Purpose: The L1 cell adhesion protein is overexpressed in tumors, such as neuroblastomas, renal cell carcinomas, ovarian carcinomas, and endometrial carcinomas, and represents a target for tumor diagnosis and therapy with anti-L1-CAM antibody chCE7. Divalent fragments of this internalizing antibody labeled with 67/64Cu and 177Lu were evaluated to establish a chCE7 antibody fragment for radioimmunotherapy and positron emission tomography imaging, which combines high-yield production with improved clearance and biodistribution properties.

Experimental Design: chCE7F(ab′)2 fragments were produced in high amounts (0.2 g/L) in HEK-293 cells, substituted with the peptide-linked tetraazamacrocycle 3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-triglycyl-l-p-isothiocyanato-phenylalanine, and labeled with 67Cu and 177Lu. In vivo bioevaluation involved measuring kinetics of tumor and tissue uptake in nude mice with SK-N-BE2c xenografts and NanoPET (Oxford Positron Systems, Oxford, United Kingdom) imaging with 64Cu-3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-triglycine-chCE7F(ab′)2.

Results: The 177Lu- and 67Cu-labeled immunoconjugates reached maximal tumor accumulation at 24 hours after injection with similar levels of 12%ID/g to 14%ID/g. Blood levels dropped to 1.0%ID/g for the 177Lu fragment and 2.3%ID/g for the 67Cu fragment at 24 hours. The most striking difference concerned radioactivity present in the kidneys, being 34.5%ID/g for the 177Lu fragment and 16.0%ID/g for the 67Cu fragment at 24 hours. Positron emission tomography imaging allowed clear visualization of s.c. xenografts and peritoneal metastases and a detailed assessment of whole-body tracer distribution.

Conclusions:67/64Cu- and 177Lu-labeled recombinant chCE7F(ab′)2 revealed suitable in vivo characteristics for tumor imaging and therapy but displayed higher kidney uptake than the intact monoclonal antibody. The 67Cu- and 177Lu-labeled immunoconjugates showed different in vivo behavior, with 67/64Cu-3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-triglycine-F(ab′)2 appearing as the more favorable conjugate due to superior tumor/kidney ratios.

