site-related differences in the localization of the monoclonal antibody ox7 in sl2 and sl1 lymphomas

Cancer Immunol Immunother (1986) 22: 169-175 ancer mmunology mmunotherapy

Springer-Verlag 1986

Site-related differences in the localization of the monoclonal antibody OX7 in SL2 and SL1 lymphomas

Howard Sands, Peter L. Jones, William P. Neacy, Sudhir A. Shah, and Brian M. Gallagher

Biomedical Products Department, Immunopharmaceutical Research and Development Department, E. I. duPont de Nemours and Co. (Inc.), 331 Treble Cove Road, North Billerica, Massachusetts 01862, USA

Summary. The uptake of a monoclonal antibody (OX7) by murine lymphomas (SL1, SL2) growing in two sites in the mouse were compared. SL2 tumors grown in the subrenal site showed greater specific antibody uptake than did the same tumor grown in the subcutaneous site. Major differences in membrane bound antibody, in vitro antibody binding patterns, and gamma scintillation camera imaging were also observed between the two sites. These differences may be due to the greater blood flow measured in tumors growing in the subrenal capsule than those growing at the subcutaneous site. The differences observed in antibody uptake of the same tumor growing in two different sites raises questions concerning the choice of animal model systems that can be used to predict clinical utility.

Introduction

The use of monoclonal antibodies as tumor-specific carri- ers of drugs and therapeutic and diagnostic radionuclides has been proposed [1, 3] and recently reviewed [7]. There have been numerous experimental studies to test the valid: ity of the antibody delivery concept [5, 6, 7, 9, 13]. Most have utilized the xenograft model of the subcutaneous human tumor residing in the athymic mouse [5, 6, 9] whereas others have used homograft models, such as subcutaneous guinea pig tumors [11, 20]. In most of the studies carried out using these systems, relatively large tumors in compar- ison with body weight have been utilized. Bogden has proposed the use of tumors residing under the renal capsule of the mouse as a model for the study of the cancer chemotherapeutic potential of a drug [4]. The small tumor size and its location make the subrenal tumor an excellent model for the study of antibody localization. We have previously reported excellent tumor imaging by monoclonal antibodies to murine lymphomas residing in the subrenal capsule of mice [17]. In this report we have expanded these studies and compared the ability of an antibody to localize and image the same tumor located in either the subcutaneous or subrenal site. In addition, the comparative histopathology, antibody distribution within these tumors, anti- genic heterogeneity, antibody membrane binding, and blood flow to SL1 and SL2 mouse lymphoma tumors

Offprint requests to: H. Sands

growing in the subrenal capsule have been compared to those growing subcutaneously.

The murine lymphoma system consisted of two lymphoma cell lines, SL1 (THY 1 .1 - ) and SL2 (THY 1.1 +), a mouse strain (AKR/Cumberland) which lacks the THY 1.l antigen, and a monoclonal IgG I antibody (OX7) directed against the THY 1.1 antigen. Anti-horseradish per- oxidase (anti-HRP), also an IgG1, served as a control. The SL1 and SL2 cells can be carried in tissue culture and were capable of producing tumors in the AKR/Cumber land mice and tumor growth could be controlled by varying cell burden. The SLI tumor served as an antigen negative control. The presence of SL2 lymphomas (THYI.1 + ) in the AKR/Cumber land mice (THY 1.1-) , represents a model of a "tumor specific" antigen in that only the lymphoma cells express the target antigen [10, 16].

Materials and methods

Preparation of radiolabeled OX7 and anti-HPR. OX7 was obtained from, Dr. A. F. Williams, Oxford (UK) and the anti-HRP was prepared by standard hybridoma tech- niques [121. Antibodies were radioiodinated using the chloramine T method [10]. Briefly, 2.5 mg of antibody was vortexed on ice for 1 min with 2.5 mCi of radiolabeled NaI (DuPont /NEN Products N. Billerica, Mass.) in the presence of 75 ~tg of chloramine T. The reactivity was quenched with 75 p~g of sodium metabisulfite and the free iodine removed using a I0 ml Sephadex G-25 column. In- corporation of either 125I or 131I was 85% to 93% and the final specific activities were 0.8 to 0.9 mCi/mg.

