In search for a new therapeutic approach for metastasized renal cell carcinoma (RCC), we evaluated the cytotoxicity of a novel prodrug chemoimmunoconjugate with monoclonal antibody (mAb) 138H11 and the DNA-cleaving enediyne calicheamicin θI1(Camθ) in vitro and in vivo. Previously, mAb 138H11, produced against human renalγ-glutamyltransferase, stained over 99% clear cell and papillary RCC on frozen sections, showing a membranous expression of the target antigen. In contrast, in normal kidneys γGT was restricted to the brush-border in the lumen of proximal tubules and not accessible to the circulation. Thus, human tumor-bearing kidneys perfused in an extra-corporeal system with 99mTc-138H11 revealed a high, specific uptake into the tumor. In this study,fluorescence-activated cell sorting analysis showed binding of mAb 138H11 to RCC cell lines, whereas squamous cell carcinoma lines,fibroblasts, and the murine RENCA were negative. XTT cell proliferation assays revealed efficient killing of the Caki-1 cell line by the 138H11-Camθ conjugate using SPDP(EC50 = 5 × 10−11m) as a covalent linker. For in vivotesting, five groups of eight nude mice each were injected with 2.5 × 106 Caki-1 cells s.c. and treated with the following: (a) PBS; (b) 138H11;(c) Camθ; (d) a mixture of 138H11 and Camθ; and (e) 138H11-Camθ conjugate. Treatment started on day 1 after tumor induction and was repeated three times. The data show a highly significant inhibition of tumor growth with the 138H11-Camθ conjugate versus PBS(P = 0.004). Only mice treated with 138H11-Camθ showed a tumor shrinkage to minimal residues. In a second experiment, lower doses of the 138H11-Camθ conjugate were compared with an antineuroblastoma mAb (ch14.18), confirming targeted killing of RCC by the 138H11-Camθ conjugate at tolerable toxicity in vivo. In conclusion, these combined results encourage further studies for targeted therapy of metastatic RCC with mAb 138H11 conjugates.

RCCs3are highly malignant tumors that originate from the tubular epithelial cells in the kidney. They account for ∼3% of all human tumors. In the United States alone, approximately 30,000 new cases and 12,000 deaths each year attributable to RCC have been reported from 1996 through 1998 (1, 2, 3, 4). In Germany, RCC belongs among the 12 most frequently diagnosed malignant tumors with an estimated 6,612 deaths in 1995, according to the tumor register in Munich.4The incidence is drastically increasing, and for the year 2000, a worldwide mortality of 100,000 is expected (2, 3, 4). Once the tumor has spread beyond the kidney, patients have a very poor prognosis. The 5-year survival rate for the Robson states III and IV are 15–35% and 0–10%, respectively (4). When local recurrence occurs, most patients die within a few months. As tumors restricted to the kidney rarely cause symptoms, up to 30% of patients are diagnosed with metastatic disease, and another 40% of patients experience a relapse after nephrectomy (3). For these patients, there is no effective treatment available currently. Conventional chemotherapy is ineffective due to several mechanisms of high multidrug resistance of RCC, and radiotherapy is only useful for palliation of tumor symptoms (2, 5).

It has been known for a long time that RCC sometimes can regress spontaneously. This has been attributed to immune mechanisms(6, 7, 8). From this experience, systemic immunotherapy using IFN-α, Interleukin-2, or a combination of both was developed. However, results of large clinical trials have shown overall objective responses <20% and of short duration, accompanied with severe toxicities (8, 9, 10, 11).

We developed the mAb 138H11 against human γGT (EC 2.3.2.2) for possible use in differential diagnosis and targeted therapy of RCC(12, 13, 14). This mAb stained over 99% of primary clear cell and papillary RCCs on frozen sections. These two tumor types represent over 90% of the malignant RCC. Oncocytomas (considered benign),chromophobe, and Duct Bellini carcinomas were all negative(12). mAb 138H11 has also proven useful for the differential diagnosis of metastases when the primary tumor is not known (15). In immunoscintigraphy, 99mTc-labeled 138H11 demonstrated specific uptake into RCC in ex vivo perfused human tumor-bearing kidneys(13). The enrichment of the labeled mAb in the tumor was up to 20-fold compared with the kidney cortex tissue.