At the present time, more than 10 antibody drugs are approved by the Food and Drug Administration for therapy of a variety of human diseases, including cancers (1). A few antibodies have been approved in radiolabeled form for tumor imaging and therapy, and this number may increase as new cancer-associated target antigens are found. The cell surface protein L1-CAM is a target antigen that plays a role in cell proliferation (2) and is emerging as a promising marker for cancers, such as neuroblastoma, renal cell carcinomas (3), and melanomas (4), as well as for ovarian and endometrial carcinomas (5). The chimeric monoclonal antibody chCE7 binds with high affinity to L1-CAM, is internalized into target tumor cells, and has been useful in radioimmunodiagnosis of metastatic neuroblastoma (6). For effective radioimmunotherapy, optimal tumor targeting is necessary to achieve therapeutic levels of the chosen radionuclides at the tumor site. Divalent antibody fragments, such as “minibodies” (7, 8), domain-deleted antibodies (911), or F(ab′)2 fragments (12), are tumor-targeting agents that combine high accumulation at the tumor site comparable with intact antibodies, with more rapid clearance from the blood. However, in contrast to intact antibodies, antibody fragments have not been used frequently for radioimmunotherapy. In some cases, especially with small-sized monovalent fragments, this is due to unfavorable biodistributions with low tumor uptake and accumulation of radioactivity in the kidneys, but there are also disadvantages related to large-scale protein production of fragments. Monovalent and multivalent antibody fragments produced in bacteria are often characterized by poor solubility and tend to form aggregates, complicating their purification and further handling. Production of F(ab′)2 fragments by proteolytic cleavage of intact antibody leads to low yields. Production in mammalian cells avoids such problems, but low-producer cell lines make antibody production time consuming and expensive. We developed a system for producing high amounts of antibody fragments in mammalian cells (13), and in this study, we evaluate engineered F(ab′)2 fragments of anti-L1 monoclonal antibody (mAb) chCE7 with a view to their application in radioimmunotherapy and positron emission tomography (PET) imaging. In contrast to monovalent mAb chCE7 fragments, divalent fragments are internalized into target tumor cells and radioactivity from 67Cu-labeled chCE7F(ab′)2 is retained intracellularly similarly to intact mAb chCE7 (14, 15), indicating the advantage of combining residualizing metallic nuclides with internalizing immunoconjugates (1618). Radiometal-labeled antibodies have advantages over 131I-labeled antibodies in terms of radiation doses delivered to tumors; consequently, 67Cu-labeled (1925) and, more recently, 177Lu-labeled intact antibodies have shown promising results in animal models (18, 26) and in clinical trials (27). The choice of therapeutic nuclides for radioimmunotherapy depends on the size of targeted tumors. Both Monte Carlo simulations (28) and data from actual patient studies (29) show that the high energy of 90Y and its long particle range make it suitable for irradiating large metastases, whereas medium-energy β emitters, such as 67Cu and 177Lu, are better suited for the irradiation of small-sized, disseminated metastases. The main physical difference between 67Cu and 177Lu nuclides, which emit β particles of similar energy, is their half-life of 2.6 and 6.7 days, respectively. A disadvantage of antibody fragments labeled with metallic radionuclides, including radiocopper, consists in the high uptake of radioactivity in the kidneys, due to retention of radiolabeled metabolites (3032). For 177Lu and 67Cu labeling of F(ab′)2 fragments, we selected a peptide-linked chelator, which we had found to reduce kidney uptake of radiocopper-labeled antibody fragments (32). The kinetics of uptake in tumor xenografts and in normal tissues of 67Cu and 177Lu conjugates labeled with the triglycine-linked chelator were compared, and radiation doses to organs, such as kidney and liver, were evaluated. The purpose of this study was to investigate tumor and normal tissue uptake of 67Cu- and 177Lu-labeled recombinant F(ab′)2 fragments of anti-L1-CAM antibody chCE7 in SK-N-BE2c tumor-bearing mice to characterize their potential for treatment of L1-CAM-positive tumors. In addition, ultrahigh resolution PET imaging was done with 64Cu-3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA)-triglycine–labeled F(ab′)2 fragments in two different murine tumor models to evaluate their potential for tumor imaging and to obtain a detailed regional distribution pattern in small structures, such as lymph nodes (33). The data support efforts to initiate clinical investigations with 67Cu- and 64Cu-labeled chCE7 antibody fragments.

Chemicals and solvents used were from Fluka (Buchs, Switzerland) unless stated otherwise. Fast protein liquid chromatography was done on a Pharmacia (Amersham Biosciences, Duebendorf, Switzerland) instrument (Pharmacia/LKB HPLC-pump 2248, Pharmacia/LKB controller LCC-2252 and Pharmacia/LKB UV detector UV-MII). A Berthold (Regensdorf, Switzerland) HPLC-LB 506A online radioactivity detector was used.

Cells and antibodies. HEK-293 cells were from the German Collection of Microorganisms (Braunschweig, Germany), SK-N-BE2c human neuroblastoma cells were from Instituto Nazionale per la Ricerca sul Cancro (Genova, Italy), and SKOV3 human ovarian carcinoma cells were a gift from Prof. P. Altevogt (German Cancer Center, Heidelberg, Germany). HEK-293 and SKOV3 cells were maintained in DMEM (4.5 g/L glucose) and SK-N-BE2c cells in MEM/Ham's F-12 (1:1) and 1% nonessential amino acids. All media were supplemented with 10% FCS, 2 mmol/L glutamine, 100 units/mL penicillin, 100 μg streptomycin, and 0.25 μg/mL fungizone. All media and additives were obtained from BioConcept (Allschwil, Switzerland).

chCE7 fragments. mAb chCE7 is a high-affinity, internalizing chimeric monoclonal antibody of the IgG1 subtype (human κ light chain and human γ1 heavy chain), which recognizes L1-CAM. Intact mAb chCE7 and chCE7 fragments were produced in HEK-293 cells and purified from cell culture supernatants as described (13).

