Lab., Grand Island, N.Y.) (Tashjian et al., 1968). Mouse monoclonal antibodies T.43. T43 (UROlO, IgGI) antibody hybridoma was generated by immunization of BALB/c x C57Bl/6 Fl female mice with T24 cells (a human bladder cell line) and fusion of spleen cells with MOPC-21 NS/l cells. It recognizes a glycoprotein of 85,000 Da (Fradet et al., 1984, 1986). In an indirect immunofluorescence assay T43 bound to 100% T47D clone 11 cells; GH3 cells were negative. CU18, A hybridoma secreting monoclonal antibody CU18 (IgGl) was produced by fusion of NS/l cells with spleen cells from BALB/c mice immunized with the 1.16-1.19 g/mL sucrose density gradient isolate from T47D cells. U 18 has been shown to bind to a high molecular weight glycoprotein (BCA-225) on T47D cells and in culture supernatant (MesaTejada ef al., 1988). URO4. Antibody UR04 (S-27, IgGl) was raised against cell surface antigen of renal cancer cell line SK-RC-7. It recognizes a glycoprotein of 120,000 Da (Ueda et al., 1981) and it does not bind to either T47D cells or GH3 cells. All mouse monoclonal antibodies were purified from ascites by protein A affinity chromatography using Afhgel protein A MAPS II kit (Bio-Rad, Richmond, Calif.). Monoclonal antibodies T43 and UR04 were supplied by Dr T. Parish, Cambridge Research Laboratories, Cambridge, Mass. and CU18 was made at Columbia University. Preparation of F(ab’)z fragments
F(ab’), fragments were prepared according to the method of Parham by pepsin digestion (Parham, 1983). F(ab’), fragments were obtained in 60-80% yield and migrated as a single band at about 100,000 Da in 7.5% SDS_PAGE/7M urea. Derivatization of antibodies and their F(ab’), ments with DTPA cyclic anhydride
frag-
Purified IgGs were treated in 0.1 M NaHCO,, pH 8.2, with DTPA cyclic anhydride (1 mg/mL in
et ai.
anhydrous DMSO, Aldrich Chem. Co., Milwaukee, Wis.) at room temperature in a protein/DTPA ratio of 1:5, l:lO, 1:20, 1:40 and 1:lOO (mol/mol) for 1 h (Paik et al., 1983). Excess DTPA was then removed by dialysis. The purity of the conjugates was analyzed by 7.5% SDS-PAGE/7 M urea. The extent of derivatization was determined by TLC method (Paik et al., 1985) and InCl,/“‘In dialysis method (Hnatowich et al., 1982). l-2 DTPA groups/IgG were usually introduced with a protein/DTPA ratio of 1:20. T43 F(ab’), DTPA conjugate was prepared similarly using a protein/DTPA ratio of 1:20. “‘In labeling of the DTPA conjugates
DTPA conjugates (1 mg/mL) in 0.1 M NaOAc, pH 5.0, were reacted with “‘InCl, (NEN, Boston, Mass.) in a ratio of l-2 mCi/mg protein at room temperature for 1 h and then fractionated on a column of Sephadex G-50 using PBS pH 7.4 as eluant. The specific activities of l-2 mCi/mg were usually obtained and radiochemical purities were greater than 90% (by TLC). Immunoreactivity determination of the conjugates by ELBA
T47D cells (25,000 cells/well) were cultured in a 96 well round bottom plate (Costar) and fixed with 0.1% glutaraldehyde for 1 h at 4°C. The wells were blocked with 2% BSA in phosphate buffered saline (PBS) for 1 h at 37°C. The conjugates and underivatized antibody samples were then added by serial 1:2 dilution to the wells ranging from 25 to 0.78 pg/mL in PBS pH 7.6/l% BSA/O.OS% Tween 20 and incubated for 2 h at 37°C. After washing, biotinylated anti-mouse IgG made in horse (1 pg/mL, Vector Lab., Burlingame, Calif.) was added and incubated for 1 h at 37°C. The wells were washed and incubated with avidin-biotinylated horseradish peroxidase Complex (Vector Lab., Burlingame, Calif.) at 1: 800 dilution in PBS for 0.5 h at 37°C. Finally, after washing the plates, color was developed using o-phenylene diamine (OPD) as chromogen in McIlvan’s buffer pH 6.0. The wells were read after 0.5 h at 492 nm in an ELISA Plate reader (Titertek Multiscan, Flow Lab., McLean, Va). Determination conjugates