This specific tumor uptake can be explained by the localization of the target antigen. In a normal kidney, γGT is confined mostly to the brush border membrane of the proximal tubules (12). Similarly, in the liver, γGT is located at the luminal part of the bile canaliculi. Thus, in normal organs, the 138H11-antigen is not accessible via the circulation. In contrast, in primary and metastatic RCC, the enzyme is expressed over the entire tumor cell surface, thus becoming accessible to mAb 138H11 in the circulation(15, 16). In conclusion, a targeting effect with mAb 138H11 is attributable to a different antigen localization on tumor cells compared with normal organs. This is unlike most other antibody targeting strategies, which rely mostly on a higher antigen concentration in tumor cells compared with normal tissues(17, 18).

Although tumor therapy with mAbs alone may result in impressive clinical responses (17, 19), increasing the response rate requires the improvement of the antibody-effector function. This can be done, for example, by creating immunotoxins, bispecific antibodies, and cytokine fusion proteins or by the chemical coupling of cytotoxic drugs(20, 21, 22). We decided to use the latter strategy and coupled Camθ to mAb 138H11 (21). Camθ was rationally designed on the basis of calicheamicinγ I1(23). Both compounds belong to the chemical group of enediyne antibiotics:naturally occurring substances that are able to damage DNA by double-strand cleavage (22, 24). Camθ is several orders of magnitude more potent than other chemotherapeutics (21, 25), including the natural calicheamicinγ I1(20, 23). In this study, we evaluated for the first time the potential of a novel chemoimmunoconjugate of mAb 138H11 and Camθ in vivo.

mAb 138H11 Production and Purification

138H11 was produced in the miniperm system (Heraeus Instruments, Inc., Hanau, Germany) as described(21). The hybridoma cells were grown in RPMI with 10% FCS(Seromed, Berlin, Germany; Ref. 14). No antibiotics were added to immediately detect bacterial contaminations. The cell titer was counted every day and the supernatant was harvested 2 days after stagnation or if <80% of the cells were viable. mAb from cell culture supernatant was purified using Protein G affinity chromatography (FPLC; Pharmacia, Freiburg, Germany). Samples were loaded onto the column (Protein G Fast Flow; Pharmacia) using binding buffer (Pierce, St. Augustin, Germany). Bound mAb 138H11 was eluted with elution buffer (Pierce) and transferred into PBS (pH 7.4)by gel filtration (PD 10; Pharmacia). Aliquots were analyzed for purity with a G250 HPLC column, with native PAGE and Western blot using protocols as described previously (26).

FACS

Trypsinized cells (5 × 104in PBS) were incubated with 50 μg of purified mAb for 60 min at 4°C, washed with 1 ml of PBS containing 5% FCS, and then pelleted at 1000 × g for 5 min. This was followed by incubation for 60 min at 4°C with FITC-labeled goat antimouse-IgG(GAM-FITC; Becton Dickinson, Heidelberg, Germany). Cells were washed once and fixed with 1% paraformaldehyde/PBS prior to FACS analysis(FACScan, Becton Dickinson). Cells incubated with irrelevant mAbs,ch14.18 (17), or secondary antibody only were used as negative controls.

Conjugation of Camθ to mAb 138H11 and ch14.18

The principle used to conjugate Camθ to 138H11 involved the activation of the amino group with SPDP (Pierce), followed by disulfide exchange with iminothiolane-modified mAb 138H11 (Fig. 1). For activation, Camθ (500 μg in 500 μl of DMF) was reacted with 10 μl of SPDP stock (2.2 mg in 200 μl DMF) at 4°C for 6 h and stored in small aliquots at −80°C until further use. mAb 138H11 [10 mg in 2 ml of PBS (pH 8.3)] was reduced by adding 10 μl of iminothiolane (Pierce; 4.4 mg dissolved in 1 ml DMF) for 1 h under nitrogen. The reduced antibody was purified by size exclusion chromatography using a NAP-5 column (Pharmacia)preequilibrated with PBS. The conjugation of activated Camθ(mr ∼1,464) with thiolated mAb 138H11 (mr ∼155,000) was done at RT for 1 h under nitrogen using a 2:1 molar ratio. Finally, solutions were filter-sterilized. The purification grade was checked by HPLC analysis. The number of drug molecules/mAb were two on the average as calculated from the ratio of A303/A280 in the HPLC spectrum. The dosage of the 138H11-Camθ conjugate for cytotoxicity and in vivo experiments was calculated according to the total amount of Camθ. The conjugation of Camθ with ch14.18 was done as described above.