Ligand substitution of monoclonal antibody chCE7 and chCE7F(ab′)2. DOTA-l-p-isothiocyanato-phenylalanine was purchased from Macrocyclics (Dallas, TX). Bifunctional 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetate and triglycine–linked DOTA were synthesized according to published procedures (32). An aqueous solution (60 mmol/L; 7.5-30 μL) of the ligands (DOTA-l-p-isothiocyanato-phenylalanine and DOTA-triglycyl-l-p-isothiocyanato-phenylalanine) was added to 200 to 400 μL of 0.1 mol/L sodium phosphate buffer (pH 8) containing 1 mg (0.01 μmol) of chCE7F(ab′)2. The pH was adjusted using Na3PO4 (saturated solution) to 9 to 10 and the reaction mixture was incubated overnight (16 hours) at 4°C. Excess ligands was then removed by centrifugation-dialysis (Vivascience, Winkel, Switzerland) and buffer exchanged into 0.1 mol/L sodium citrate buffer (pH 5.5) for 67Cu labeling and 0.25 mol/L ammonium acetate (pH 5.5) for 177Lu labeling. The immunoconjugates were concentrated by centrifugation-dialysis to 1 to 2 mg/mL and stored at 4°C. The number of chelators coupled per F(ab′)2 molecule was estimated with an isotope dilution assay as described before (33).

Radiolabeling.67Cu was produced in-house by irradiating natZn with protons at the 72 MeV accelerator of the Paul Scherrer Institute (Villigen, Switzerland) as described before (34) and used for radiolabeling 1 day after production. For the PET imaging experiments, 64Cu was produced at Paul Scherrer Institute as described before (33). 177Lu was from IDB (Petten, the Netherlands) and used for radiolabeling 7 days after production.

Two hundred to 400 μg (250 μL) of the immunoconjugates in a total volume of 500 μL of 0.1 mol/L sodium acetate buffer (pH 5.5) or in 0.25 mol/L ammonium acetate buffer (pH 5.5) were reacted with 18.5 to 30 MBq (500-800 μCi) of neutralized 67Cu solution or with 50 to 80 MBq 177Lu solution. Labeling with 67Cu was done for 30 minutes at 22°C and labeling with 177Lu was done for 20 minutes at 37°C. After incubations, EDTA was added to a final concentration of 5 mmol/L for 5 minutes to complex unchelated copper or lutetium. Purification of the labeled antibodies was achieved by fast protein liquid chromatography size exclusion chromatography on a Superdex 200 column (Amersham Biosciences) in PBS buffer [0.1 mol/L NaCl, 0.05 mol/L sodium phosphate (pH 7.4)] with a flow rate of 1.0 mL/min. The F(ab′)2 peak eluted with a retention time of 13 minutes.

Quality control of radiolabeled preparations. Immunoreactivity of labeled antibody conjugates was measured by cell-binding assays as described (35) and data were analyzed by the Lindmo and Bunn (36) method. Stability of the labeled antibodies after incubation in human plasma at 37°C was analyzed by fast protein liquid chromatography size exclusion chromatography on a Superdex 200 column in PBS buffer.

Animal studies. Animal studies were done in compliance with the Swiss laws on animal protection. Housing and animal husbandry was according to local law on animal protection. Nude mice (CD1-nu) from Charles River, Inc. (Sulzfeld, Germany) were used for the experiments. Growth of SKOV3 tumors was monitored in a pilot experiment by sequential PET imaging with 64Cu-4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl benzoic acid–labeled mAb chCE7 (33). Three nude mice, which had been injected i.p. with 5 × 106 SKOV3 human ovarian carcinoma cells, were imaged once weekly for a period of 3 weeks. Results showed that small metastases could be detected 2 weeks after inoculation and this time was chosen for imaging with 64Cu-chCE7F(ab′)2. S.c. tumors were generated by inoculating s.c. on one flank with 5 × 106 SK-N-BE2c human neuroblastoma cells suspended in 400 μL medium [MEM/Ham's F-12, 10% FCS, 2 mmol/L glutamine, and 1% nonessential amino acids (BioConcept), including 50% Matrigel HC (BD Biosciences, Allschwil, Switzerland)]. After 14 to 20 days when tumors had reached weights between 200 and 500 mg, 67Cu- or 177Lu-labeled immunoconjugates [100 μL, corresponding to 185 kBq (5 μCi), 5-10 μg F(ab′)2] were injected i.v. into groups of four mice. At the indicated time points, animals were sacrificed and dissected. Tumors and organs were removed and measured together with an aliquot of the injected solution in a gamma counter using an energy window between 160 and 210 keV for 67Cu and 15 to 600 keV for 177Lu. Results are expressed as % injected dose per gram of tissue (%ID/g). Statistical analysis of data was done with Student's t test (two-tailed, unequal variance).