of immunoreactive fraction
of the
True immunoreactive fractions of “‘In-labeled antibodies or F(ab’), fragments were determined by the method of Lindmo and Bunn (1986). Establishment of tumors in mice
T47D clone 11 and GH3 cells (> 95% viable) were injected subdermally into the flanks of the mice using a 25 gage needle and a tuberculin syringe (10’ T47D cells in 0.6 mL left flank, IO6 GH3 cells in 0.4 mL right flank), A pellet of 0.5 mg of estradiol (Innovative Research of America, Toledo, Ohio) was placed subdennally in the neck area through a 1 cm
Imaging breast tumor xenografts in mice and closed with silk sutures. The tumors were allowed to grow for about 3 weeks, when the T47D tumors were approx. 4-8 mm in diameter. incision,
Injection of “‘In -labeled IgG and F(ab ‘)2 fragments into nude mice bearing tumor xenografts
“‘In-labeled IgG and F(ab’), fragments were sterilized by filtration through 0.45 pm filter (Acrodisc, Gelman, Ann Arbor, Mich.). 15-30 flCi of “‘In/15 pg of IgG or F(ab’)* in 100~1 PBS were injected per mouse into tail vein. Groups of 3-6 mice were used for each antibody in each experiment.
297
linked products with the increasing DTPA concentrations (data not shown). The immunoreactivities of T43 DTPA-conjugates before and after “‘In labeling were unchanged in ELISA assay showing no further reduction in immunoreactivity after “‘In labeling. However, CU18 showed more sensitivity to both DTPA conjugation and “‘In labeling. Figure l(A) and (B) shows the immunoreactivities of T43 and CU18 conjugates. The immunoreactivities of T43
A
(T431DTPA)hlll
Scanning and dissection of injected mice
Mice were scanned at intervals ranging from 48 to 144 h following radioconjugate injection. The mice were anesthetized with ketamine and xylazine for the duration of the scan. All images were obtained using a small-field-of-view 37 photomultiplier tube Anger camera with a pinhole collimator (Searle Pho-Gam HP). The single pulse height analyzer was set to the upper principle photopeak of “‘In (247 keV) with a 20% window width. The data were acquired by a dedicated nuclear medicine minicomputer system (MDS A*, Ann Arbor, Mich.) in a 128 x 128 word mode image matrix. Acquisition times were either 5 or 10 min per image depending on the count rate. Data analysis included qualitative visual assessment of the video display of contrast enhanced antibody images by a consensus of two or three observers. The mouse body outlines were clearly evident, and blood pool subtraction images were not necessary. Quantitative comparisons of target and non-target count ratios were obtained from manually drawn regionsof-interest on the images. After scanning the animals were sacrificed. The blood was collected by transecting the jugular vein. The entire T47D breast and GH3 pituitary xenografts were excised. Portions of other organs, including heart, lung, liver, spleen, kidney, intestine, muscle and bone were excised. The tissues were weighed and the radioactivity was counted gamma counter (Packard Multi-Prias 1).
1
10
100
ug/mL
I
.”