Cytotoxicity Assays

Tumor cells were seeded in 200 μl of RPMI with 10% FCS at a density of 1–5 × 104 cells/well in 96-well plates (Falcon, Becton Dickinson) and allowed to adhere for 24 h at 37°C and 5% CO2. Then the agents:(a) activated Camθ-SPDP; (b) the mixture of mAb 138H11 and Camθ (both in concentrations corresponding to the conjugate; (c), the 138H11-Camθ conjugate; (d)the Camθ-ch14.18 conjugate; (e) 138H11; (f)ch14.18; or (g) PBS, each in a total volume of 200 μl,were added to the wells at 1:10 serial dilutions. The plates were incubated for 72 h at 37°C and 5% CO2. Visual scoring of cell viability was performed at 24, 48, and 72 h after drug application. Cytotoxicity assays were performed using a vital stain XTT assay. 10 μl of a 1.5-mg Phenazin(Sigma-Aldrich, Deisenhofen, Germany)/ml aqua bidest solution were added to 10 ml of a 5-mg XTT (Sigma-Aldrich)/ml RPMI stock solution. This freshly prepared mixture was sterile-filtrated, and 40 ml were added to each well with the cells to be tested. Absorption was measured at 450 nm after 2 h of incubation at 37°C with 5%CO2. Absorption/concentration profiles were obtained using the following 4-parameter fitting equation:

\[f(x)\ {=}\ \frac{A\ {-}\ D}{1\ {+}\ \frac{x}{C^{B}}}\ {+}\ D\]

where A and D describe the left and right asymptote of the sigmoid curve, respectively. B describes the slope, and C describes the inflection point of the curve. The inflection point corresponds to approximately the half-maximal effective dose, EC50(21).

Treatment of Human RCC Xenografts in Nude Mice

High Dose.

Five groups of eight female NMRI-nu/nu mice (“Swiss”; M & B, Ry,Denmark) each were implanted with 2.5 × 106 Caki-1 cells s.c. The mice were treated with:(a) PBS; (b) 138H11; (c) Camθ (1μg/kg); (d) the mixture of 138H11 and Camθ (1 μg/kg);and (e) the 138H11-Camθ conjugate corresponding to a 20μg/kg dose of Camθ.

Low Dose.

Three groups of six mice each xenografted as above were treated with:(a) PBS; (b) the 138H11-Camθ (10 μg/kg)conjugate; and (c) the ch14.18-Camθ (10 μg/kg)conjugate.

In both experiments (Fig. 4 and 5), treatment started one day after tumor inoculation in agreement with many accepted experimental models(20, 27, 28) and was repeated every other day for three times. Mice were observed, weighed, and measured individually throughout the experiment. Each tumor was measured by a caliper in two dimensions, and the tumor volume was calculated using an established formula:

\[\mathrm{Volume\ {=}\ width}^{\mathrm{2}}\mathrm{\ (mm}^{\mathrm{2}}\mathrm{)\ {\times}\ length\ (mm)/2}\]

Tumor volumes of control and treatment groups at indicated time points were analyzed using student’s t test (SigmaPlot;SPSS Science, Erkrath, Germany; Fig. 4) or the Mann-Whitney rank-sum test (SigmaStat, SPSS SCIENCE; Fig. 5).

In Vitro Characterization of the Antibody Conjugates

Using the miniperm fermenter for hybridoma growth and FPLC protein G affinity purification, ∼120 mg of mAb 138H11 were purified. Immunohistochemistry, gelelectrophoresis, and HPLC gel chromatography revealed biological activity and a single band or peak, respectively(not shown). Specific binding of purified mAb 138H11 as well as the 138H11-Camθ conjugate to living RCC cells was determined in FACS analysis. All analyzed human RCC cell lines such as Caki-1, Caki-2, and A498 were positive (Fig. 2), whereas controls such as squamous cell carcinoma lines C33-a (Fig. 2)and D36-1, fibroblasts, and the mouse RCC cell line RENCA were all negative.