Positron emission tomography imaging. Radiocopper production by proton irradiation of natZn was modified to produce a larger amount of 64Cu by changing the irradiation protocol as described (33). For the PET experiment, tumor mice were imaged using the dedicated small animal PET tomograph Nano-PET (Oxford Positron Systems, Oxford, United Kingdom) based on the Hierarchical Design and Characterization technology (36). The awake animals were lightly restrained and injected with 10 MBq (267 μCi, 130 μg) 64Cu-DOTA-triglycine-F(ab′)2 via a lateral tail vein. Twenty-one to 24 hours later, the animals were anesthetized and scanned as described before (33). PET data were acquired in list mode for 60 to 90 minutes and reconstructed in a single time frame using the OPL-EM algorithm (0.5 mm bin size, 200 × 240 × 240 matrix size; ref. 37). Image files were analyzed using the dedicated software Pmod (38).

Dosimetry. Dose calculations were done with the OLINDA software (39) using organ ratios human/mouse (g/g) of 901 for the kidney and 2,134 for the liver and assuming species-independent pharmacokinetics. The integral of the time activity curve was determined from the pharmacokinetic data in Tables 3 and 4. Data were extrapolated from 72 to 500 hours either assuming only the physical decay (Tphys) of the radionuclides or estimating a biological half-life (Tbiol) based on the last three time points. The effective half-life was then calculated 1 / Teff = 1 / Tphys + 1 / Tbiol.

Labeling of recombinant chCE7 fragments with 177Lu and 67Cu.Table 1 shows the physical characteristics of 67Cu and 177Lu (Table 1) and compares some features of labeling mAbs with these nuclides (Table 2). The amount of carrier lutetium or copper present in the isotope preparations influences the specific activity that can be achieved by labeling the immunoconjugates. At the present time, the specific activity of 177Lu is higher than that of 67Cu. In our study, the specific activity of the 177Lu solution was 405 MBq/μg Lu at the time of antibody labeling (1 week after production). At this time, it was ∼4-fold higher compared with the 67Cu solution (112.5 MBq/μg Cu) at the time of antibody labeling, 24 hours after production). Carboxylated tetraazamacrocycles can be used for labeling with either Cu or Lu, as they chelate both metals rapidly under mild conditions and the resulting metal complexes show high in vivo stability (40). A difference of the resulting copper and lutetium immunoconjugates consists in the different charge of the metal complexes (negative for Cu, neutral for Lu) attached to mAb.

Table 1.

Physical properties of 67Cu and 177Lu

67Cu177Lu
β energies, keV   
    Maximal 577 498 
    Mean 141 133 
γ energies, keV (%) 93 (16) 113 (6) 
 185 (48) 208 (10) 
Half-life (d) 2.6 6.7 
67Cu177Lu
β energies, keV   
    Maximal 577 498 
    Mean 141 133 
γ energies, keV (%) 93 (16) 113 (6) 
 185 (48) 208 (10) 
Half-life (d) 2.6 6.7 
Table 2.

Some features of radiolabeling antibodies with 67Cu and 177Lu

67Cu177Lu
Nuclide availability Limited Good 
Labeling procedures Postlabeling, 30 min, 22°C Postlabeling, 20 min, 37°C 
Bifunctional chelators CPTA-NHS, TETA (BAT) DOTA-NCS 
 DO3A-NCS DOTA-triglycerine-NCS 
 DOTA-triglycerine-NCS PA-DOTA-NCS 
Labeling yields (%) 30-70 30-70 
Specific activities of mAbs (MBq/mg) 37.5-112.5 37.5-187.5 
67Cu177Lu
Nuclide availability Limited Good 
Labeling procedures Postlabeling, 30 min, 22°C Postlabeling, 20 min, 37°C 
Bifunctional chelators CPTA-NHS, TETA (BAT) DOTA-NCS 
 DO3A-NCS DOTA-triglycerine-NCS 
 DOTA-triglycerine-NCS PA-DOTA-NCS 
Labeling yields (%) 30-70 30-70 
Specific activities of mAbs (MBq/mg) 37.5-112.5 37.5-187.5 