(CU18IDTPA)ln-111 ___ Q___
B
l:o
in a
Results Derivatization of monoclonal antibodies with DTPA cyclic anhydride using d$erent protein/DTPA ratios
To determine the optima1 derivatization of monoclonal antibodies with DTPA cyclic anhydride, antibodies were derivatized using protein/DTPA ratios of 1:5, l:lO, 1:20, 1:40 and 1:lOO. The purity and immunoreactivities were assessed by SDS-PAGE and Elisa before and after “‘In labeling. The SDS-PAGE analysis of the conjugates prepared with a protein/DTPA ratio of 1: 5 to 1: 20 revealed a single band corresponding to monomeric form and no high molecular weight aggregates or cross-linked polymeric products were seen. However, the conjugates prepared using a protein/DTPA ratio of 1: 40 and 1: 100 showed formation of high molecular weight cross-
1
10
10 0
uglmL
Fig. 1. ELISA assays of monoclonal antibodies (A) T43 and (B) CU 18 and their DTPA conjugates prepared with different protein/DTPA ratios and “‘In labeling are shown (see Materials and Methods). Underivatized antibodies were used as a control in each assay. Lower immunoreactivities were seen with increasing DTPA conjugation of CU18 as compared to control antibody. T43 showed less damage at lower protein/DTPA ratios.
298
ai.
SHRIS~AILAMYEMUL~~
conjugates prepared with protein/DTPA ratios of 1: 5, 1: 10 and I:20 were similar to underivatized antibody, but CU18 conjugates prepared with the same protein/DTPA ratios had lower immunoreactivities. The conjugates prepared with protein/DTPA ratios of 1: 40 and 1: 100 showed even more reduction in immunoreactivities for both antibodies. The true immunoreactive fractions for these conjugates were determined and compared. T43. Figure 2 shows plots of total applied antibody concentration/specific bound antibody concentration versus inverse cell concentration (mL x lo+) of “‘In-labeled DTPA conjugates of T43. The conjugate prepared using a protein/DTPA ratio of 1: 5 had an immunoreactive fraction of -9O%, which was similar to the immunoreactivity observed in ELBA assay; however, the conjugates prepared using ratios of l:lO, 1:20, 1:40 and 1:lOO contained 31, 25, 22 and 21% immunoreactive fractions, respectively. CU18. The “‘In-labeled DTPA conjugates of CU18 showed negligible binding to T47D cells at the concentration (40-800 ng/mL) employed in this assay and the immunoreactive fractions of less than 10% were obtained. The immunoreactivities of both of these conjugates determined by ELBA were higher than the true immunoreactive fractions determined at infinite antigen excess by linear extrapolation. The higher immunoreactivities observed in ELISA might be due to the presence of aggregates, and some underivatized antibody. In these preliminary evaluations, antibody CU18 conjugates showed reasonable reactivity in ELISA, but the immunoreactive fractions determined at antigenie excess were less than 10% and for this reason CU18 was withdrawn from further studies. A proteinjDTPA ratio of 1: 20 was used for the preparation
30
-)
143
W
I1
----o---
T43 F(ab32/DTPA/lnl UR04 F(ab’)P
F(ab.12
_
UR04
I 1I
F(ab’)P/DTPA/ln
I .o 0.0 0.6 N
s d d
0.4 0.2 0.0 I
I
o0.0
q
T43
0.1 inverse
2
111
F(ab’)P/DTPA/ln-
0.2 of
ug/mL
cell
0.3
0.4
concentration
0.5
I
0.6 (mL/
7
10’6)
Fig. 3. (A) ELISA assay of T43 F(ab’), and UR04 F(ab’), and their DTPA conjugates prepared with a protein/DTPA of 1: 20 and “‘In labeling. (B) Immunoreactivity plot of “‘In-labeled T43 F(ab’), DTPA conjugate. Immunoreactivity fraction = 42%.
of T43 antibody-DTPA conjugates. 1-2 DTPA groups/antibody molecules were normally introduced at this ratio and specific activities of l-2 mCi/mg were obtained. T43 F(ab’)* conjugate prepared similarly with this ratio of protein/DTPA and subsequent “‘In labeling showed an immunoreactive fraction of 42% (Fig. 3). Serum stability
0 0 Invww
7 of cell
2
3
4
eoncentmllon (mUlO%)
Fig. 2. Immunoreactive fractions of “‘In-labeled T43 DTPA conjugates prepared with different protein/DTPA ratios were determined at infinite antigen excess by linear extrapolation to the ordinate. Immunoreactive fractions of 90, 31, 25, 22 and 21% were obtained for the conjugates with protein/DTPA ratios of 1: 5 to 1:100, respectively.