Following iodination, the immunoreactivity of the antibody was assessed using a live cell binding assay. La- beled antibody (0.9 lxCi) was added to a suspension of either SL1 or SL2 cells. The cell number ranged from 3 × 104 to 3 × 106/0.1 ml. The cells were suspended in 0.3 ml buffer for 30 min at room temperature followed by two washes and harvesting the cells in a Beckman J2-21 centrifuge at 200 g for l0 min. The amount of antibody bound to the cells was determined by counting the cell pellet in a gamma scintillation counter. A typical result is shown in Fig. 1. The radioiodinated OX7 demonstrated a high degree of specificity for SL2 cells over SL1 (over 70% of the radioiodinated OX7 bound to 106 SL2 cells during a single 30 min incubation while less than 10% bound to SL1 cells) and thus retained the vast majority of its immunoreactivity.

170

8 O

60 0

40

'1 20

Fig. 1.

I 4

In

~ o Q u o SL2 / SLI

I I I

5 6 7

Log Cell Number

vitro assay of radioiodinated OX7 using SL2 (THYI.1 +) and SL1 (THYI.1 - ) cells

I I i I I I

48OO

,o == 8 2400

y v~ Normal Kidney

t 2 0 0 t I I

0 I I I

0 1 2 3 4 5

Minutes Post 8eRb Injection

Fig. 2. Time course of 86Rb uptake by mouse tissues

Animals, cell lines', tumors. AKR/Cumberland mice (THY 1 .1- ) were obtained from Cumberland View Farms Clin- ton, Tenn. SL1 and SL2 cells were grown in RPMI 1640 containing 10% fetal bovine serum. Cells were harvested by centrifugation and resuspended in RPMI medium. Sub- cutaneous tumors were formed by the injection of 1 x 10 7 cells. Subrenal tumors were formed by the implantation of a 1 mm 3 piece of a subcutaneous tumor according to the method of Bodgen et al. [4]. In some experiments the animals carried two tumors, one SL1 and SL2 in either the subcutaneous or subrenal site.

Antibody was injected i. v. 7 days following subrenal implantation and 14 days following subcutaneous injection of cells. At that time subrenal tumors were approximately 40-60 mg and subcutaneous tumors approximately 200-400 mg, respectively.

Pharmacokinetics, localization index. Tumor-bearing animals were injected i. v. in the tail vein with 5 p~Ci each of OX7 and anti-HRP. The animals were sacrificed by cervi- cal dislocation 48 h after antibody injection and samples of blood and tumor were obtained, weighed, and their content of 131I and ]25I determined by counting them in a gamma counter. Antibody uptake is reported as the percent of the injected dose/g tumor (I. D./g). The localization index was determined by the method of Moshakis et al. [15]:

L.I. = 1251 OX7 in tumor/13q anti-HRP in tumor

125I OX7 in blood/131I anti-HRP in blood

Scintillation imaging. Tumor-bearing mice were injected i. v. through the tail veil with 100 ~tCi of either 125I OX7 or 1311 anti-HRP. Scintillation imaging was done at 4, 24, and 48 h after antibody injection using a Searle Pho-Gamma HP gamma camera with a Medx-37 upgrade. Images were composed of 100,000 counts and were stored and analysed using a DEC PDP 11/34 computer. Thyroid uptake of any free radioiodine was not blocked with NaI.

Antibody bound to a crude membrane preparation. To deter- mine the percentage and absolute amount of antibody found to a crude membrane fraction of tumors, a proce- dure essentially the same as described for the pharmaco- kinetic studies was followed, in which a sample of the tumor was homogenized using a Polytron (Brinkman) homo- genizer. The homogenate was centrifuged at 200 g for

10 min to remove large fragments and then centrifuged at 50,000 g for 2 h using a Beckman centrifuge. The percentage of the total radioiodine found in the pellet was then determined. Using this value, the percent I. D./g, and the specific activity of the antibody, the ng of antibody bound/rag tumor was calculated.

Blood flow. The relative blood flows to the tumors and normal tissues were determined using the 86Rb method of Gullino [8]. It was first determined that the SdRb content of tumor, kidney, and muscle reached equilibrium 2 rain after an i. v. tail vein injection of 75 p~Ci of 86Rb (Fig. 2). This was done by placing a miniature cadmium-telluride probe (RMD Inc. Marlboro, Mass.) over the tumor or tissue to be monitored and recording the relative change in count rate in the tissue. Since values reached a plateau within 2 rain after injection, this time was chosen for animal sac- rifice. The relative blood flow values are reported as percent I. D. 86Rb/g tissue.