Abbreviations: CPTA, 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl benzoic acid; TETA, 6-(p-nitrobenzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid; DOTA-NCS, DOTA-L-p-isothiocyanato-phenylalanine; DO3A-NCS, DO3A-L-p-isothiocyanato-phenylalanine; DOTA-triglycerine-NCS, DOTA-triglycyl-L-p-isothiocyanato-phenylalanine; PA-DOTA, 1-(1 carboxy-3-(p-nitrophenyl)prophyl)-1,4,7,10-tetraazacylclododecane-4,7,10-triacetate.

mAbs chCE7 and chCE7F(ab′)2 were produced in high yields (0.1-0.2 g/L) in HEK-293 cells. Recombinant F(ab′)2 fragments were engineered as histidine-tagged proteins and purified by a two-step procedure using Ni-affinity chromatography followed by size exclusion chromatography. Yields after purification were ∼50% because of the coexpression of monovalent fragments (13). The DOTA- and triglycine-linked DOTA chelators were coupled to antibodies as isothiocyanato-derivatives (Fig. 1). Substitution of antibody fragments with the chelators and conditions for radiolabeling were similar for the two nuclides. Labeling yields of the 177Lu-DOTA-F(ab′)2 ranged between 40% and 70% and labeling yields of the DOTA-triglycine–linked F(ab′)2 were consistently lower (6-30%) with both 67Cu and 177Lu. When the molar excess of DOTA-triglycyl-l-p-isothiocyanato-phenylalanine for conjugation was increased, the resulting conjugates did not show increased labeling yields; therefore, a molar excess of 45 was employed. About two DOTA-triglycine chelators were found to be attached per F(ab′)2 using these conditions, which resulted in conjugates with good biodistributions. After labeling, a final concentration of 5 mmol/L EDTA was added and the conjugates were purified by fast protein liquid chromatography size exclusion chromatography to remove unbound radioactivity as well as small fragments. Immunoreactivity of 67Cu- or 177Lu-labeled fragments, as determined by cell-binding assays, was between 80% and 100%. Stability in human plasma at 37°C was analyzed by fast protein liquid chromatography size exclusion chromatography. Labeled fragments were stable in human plasma at 37°C for at least 48 hours, and no evidence of aggregation, fragmentation, or release of radioactivity was found (data not shown).

Fig. 1.

Chelators used for 177Lu and 67Cu labeling of antibody fragments. A, DOTA-l-p-isothiocyanato-phenylalanine; B, DOTA-triglycyl-l-p-isothiocyanato-phenylalanine.

Fig. 1.

Chelators used for 177Lu and 67Cu labeling of antibody fragments. A, DOTA-l-p-isothiocyanato-phenylalanine; B, DOTA-triglycyl-l-p-isothiocyanato-phenylalanine.

Close modal

Effect of different chelators on biodistributions of 177Lu-labeled mAb chCE7 and its F(ab′)2 fragments in SK-N-BE2c tumor-bearing nude mice. chCE7 fragments were substituted using different amounts of the DOTA chelate to determine optimal labeling conditions. When the molar excess of DOTA/F(ab′)2 was increased from 45 to 180, the number of chelators attached per F(ab′)2 increased from 3 to 5. The immunoreactivity as measured by a cell-binding assay was found to be fully retained; however, biodistributions were strongly affected. Figure 2 shows biodistributions of 177Lu-DOTA-chCE7F(ab′)2 substituted with three and five chelators 24 hours after injection. The conjugate with the higher substitution ratio was characterized by drastically lower levels of radioactivity in the kidneys, high liver uptake, and low tumor uptake. In contrast, the conjugate with the lower substitution ratio shows higher tumor uptake and lower levels of activity in the liver. This conjugate is characterized by ∼3-fold higher levels of radioactivity in the kidneys than in the tumor. When the DOTA-triglycine chelate we had employed previously for radiocopper labeling of antibody fragments was used for 177Lu labeling, about two chelators were attached per F(ab′)2. In contrast to previous results with 67Cu-DOTA-triglycine-F(ab′)2, no significant difference in radioactivity present in the kidneys was found 24 hours after injection with the 177Lu-DOTA-triglycine conjugate (35.9 ± 8.45%ID/g compared with 34.48 ± 8.85%ID/g).