SDS-PAGE and autoradiography analyses of the radioconjugates incubated in mouse sera showed that all the “‘In radioactivity was associated with the protein bands corresponding to IgG or F(ab’),. Thus, the l”In-labeled immunoconjugates were stable in mouse sera at 37°C for at least 48 h. Biodistribution The radioactivity distribution in various tissues in tumor bearing mice at 48 and 72 h after injecting “‘In-labeled DTPA conjugates of T43, and UR04
Imaging breast tumor xenografts in mice
299
L”
n q
18
Br. Tumor Pit Tumor
16 -
16 14
T
._
•j
Blood
•3
Liver
n q
18 -
14 12 -
Br. Tumor Pit. Tumor
•j
Blood
a
Liver
10 -%
IO
8 6 4 2 n UR04 (n=3)
T43 (n=lS)
Blodlrtrlbution
at 48h
T43
UR04 (n=3)
(n=ll)
Blodlstributlon
at 72h
Fig. 4. Tissue distributions of “‘In-labeled T43 and UR04 conjugates in nude mice bearing human breast tumor as targeted and rat pituitary tumor as co’ntrol tumor xenografts. Higher accumulation of T43 was seen in breast tumor at 48 h. n = number of mice.
antibodies is presented in Fig. 4. Antibody T43 readioconjugate showed higher accumulation in breast tumor than other tissues at 48 h. The radioactivity associated with the breast tumor was 11.l -+_6.0% of the injected dose per g tissue (ID/g tissue); however, the control antibody UR04 showed 4.0 +_3.1% ID/g tissue. The radioactivity in pituitary tumor was three times less than breast tumor. Liver and kidneys were the relatively high radioactivity accumulation sites. There were about equal amounts of radioactivity (7.3-7.9% ID/g tissue) in these organs. Moderate accumulation of radioactivity was
‘OY
l
4 J
seen in spleen (about 4% ID/g tissue). Lung and bone also showed -4% uptake. The sites of least radioactivity localization were heart, muscle and intestine (- 1.5% ID/g). After 72 h there was considerable clearance of T43 radioconjugate from the circulation which resulted in a better tumor/blood ratio. There was a gradual decrease in radioactivity in other organs except liver and kidneys, where the radioactivity persisted for longer time. Figure 5 shows the tissue distribution of T43 F(ab’), and UR04 F(ab’)z in tumor bearing animals at 48 and 72 h. Compared to the control antibody
Er. Tumor
Tq q
T43 Flob’)2 Biodistribution
(n-81
Liver
Kidney
UR04 F(ob’)2 (n-3)
of F (ob’)2
ot
48h
T43 F(ab’)P (n=6)’ Biodistribution
UR04 Fob’l2(n=21 of F(ab’)2
at 72h
Fig. 5. Tissue distributions of “‘In-labeled T43 F(ab’), and UR04 F(ab’), conjugates in nude mice bearing human breast tumor as targeted and rat pituitary tumor as control tumor xenografts. Higher % ID/g tissue in breast tumor was seen with T43 F(ab’), than UR04 F(ab’),. Kidneys were the site of clearance for these radioconjugates. n = number of mice.