Autoradiography. SL1 and SL2 tumors were removed from mice 24 h after an i.v. injection of 25 p, Ci 125I-OX7 and were fixed in 10% buffered formalin and embedded in par- affin. Sections of 6 p,m were cut on a microtome at room temperature, deparaffinated, and coated with Ilford K5 autoradiographic emulsion (Polysciences, Warrington, Pa.) at 50 °C under safelight illumination. Slides were stored at room temperature in a light-proof box in the presence of drierite for 7 days and then developed using Kodak DI9 at 4 °C. Tissues were then stained with hematoxylin and eosin.

Histopathology. Standard hematoxylin-eosin stained sections of both subrenal and subcutaneous tumors were ana- lyzed by a Board Certified veterinary pathologist.

R e s u l t s

Pharmacokinetics, localization index

There were dramatic differences in the ability of OX7 to accumulate in SL2 tumors residing in two different sites (Table 1). Tumor accumulation of antibody was greatest in the subrenal SL2 tumor. Control tumor (SLI) and control antibody (anti-HRP) in both SL1 and SL2 tumors gave values of ~ 2% I. D,/g of tumor tissue, thus indicating excellent specific uptake of antibody. The nonspecific uptake of

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Fig. 3. Gamma camera scintillation images of SL1 and SL2 tumors. Arrows indicate location of tumors. SLI tumors are on the left and SL2 tumors on the right. Subcutaneous tumors are shown in A and B. Subrenal tumors are shown in C and D. Images produced by the control antibody (anti-HRP) are shown in A and C. Images produced by the specific antibody are shown in B and D. "T" indicates the location of the thyroid

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Table 1. In vivo values for subrenal and subcutaneous SL1 and SL2 tumors 48 h after antibody injection

Subcutaneous Subrenal

SL1 SL2 SL1 SL2

% 1.D./g OX7 1.92+ 1.12 6.24+3.59 2.94+ 1.05 22.44+ 7.64 anti-HRP 2.13+ 1.16 2.26+ 1.14 4.26+2.60 2.96+ 0.18 ng OX7bound/mg 29.46+3.13 97.66+7.56 26.49+2,29 688.86+ 121.46 ng anti-HPR bound/mg N.D. N.D. 21.92+4.68 28.43 + 13.62 L.I. 1.04 + 0.07 3.07 +_ 1.28 1.55 + 0.47 19.21 + 16.87

Data represent the mean + SD of the values from three animals N. D. = Not determined

OX7 by subrenal and subcutaneous SL1 tumors was ident- ical. The specific uptake of OX7 by the subrenal SL2 tumors was, however, several fold greater than that of the subcutaneous SL2 tumors.

The differences in the accumulat ion of OX7 in the two implant sites were made more dramat ic when expressed as the local izat ion index (L. I.) [15]. This permits the compar- ison of specific and nonspecif ic accumulat ion in tumors and organs taking into account the relative b lood concen- t rat ions of the two antibodies. Therefore an L. I. of 1.0 indicates no specific an t ibody uptake. The L. I. for the subcutaneous SL2 tumor was approximate ly 3 while the L. I. for the subrenal SL2 tumor was approx imate ly 15 (Table 1).

less then that of well perfused organs such as heart, lung, and kidney. Despite the higher b lood flow to these normal organs they demonst ra ted no specific an t ibody accumulation. We have previously shown that both the SL1 and SL2 tumors grown at the subrenal site have the same relative b lood flow, yet only the SL2 tumor showed marked OX7 uptake [17]. Therefore, the differences observed in the up-

Table 2. Relative blood flow as determined by 86 Rb in tissues of AKR/Cumberland mice

Tissue 86 Rb (% I.D./g + SD)

Antibody bound to a crude membrane preparation Subcutaneous tumor

In Table 1 the da ta are also repor ted in terms of the ng of Heart an t ibody bound to a crude prepara t ion of tumor mere- Kidney branes. A significantly higher specific an t ibody b inding to Muscle the subrenal SL2 tumor membranes compared to subcu- Skin taneous SL1 and SL2 tumor membranes or subrenal SL1 Lungs membranes was observed, n =