Fig. 2.

Biodistributions 24 hours after injection of different 177Lu-chCE7F(ab′)2 conjugates (1.5-3.0 MBq, 13 μg) in tumor-bearing nude mice: effect of the number of chelators coupled to F(ab′)2. White columns, 177Lu-DOTA-chCE7F(ab′)2 [five chelators/F(ab′)2]; black columns, 177Lu-DOTA-chCE7F(ab′)2 [three chelators/F(ab′)2]; striped columns, 177Lu-DOTA-triglycine-chCE7F(ab′)2 [two chelators/F(ab′)2]. Data are %ID/g ± SD (n = 3) for the DOTA-conjugates (n = 8) for the DOTA-triglycine conjugate.

Fig. 2.

Biodistributions 24 hours after injection of different 177Lu-chCE7F(ab′)2 conjugates (1.5-3.0 MBq, 13 μg) in tumor-bearing nude mice: effect of the number of chelators coupled to F(ab′)2. White columns, 177Lu-DOTA-chCE7F(ab′)2 [five chelators/F(ab′)2]; black columns, 177Lu-DOTA-chCE7F(ab′)2 [three chelators/F(ab′)2]; striped columns, 177Lu-DOTA-triglycine-chCE7F(ab′)2 [two chelators/F(ab′)2]. Data are %ID/g ± SD (n = 3) for the DOTA-conjugates (n = 8) for the DOTA-triglycine conjugate.

Close modal

Comparative biodistribution of 177Lu-DOTA-triglycine-F(ab′)2 and 67Cu-DOTA-triglycine-F(ab′)2 in SK-N-BE2c tumor-bearing nude mice. The DOTA-triglycine–linked F(ab′)2 conjugates were compared in 67Cu- and 177Lu-labeled form. Similar amounts (7.5-10 μg) of the conjugates were injected in nude mice with human neuroblastoma (SK-N-BE2c) xenografts and biodistributions were measured starting 2 hours after injection up to 72 hours. The results are summarized in Table 3 (177Lu) and Table 4 (67Cu). Both conjugates reached maximal tumor accumulation with similar levels of 12%ID/g to 14%ID/g at 24 hours after injection; differences were not statistically significant (P = 0.869). At 24 hours after injection, both conjugates showed ∼3%ID/g uptake in control tumors (PC3 human prostate carcinoma cells), which do not express L1-CAM (data not shown). Figure 3 shows clearance of 67Cu- and 177Lu-labeled F(ab′)2 from tumor and blood (Fig. 3A) and from the kidneys (Fig. 3B). Blood levels of the two conjugates dropped with a similar rate. Activity present in the blood reached at 24 hours 1.00 ± 0.79%ID/g for the 177Lu conjugate and 2.30 ± 0.43%ID/g for the 67Cu conjugate, the difference being significant (P = 0.001). The most conspicuous difference of the two conjugates was the clearance of radioactivity from the kidneys. Up to 8 hours, similar activities were observed in the kidneys, but at 24 hours and later 67Cu-F(ab′)2 showed about half as much activity in the kidneys compared with 177Lu-F(ab′)2 (significance levels at 24 hours: P = 0.00049, 48 hours: P = 0.0153, and 72 hours: P = 0.0083). Tumor/kidney ratios at 24 hours were 0.76 ± 0.22 for 67Cu-F(ab′)2 and 0.38 ± 0.15 for the 177Lu-F(ab′)2 (significant difference, P = 0.0005). Radioactivity in the liver was higher with the 67Cu-F(ab′)2 than with the 177Lu-F(ab′)2 at all of the time points. At 24 hours, differences were not significant (P = 0.243) but became significant at the 72-hour end point (P = 0.0311).

Table 3.