%I~ISHAILAM YEMUL
300
UR04 F(ab’),, the specific antibody T43 F(ab’), showed 2-fold higher localization in breast tumor at 48 h. There was 8.2 + 2.4% ID/g breast tumor tissue accumulation of T43 F(ab’), , whereas UR04 F(ab’), accumulated up to 4.2 k 1.2% ID/g tissue. Much less radioactivity was found in other organs except liver, spleen and kidneys. For both the fragments, kidneys were the site of highest radioactivity accumulation (up to 42.1% ID/g). At 72 h most of the antibody fragment radioconjugates had cleared from the circulation. The concentration of the immunoconjugate localized in the breast tumor remained unchanged. Thus at 72 h higher breast tumor/blood counts were obtained. Kidneys continued to be the site of radioactivity retention with 27.0 + 15.4% ID/g tissue. Breast tumor/blood ratios of the radioactivity of the antibodies studied are shown in Table 1. Antibody T43 showed a greater tumor/blood ratio than UR04. With increasing time the tumor/blood ratio of T43 had increased as the radioconjugate had cleared from the circulation. The F(ab’), fragment showed a higher tumor/blood ratio than the whole antibodies. The ratio as high as 14.3 was obtained at 72 h. Even higher tumor/blood ratios were obtained at 120 and 144h. The scintigrams of the mice injected with radioconjugates of T43 and F(ab’), are shown in Fig. 6. At 48 h the breast tumor in the left flank was discernible in the mice injected with the whole antibody radioconjugate despite the high background of the liver. The nontarget pituitary tumor in the tight flank was relatively larger and well vascularized than the breast tumor but it was not detected in these images. The fragments produced less background compared to the whole antibody and the breast tumor was clearly visible at 48 h. The images at 120 h showed reduction of counts in background as well as in tumor site, but there were sufficient counts to visualize the tumor.
Discussion In this paper we have described the effect of DTPA derivatization on immunoreactivities of a monoclonal antibody, T43, directed to T47D cell membrane antigen, and a second monoclonal antibody, CU18, directed to an antigen (BCA-225) expressed on T47D cell membrane as well as in the cytoplasm (Mesa-Tejada et al., 1988). A nonspecific monoclonal antibody, UR04, that did not bind to T47D and GH3 cells was used as a control.
el al.
The immunoreactivities of the conjugates of the antibodies prepared by DTPA cyclic anhydride coupling method were affected by the extent of the derivatization of lysine amino groups in the antigen binding region. Other investigators (Kulmann and Steinstrasser, 1988) have reported similar effects on immunoreactivities of antibodies with DTPA derivatization. The loss of immunoreactivity was thought to be due to cross linking and the steric hindrance caused by the DTPA groups introduced in the antigen binding sites. The damaging effect of increased derivatization on immunoreactivity was much more pronounced in CU18, which suggested the presence of highly exposed lysines in antigen binding sites. T43 and CU18 showed different degrees of sensitivity to DTPA derivatization. Such variation in deleterious effect of conjugations from antibody to antibody was also observed by other workers (Paik et al., 1983). When we compared the immunoreactivities determined by ELISA with the true immunoreactive fraction determined by the methods of Lindmo et al. (1986), higher immunoreactivities were observed in ELISA. This may be attributed to the presence of some underivatized antibody molecules in the conjugate preparations. The DTPA cyclic anhydride derivatization produces a range of conjugation products with different degrees of substitution in addition to the presence of some underivatized antibody molecules. Since the reaction is thought to obey Poisson statistics, after the introduction of an average of l-2 groups per protein molecule there will be 1535% unmodified protein molecules in the conjugate prep aration (Petrella et al., 1987). The purification of the DTPA conjugates by dialysis and size exclusion chromatography does not remove undervatized antibody molecules, and ion exchange chromatography or affinity chromatography might be required for further purification. In our in vivo model the nude mice were transplanted with T47D (human breast carcinoma cell line) and GH3 (rat pituitary tumor cell line) xenografts. Monoclonal antibody T43 localized in the tumor site better than the control antibody UR04. At 48 h a tumor/blood ratio of 3.9 was obtained for T43, whereas UR04 showed a ratio of 1.3 (P < 0.01). Liver, spleen and kidney were the organs that sequestered radioconjugates very efficiently. Such sequestration by a reticuloendothelial system has been reported for most IgG radioconjugates and major uptake by liver seems to be a disadvantage with the use of radioimmunoconjugates. We have observed liver uptake between 3-15%, whereas others
Table 1. Breast tumor/blood ratios Antibody
1 2 3 4
T43 UR04 T43 F(ab’), UR04 F(ab’),
*ND = not determined.