The 138H11-Camθ conjugate was active with an EC50 of 4.5 × 1011 in the cytotoxicity assay using the Caki-1 cell line. This cytotoxicity was nearly two logs higher than that observed for free or activated Camθ and a mixture of mAb and Camθas shown by a strong left shift of the curve in Fig. 3. The latter control suggests that the increased toxicity of the conjugate is due to a synergistic targeting effect and not just additive. In contrast, on a neuroblastoma cell line (SKN-ML), the cytotoxicity of the 138H11-Camθ conjugate was comparable with unconjugated Camθ (Table 1). Used as a negative control, the chimeric antineuroblastoma antibody ch14.18-Camθ conjugate (20) showed the opposite reaction, demonstrating a strong increase in cytotoxicity on its target cell line SKN-ML but not on RCC (Table 1).

Effect of RCC Treatment with Camθ-antibody Conjugates in Nude Mice

In the first experiment (Fig. 4), the 138H11-Camθ conjugate was applied at a dose corresponding to 20μg Camθ/kg mouse body weight. Free Camθ was given at a dose of 1μg/kg only because of its known high toxicity (20). The mice treated with the conjugate showed a strong reduction in total tumor volume with a shrinkage to minimal residues (Fig. 4). This shrinkage was highly significant in the t test on day 26 compared with the PBS control (P = 0.004). Camθ, mAb 138H11, and a mixture of both also showed some initial tumor reduction, but here the tumors continued growth shortly after the treatment was stopped. The reduction of tumor volume measured during the first days is at least partially due to the fact that the monitoring started 1 day after injection of the tumor cells. Thus, the calculated initial tumor volume includes local swelling and the injected buffer. However, when treatment started, the tumors already represented palpable masses similar to established tumors.

In a second experiment (Fig. 5), the 138H11-Camθ conjugate was applied at a 50% lower dose corresponding to 10 μg Camθ/kg and compared with a group treated with a ch14.18-Camθ conjugate. After an initial tumor shrinkage in all mice similar to that in the first experiment, the tumors of the PBS- and ch14.18-Camθ conjugate-treated mice started to grow strongly. In contrast, in the group of mice treated with the 138H11-Camθ conjugate, the tumors continued to shrink. They did not start regrowing before day 15. At this time point, the 138H11-Camθgroup was highly statistically different from the PBS-treated group using the t test (P = 0.004 on day 14). The 138H11-conjugate group continued to be statistically different in the t test from the ch14.18-conjugate group until the end of the experiment on day 25 (P = 0.024).

However, in the PBS-treated control group, three mice showed an exorbitant tumor growth starting on days 3, 9, and 11, respectively,with a tumor volume of 1598 mm3 on day 26 in the first case. Therefore we plotted the medians of tumor volumes instead of the means in Fig. 5 and used the nonparametric Mann-Whitney rank-sum test for statistical evaluation on day 26. The 138H11-Camθ group was significantly different from both the ch14.18-Camθ-treated(P = 0.011) as well as the PBS-treated(P = 0.035) group. In contrast,ch14.18-Camθ conjugate-treated mice were not significantly different from the PBS control group (P = 0.945).

This clearly shows the significant difference between the 138H11-conjugate and the control antibody-conjugate in the treatment of experimental RCC, confirming the targeting effect due to 138H11 as already observed in vitro (Table 1) and in Fig. 4. Although some tumors in the 138H11 conjugate group started to grow again around days 15 to 20, until this point in time there was a clear reduction in tumor size and a stoppage of tumor growth, in contrast to the controls. In future experiments, the regrowth of tumors may be prevented by differing schedules of treatments, e.g.,another injection of the 138H11-Camθ conjugate at day 15. In conclusion, the second experiment (Fig. 5) strongly supports the results of the first experiment (Fig. 4).

Toxicity of Free and Conjugated Camθ

Toxicity was monitored as a function of change in the individual and total body weight of mice in each treatment group (Fig. 6). Initially, a mouse given 138H11-Camθ doses equivalent to 20μg Camθ/kg every other day to determine the MTD did not show symptoms of toxicity before the sixth injection. However, in the first experiment with four injections of the 138H11-Camθ conjugate at the high dose (20 μg/kg), this treatment group showed an ∼23% weight reduction within 12 days after treatment (Fig. 6a). Five of eight mice died on days 9, 9, 20, 20, and 26, respectively. Comparing these data with the apparent MTD and those published previously (20),the high death rate was unexpected. In the neuroblastoma model, even at a 50% weight loss (30 μg/kg dose) only 2 of 4 mice died, indicating that the 30 μg/kg dose was just beyond the MTD (20). In contrast, at a 10 μg/kg dose, mice with an initial decrease of∼20% in body weight comparable with our experiment started recovery 2 weeks after the last injection. In the neuroblastoma model, the∼20% weight reduction was very well tolerated by the animals(20), encouraging us to continue the first experiment until day 26.