Biodistributions of 177Lu-DOTA-triglycine-chCE7F(ab′)2 in nude mice with human neuroblastoma (SK-N-BE2c) tumor xenografts

Tissue2 h4 h8 h24 h48 h72 h
Tumor 7.81 ± 1.86 9.54 ± 1.86 12.94 ± 3.67 14.43 ± 5.6 9.47 ± 1.70 7.56 ± 1.96 
Blood 22.49 ± 1.10 10.89 ± 1.87 6.8 ± 0.96 1.00 ± 0.79 0.19 ± 0.06 0.28 ± 0.32 
Heart 8.02 ± 1.88 4.57 ± 0.64 4.36 ± 0.9 2.71 ± 0.68 2.07 ± 0.64 1.57 ± 0.42 
Spleen 6.79 ± 1.40 2.44 ± 0.52 4.91 ± 2.00 4.65 ± 3.15 5.20 ± 1.34 3.88 ± 2.02 
Kidney 19.41 ± 1.75 15.05 ± 1.68 27.52 ± 5.95 34.48 ± 8.85 23.08 ± 8.04 17.22 ± 3.06 
Stomach 1.02 ± 0.14 0.92 ± 0.43 1.16 ± 0.25 0.55 ± 0.25 0.47 ± 0.32 0.36 ± 0.11 
Intestine 2.36 ± 0.24 1.78 ± 0.89 2.14 ± 0.32 1.24 ± 0.33 0.90 ± 0.22 0.73 ± 0.29 
Liver 7.55 ± 0.60 5.89 ± 1.14 8.18 ± 2.15 8.70 ± 4.26 7.17 ± 1.47 5.44 ± 1.15 
Muscle 1.64 ± 0.76 1.10 ± 0.37 1.75 ± 0.58 1.39 ± 0.56 2.12 ± 0.53 1.73 ± 0.81 
Tissue2 h4 h8 h24 h48 h72 h
Tumor 7.81 ± 1.86 9.54 ± 1.86 12.94 ± 3.67 14.43 ± 5.6 9.47 ± 1.70 7.56 ± 1.96 
Blood 22.49 ± 1.10 10.89 ± 1.87 6.8 ± 0.96 1.00 ± 0.79 0.19 ± 0.06 0.28 ± 0.32 
Heart 8.02 ± 1.88 4.57 ± 0.64 4.36 ± 0.9 2.71 ± 0.68 2.07 ± 0.64 1.57 ± 0.42 
Spleen 6.79 ± 1.40 2.44 ± 0.52 4.91 ± 2.00 4.65 ± 3.15 5.20 ± 1.34 3.88 ± 2.02 
Kidney 19.41 ± 1.75 15.05 ± 1.68 27.52 ± 5.95 34.48 ± 8.85 23.08 ± 8.04 17.22 ± 3.06 
Stomach 1.02 ± 0.14 0.92 ± 0.43 1.16 ± 0.25 0.55 ± 0.25 0.47 ± 0.32 0.36 ± 0.11 
Intestine 2.36 ± 0.24 1.78 ± 0.89 2.14 ± 0.32 1.24 ± 0.33 0.90 ± 0.22 0.73 ± 0.29 
Liver 7.55 ± 0.60 5.89 ± 1.14 8.18 ± 2.15 8.70 ± 4.26 7.17 ± 1.47 5.44 ± 1.15 
Muscle 1.64 ± 0.76 1.10 ± 0.37 1.75 ± 0.58 1.39 ± 0.56 2.12 ± 0.53 1.73 ± 0.81 

NOTE: Groups of four mice were injected with 124 kBq (7.5 μg) 177Lu-DOTA-triglycine-chCE7F(ab′)2, and radioactivity in tumor and normal tissues was measured at the indicated time points. Data are presented as %ID/g ± SD (n = 4) and %ID/g ± SD (n = 8) for the 24-hour point.

Table 4.

Biodistributions of 67Cu-DOTA-triglycine-chCE7F(ab′)2 in nude mice with human neuroblastoma (SK-N-BE2c) tumor xenografts