48 b 3.92 1.3 a.79 8.54
+ f f +
1.57 1.22 5.43 3.75
72 b 4.57 1.9 14.26 9.44
f * f +
2.16 0.87 4.13 0.35
120b
144h
9.35 + 0.29 ND* 15.4 * 5.5 ND
16.75 _+ 1.24 ND 53.48 + 6.3 ND
Fig. 6. Examples of gamma camera images of nude mice with human beast tumor xenograft (T47D) in the left flank and rat pituitary tumor xenograft (GH3) in the right flank, after injecting with whole antibody or F[ab’jz fragment are shown. Breast tumors were visualized with T43 and T43 F(ab’)2 at 48 h. Pituitary tumors were not seen at 48 and 120 h in these scintigrams.
301
Imaging breast tumor xenografts in mice
have reported bodies
higher than 20% for different anti(Kalofonos et al., 1988). The mechanism for
liver uptake is believed to be multifactorial, e.g. transchelation of “‘In to transferrin and/or interaction of Fc part of IgG with hepatocytes receptors (Halpem el al., 1983; Hopf et al., 1976). Although the detection of lymph node and distal metastates may not be affected by such high liver background, the abdomenal observations and liver metastates might be obscured. However, recently single photon emission computed tomography (SPECT) technique has been employed to detect liver metastates using ‘*‘Inlabeled antibodies with considerable success (Halpern et al., 1988; Abdel-Nabi et al., 1988). Improved localization of F(ab’), compared to whole IgG has been reported by several workers (Moseley et al., 1988; Andrew et al., 1988). Similarly, in our studies T43 F(ab’), showed better contrast than T43. Due to much rapid clearance of F(ab’), from the circulation tumor/blood ratios as high as 8.79 were obtained at 48 h. Although the control antibody UR04 F(ab’), showed similar tumor/blood ratios at 48 h, the % ID/g in the tumor was 2-fold lower than T43 F(ab’), and for this reason the target tumors were visible only with T43 F(ab’),. Even higher ratios were seen for T43 F(ab’), at later time points (see Table 1). The rapid clearance of F(ab’), from the circulation is thought to be due to the absence of the Fc portion of the IgG molecules and/or the concurrent reduction in molecular size (Holten et al., 1987). The fragments were captured by liver, spleen and kidneys. The site of highest radioactivity accumulation was the kidneys. The role of kidneys in the excretion and catabolism of IgG and IgG fragments was studied previously (Wochner et al., 1967). The kidneys were shown to be the site of glomerular filtration and catabolism of the IgG fragments but not the intact IgG. A recent comparative study of tumor localization using “‘I and “‘Inlabeled anti-CEA antibody Cl98 fragments showed similar higher uptake in the kidneys of nude mice (Andrew et al., 1988). Although radioiodine from ‘3’I-labeled antibody fragments is removed much more rapidly from the kidneys, radioactivity of “‘Inlabeled antibody fragments is retained for a prolonged time. The reasons for such preferential retention of “‘In in the kidney are not clear. These studies demonstrated that a 4mm human breast tumor xenograft could be visualized at 48 h with tumor/blood ratios of 3.92 & 1.57 for the whole antibody, T43, and 8.79 + 5.43 for its F(ab’)z fragments. With increasing time even higher tumor/blood ratios were observed, and so 48-72 h appears to be suitable time for imaging. We now plan to extend the applicability of T43 F(ab’), radioconjugates to humans. Since CU18 has very high specificity to breast carcinoma, we are investigating derivatizations using very selective heterofunctional chelating agents and use of these conjugates in different tumor xenografts/nude mice models.
303
Acknowledgements-We
thank DC Tom Parish for supplying the antibodies T43 and UR04, Irene Ho and Eileen Kelley for technical assistance. This research was supported in part by Johnson & Johnson Special Biological Projects, Irving A. Hansen Memorial Foundation, Florence and Herbert Irving Foundation and NIH Grant 5ROl CA 32984-07.
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