Scintillation imaging

G a m m a camera scinti l lat ion images resulting from the accumulat ion of 125I-OX7 or ]31I-anti-HRP are shown in Fig. 3 and confirm the quanti tat ive results. Subcutaneous SL2 tumors of 200-400 mg could be visualized with 1251- OX7 (Fig. 3 B). This " tumor associated" an t ibody failed to visualize the non target SL1 tumors. Nei ther SL1 nor SL2 tumors could be imaged with 131I-anti-HRP (Fig. 3 A). The accumulat ion of rad io iod ine in the thyroid was indicat ive of an t ibody dehalogenat ion. Despite the fact that the subrenal tumors were much smaller than the subcutaneous tumors (40 mg vs 200-400 mg) and the fact that the visceral locat ion of the subrenal tumors presents a higher back- ground, specific images of the SL2 tumor were obtained. This is indica ted by the clear asymmetry of the 125I-OX7 uptake in mice bear ing subrenal SL2 tumors (Fig. 3 D). This asymmetry was not seen when 13q-anti-HRP was injected (Fig. 3 C).

Blood flow

Since ant ibody accumulat ion in a tumor is ul t imately de- penden t upon an t ibody del ivery via the b lood, the relative b lood flow to tumors was de termined using the 86Rb method (Table 2 and Fig. 4). The relative b lood flow to the SL1 and SL2 subcutaneous tumors was equal and several fold

SL1 SL2

2.25 ___ 0.4O 14.52+2.37 49.86+5.18

1.16+0.38 1.03+0.18 9.58 ___ 0.64 4

2.20 + 0.35 13.89 + 1.73 62.81 +9.36

0.96+0.12 1.72+0.31

12.61 ___0.79 6

14

12

E 10

g_

2 0 - 5 0

;;~S,C. IS.E.M"

5 1 - 1 0 0 101-20 ( 2 0 1 - 3 0 0

Tumor Weight: mg range

5 0 0 - 1 0 0 0

Fig. 4. Effect of tumor size on relative blood flow of SL1 and SL2 tumors. Data from SL1 and SL2 tumors were pooled. The number in the bars indicates the number of tumors used to generate the mean. S. C. indicates subcutaneous tumors, and S. R. subrenal tumors. *=p<0.05 compared with 20 to 50rag. **=p>0.05 compared with 51 to 100 mg

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take of OX7 between SL1 and SL2 subrenal tumors (Table 1) apparent ly is a reflection of immunospeci f ic localization. Differences in relative b lood flow between subrenal and subcutaneous tumors could account for the differences in the uptake of OX7 by SL2 tumor seen at these sites [17] (Fig. 4).

Another difference observed between the subrenal and subcutaneous tumors is shown in Fig. 4. For this figure the da ta from all SL1 and SL2 tumors were combined and then grouped according to tumor weight. The large differ-

ence between the b lood flow to subrenal and subcutaneous tumors was clearly shown. In addi t ion, a dist inct decrease in relative b lood flow per weight of tumor with increasing size of the subrenal tumors but not subcutaneous tumors was observed.

Autoradiography Autorad iography was per formed on tumor sections from mice after i. v. adminis t ra t ion of 125I-labeled OX7. The pat-

Fig. 5. Autoradiograph of the binding of OX7 in to SL 1 and SL2 tumors 24 h after i. v. injection of radiolabeled antibody. Magnification is 2500X. A represents the binding of OX7 in a subcutaneous SL1 tumor. B represents the binding of OX7 in a subcutaneous SL2 tumor. C represents the binding of OX7 in a SL1 subrenal tumor. D represents the binding of OX7 in a SL2 subrenal tumor

174

Fig. 6. Low power autoradiograph of the binding of OX7 in subrenal SL1 and SL2 tumors 24 h after i. v. injection of radiolabeled antibody. Magnification is 625 x. A represents the binding of OX7 in SL1. B represents the binding of OX7 in SL2. K= Kidney T=Tumor Ab = grains from radiolabeled OX7

tern of distribution and extent of antibody uptake appar- ently due to nonspecific diffusion was equivalent in subcutaneous and subrenal SL1 tumors (Fig. 5A, C). Antibody uptake by subcutaneous SL2 tumors was qualitatively higher (shown by grain density) than subcutaneous SL1 tumors, in accord with quantitative in vivo measurements (Fig. 5 B, vs 5A). In contrast, tumor sections taken from the SL2 tumor grown in the subrenal site showed that 125I- OX7 localization was significantly greater compared to either SL1 tumors at the subrenal site or SL1 and SL2 at the subcutaneous site (Fig. 5 D). Finally, antibody was distrib- uted heterogeneously in distinct zones throughout the whole SL2 tumor (Fig. 6).