However, one mouse died also at the 20-fold lower dose of free Camθ, although the other mice of this treatment group did not show signs of severe toxicity (Fig. 6a). This indicates that a 1-μg/kg dose of free Camθ is close to the MTD as shown previously(20). At 10 μg/kg of free Camθ, all mice had died in the murine neuroblastoma study (20).

It should be noted also that all other mice, including the PBS-group in experiment 1, showed a weight reduction between days 7 and 11, with rapid recovery (Fig. 6,a). This may be indicative of an unrelated event, such as a general infection, leading to a higher sensitivity for free and conjugated Camθ just during the phase when the treated-mice groups experienced the fastest weight reduction (Fig. 6 a).

This conclusion is supported by the second experiment (10 μg/kg dose), where both conjugate-treated mice groups showed two phases of a small weight reduction of less than 10% between days 0–4 and 6–10,respectively (Fig. 6 b). These weight reductions were very well tolerated and each time immediately followed by a quick recovery. Thus, the 10 μg/kg dose resulted in a much lower toxicity for both the 138H11-Camθ and the ch14.18-Camθ conjugates in the RCC model compared with the study published previously (20).

Conclusion

The effective treatment of metastasized RCC remains one of the major challenges in urological oncology, because RCC has been resistant to all conventional as well as experimental therapeutics thus far. In the current study, we evaluated the cytotoxic potential of a novel chemoimmunoconjugate of mAb 138H11 and Camθ for targeted therapy of RCC in vitro and in vivo.

We demonstrated that the conjugate of 138H11 and Camθ is highly toxic for the Caki-1 RCC cell line in vitro, with an EC50 of 4.5 × 10−11. This is ∼2 logs more effective than that of free Camθ or the ch14.18-Camθ conjugate in the same experimental setting (Table 1), indicating a stable construct on one hand and a specific targeting effect already in vitro. This is in contrast to a previous study (21, 25), where we used a pH degradable 3,3′-dithiobis[sulfosuccinimidyl propionate] (DTSSP) linker for coupling Camθ to mAb 138H11. With this linker, the cytotoxicity in vitro was reduced by approximately 1 log compared with free Camθ.

In vivo, the 138H11-Camθ conjugate was very effective in reducing the tumor size and preventing (Fig. 4) or significantly delaying (Fig. 5) the regrowth of residual tumor cells, in contrast to the controls. That this tumor-inhibitory effect is due to specific targeting by mAb 138H11 to the RCC was confirmed by comparing this conjugate with an antineuroblastoma antibody conjugate, ch14.18-Camθ. The 138H11-Camθ conjugate demonstrated a strong tumor inhibition that was statistically significant from that of ch14.18-Camθ (Fig. 5).

Overall, the 138H11-Camθ conjugate demonstrated it usefulness for targeted therapy of RCC. One problem that needs to be addressed carefully in dose-finding studies is the potential systemic toxicity of Camθ conjugates. The first clinical trial with calicheamicinγ I1 suggested that systemic toxicity can be controlled in humans (29). However, the very high efficiency in killing RCC cells with an EC50 of 4.5 × 10−11 several logs above conventional cytostatics (21) may overcome the multidrug resistance of RCC. In addition, the mAb itself may also stimulate the local immune response, an effect that is important in controlling any surviving tumor cells. As the primary tumor can be easily removed by surgery in most patients, a targeted approach with 138H11-Camθ holds promise for treatment of RCC in a minimal residual disease setting. High local doses of the 138H11-Camθ conjugate may kill small metastases and residual tumor cells.

Fig. 1.

Conjugation of mAb138H11 and Camθ. Camθ is first conjugated with the SPDP linker, and mAb 138H11 is reduced with immunothiolin (top). The final construct is shown below.

Fig. 1.

Conjugation of mAb138H11 and Camθ. Camθ is first conjugated with the SPDP linker, and mAb 138H11 is reduced with immunothiolin (top). The final construct is shown below.

Close modal
Fig. 2.

FACS analysis of mAb 138H11 on RCC cell lines Caki-1,Caki-2, and A498 and as a control on a squamous cell carcinoma line C33a. The thin line represents the background staining with the secondary FITC-labeled antimouse IgG antibody alone.

Fig. 2.