Histopathology The SL1 and SL2 tumors could not be distinguished by histological criteria. Both tumors were highly cellular, poorly differentiated, malignant neoplasms of lymphoid cells. The cells were round to polygonal with leptochro- matic, round to ovoid nuclei containing a single promi- nent nucleolus. Mitotic figures were numerous. Tumors grown in the subrenal site were histologically indistin- guishable from those grown subcutaneoulsy.

Discussion

There have been numerous reports concerning the detec- tion of tumors, both in nude mice and patients, using antibodies labeled with gamma-emitting isotopes such as 131I, 125I and lllIn [1, 3, 5, 6, 9, 13, 14, 17, 18, 19, 20]. The pre- dictive value for clinical utility of many of the animal experiments is uncertain since they have usually employed subcutaneous human tumor xenografts growing in nude mice. This is a highly artificial system in which many phy- siological parameters, such as the permeability of the mouse blood vessels growing in the human tumor [18],

may not be relevant to the clinical situation. In an attempt to overcome such problems, our laboratory has studied the localization and imaging of small murine SL2 tumors grown in the subrenal capsule of the Cumberland mouse [17]. In this report the localization and imaging character- istics of the SL2 and SL1 tumors growing under the renal capsule were compared to those growing in the subcutaneous site.

Significant differences were observed in antibody localization for the same tumor grown at two different sites. These differences include a greater absolute uptake, a higher L. I., and a greater crude membrane binding of antibodies in subrenal tumors. In vivo autoradiographic an- alysis of subcutaneous and subrenal tumors confirmed the increased specific binding of OX7 in subrenal compared to subcutaneous SL2 tumors. In addition, the "heteroge- neous" binding patterns demonstrated that in a tumor which takes up the antibody specifically not every cell in the tumor binds antibody. This heterogeneity in accumulation has great implications for the choice of the optimal antibody for diagnosis and therapy in vivo using radioiso- tope linked antibodies. The greater uptake of the OX7 by the subrenal SL2 tumors than observed for the subrenal SL1 and subcutaneous SL1 and SL2 tumors was not due to morphological differences since histopathogical evalua- tions showed that tumors grown in both sites were indistin- guishable. Differences in the size of the subrenal and subcutaneous tumors did not account for the greater subrenal OX7 uptake. In preliminary experiments in which large (200-350 rag) subrenal SL1 and SL2 tumors were used, antibody uptake and L. I.'s were comparable to the data reported here. The differences between the specific uptake by these tumors might be related to the much greater blood flow observed in subrenal tumors (fivefold higher than the subcutaneous tumor) [17]. Since these differences in specif-

175

ic uptake are so great at 48 h, it is highly unl ikely the subcutaneous tumor values will reach the level of the subrenal tumors within a reasonable per iod of time.

The differences in an t ibody uptake of the SL2 tumor residing in two different sites raises many questions and suggests the potent ia l pitfalls which may exist in making ext rapola t ions concerning the clinical utility of ant ibodies from a single model system. Many factors in addi t ion to the immunoreact iv i ty of an an t ibody and the expression of antigen on the tumor are involved in determining the absolute accumulat ion of an an t ibody by a tumor. These include immunospeci f ic factors such as an t ibody binding to cell membranes as well as non immunospec i f i c physiologi- cal factors such as b lood flow to the tumor [8] and differences in vascular permeabi l i ty [2, 18]. These addi t ional factors must be ident if ied and quant i ta ted both in the various tumor animal models and if possible in humans before these animal models can be rel iably used for predict ing the efficacy of rad io labe led ant ibodies for cancer diagnosis and therapy.

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Eckelman WC (eds) Animal Models in Radiotracer Design. Springer-Verlag, New York, pp 66-105

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Received July 16, 1985/Accepted February 4, 1986

site-related differences in the localization of the monoclonal antibody ox7 in sl2 and sl1 lymphomas

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