FACS analysis of mAb 138H11 on RCC cell lines Caki-1,Caki-2, and A498 and as a control on a squamous cell carcinoma line C33a. The thin line represents the background staining with the secondary FITC-labeled antimouse IgG antibody alone.

Close modal
Fig. 3.

XTT cell proliferation assay with the mAb 138H11-Camθconjugate and various controls on the RCC cell line Caki-1.

Fig. 3.

XTT cell proliferation assay with the mAb 138H11-Camθconjugate and various controls on the RCC cell line Caki-1.

Close modal
Fig. 4.

First in vivo experiment with RCC xenografts (Caki-1 cells) in nude mice. Shown is the total tumor volume calculated for each treatment group (eight mice) divided by the number of mice. Free Camθ (1) was given at 1 μg/kg, and in the conjugate group (2) the dose corresponded to 20μg/kg Camθ. Treatment started 1 day after tumor injection. Bars, SE. P according to student’s t test.

Fig. 4.

First in vivo experiment with RCC xenografts (Caki-1 cells) in nude mice. Shown is the total tumor volume calculated for each treatment group (eight mice) divided by the number of mice. Free Camθ (1) was given at 1 μg/kg, and in the conjugate group (2) the dose corresponded to 20μg/kg Camθ. Treatment started 1 day after tumor injection. Bars, SE. P according to student’s t test.

Close modal
Fig. 5.

Second in vivo experiment with RCC xenografts (Caki-1 cells) in nude mice. Shown is the median of the tumor volumes calculated for each treatment group (PBS, six mice;conjugates, seven mice each). In both antibody conjugate groups, the dose corresponded to 10 μg/kg Camθ. P according to Mann-Whitney rank-sum test, ∗ indicates significance.

Fig. 5.

Second in vivo experiment with RCC xenografts (Caki-1 cells) in nude mice. Shown is the median of the tumor volumes calculated for each treatment group (PBS, six mice;conjugates, seven mice each). In both antibody conjugate groups, the dose corresponded to 10 μg/kg Camθ. P according to Mann-Whitney rank-sum test, ∗ indicates significance.

Close modal
Fig. 6.

Toxicity as monitored by the total body weight calculated for each treatment group divided by the number of mice. Bars, SE. a, experiment one (see Fig. 4), b, experiment two (see Fig. 5).

Fig. 6.

Toxicity as monitored by the total body weight calculated for each treatment group divided by the number of mice. Bars, SE. a, experiment one (see Fig. 4), b, experiment two (see Fig. 5).

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by a grant of the Dr. Mildred Scheel-Stiftung/Deutsche Krebshilfe to P. F. The study contains data from the dissertation of K. K.

3

RCC, renal cell carcinoma; mAb, monoclonal antibody; γGT, γ-glutamyltransferase; Camθ, Calicheamicinθ I1; HPLC, high-performance liquid chromatography; FACS, fluorescence-activated cell sorting; SPDP, N-succinimidyl 3-[2-pyridylithio]propionate; XTT,2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide;MTD, maximal tolerated dose.

4

Internet address: www.krebsinfo.de.

Table 1

Half-maximal effective dose (EC50) of the 138H11-Camθ-conjugate and various controls on the RCC cell line Caki-1 and the neuroblastoma line SKN-ML as determined from the XTT cell proliferation assays

DrugsCaki-1SKN-ML
EC50EC50
Camθ 9 × 10 m 5 × 10 m 
Activated Camθ 2 × 10 m 1 × 10 m 
ch14.18-Camθ-conjugate 2 × 10 m 8 × 10 m 
Mix of 138H11+ Camθ 3 × 10 m 5 × 10 m 
138H11-Camθ-conjugate 5 × 10 m 2 × 10 m 
DrugsCaki-1SKN-ML
EC50EC50
Camθ 9 × 10 m 5 × 10 m 
Activated Camθ 2 × 10 m 1 × 10 m 
ch14.18-Camθ-conjugate 2 × 10 m 8 × 10 m 
Mix of 138H11+ Camθ 3 × 10 m 5 × 10 m 
138H11-Camθ-conjugate 5 × 10 m 2 × 10 m 

We thank Heike Lerch for excellent technical assistance and Profs. S. A. Loening, D. Schnorr, and M. Dietel, and Drs. O. Kaufmann,H. Lode, and M. Uttenreuther-Fischer for discussion and/or providing histological material.

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