Tissue2 h4 h8 h24 h48 h72 h
Tumor 6.26 ± 0.87 6.43 ± 1.79 12.53 ± 2.39 11.92 ± 4.77 7.04 ± 2.02 5.31 ± 0.56 
Blood 23.20 ± 2.48 12.86 ± 0.45 7.82 ± 0.89 2.30 ± 0.43 2.82 ± 0.18 1.93 ± 0.79 
Heart 9.53 ± 0.56 7.42 ± 0.25 5.25 ± 0.65 2.92 ± 0.71 3.66 ± 0.39 4.09 ± 0.52 
Spleen 5.07 ± 0.88 6.65 ± 0.61 6.26 ± 1.80 5.22 ± 1.73 4.21 ± 0.31 3.78 ± 0.84 
Kidney 22.54 ± 2.73 28.10 ± 2.89 25.53 ± 5.51 16.00 ± 5.13 11.11 ± 2.27 8.75 ± 0.85 
Stomach 1.11 ± 0.39 1.97 ± 0.39 2.18 ± 0.36 1.91 ± 1.17 1.57 ± 0.95 1.20 ± 0.04 
Intestine 3.88 ± 0.18 5.14 ± 0.37 4.22 ± 0.62 3.56 ± 1.48 3.34 ± 0.47 5.8 ± 5.1 
Liver 12.90 ± 4.96 16.79 ± 2.79 15.26 ± 5.56 11.12 ± 3.22 11.57 ± 1.87 9.90 ± 1.83 
Muscle 1.62 ± 0.22 1.79 ± 0.04 1.02 ± 0.32 0.91 ± 0.23 0.51 ± 0.35 0.84 ± 0.07 
Tissue2 h4 h8 h24 h48 h72 h
Tumor 6.26 ± 0.87 6.43 ± 1.79 12.53 ± 2.39 11.92 ± 4.77 7.04 ± 2.02 5.31 ± 0.56 
Blood 23.20 ± 2.48 12.86 ± 0.45 7.82 ± 0.89 2.30 ± 0.43 2.82 ± 0.18 1.93 ± 0.79 
Heart 9.53 ± 0.56 7.42 ± 0.25 5.25 ± 0.65 2.92 ± 0.71 3.66 ± 0.39 4.09 ± 0.52 
Spleen 5.07 ± 0.88 6.65 ± 0.61 6.26 ± 1.80 5.22 ± 1.73 4.21 ± 0.31 3.78 ± 0.84 
Kidney 22.54 ± 2.73 28.10 ± 2.89 25.53 ± 5.51 16.00 ± 5.13 11.11 ± 2.27 8.75 ± 0.85 
Stomach 1.11 ± 0.39 1.97 ± 0.39 2.18 ± 0.36 1.91 ± 1.17 1.57 ± 0.95 1.20 ± 0.04 
Intestine 3.88 ± 0.18 5.14 ± 0.37 4.22 ± 0.62 3.56 ± 1.48 3.34 ± 0.47 5.8 ± 5.1 
Liver 12.90 ± 4.96 16.79 ± 2.79 15.26 ± 5.56 11.12 ± 3.22 11.57 ± 1.87 9.90 ± 1.83 
Muscle 1.62 ± 0.22 1.79 ± 0.04 1.02 ± 0.32 0.91 ± 0.23 0.51 ± 0.35 0.84 ± 0.07 

NOTE: Groups of four mice were injected with 345 kBq (10 μg) 67Cu-DOTA-triglycine-chCE7F(ab′)2, and radioactivity in tumor and normal tissues was measured at the indicated time points. Data are presented as %ID/g ± SD (n = 4) and %ID/g ± SD (n = 9) for the 24-hour point.

Fig. 3.

Clearance of 177Lu-DOTA-triglycine-F(ab′)2 and 67Cu-DOTA-triglycine-F(ab′)2 from tumor, blood, and the kidneys. Corresponding numerical values are indicated in Tables 3 and 4. A, •, 67Cu-F(ab′)2 blood; ♦, 67Cu-F(ab′)2 tumor; ▵, 177Lu-F(ab′)2 blood; □, 177Lu-F(ab′)2 tumor. B, •, 67Cu-F(ab′)2 kidneys; ▵, 177Lu-F(ab′)2 kidneys.

Fig. 3.

Clearance of 177Lu-DOTA-triglycine-F(ab′)2 and 67Cu-DOTA-triglycine-F(ab′)2 from tumor, blood, and the kidneys. Corresponding numerical values are indicated in Tables 3 and 4. A, •, 67Cu-F(ab′)2 blood; ♦, 67Cu-F(ab′)2 tumor; ▵, 177Lu-F(ab′)2 blood; □, 177Lu-F(ab′)2 tumor. B, •, 67Cu-F(ab′)2 kidneys; ▵, 177Lu-F(ab′)2 kidneys.

Close modal

These results show that the biological behavior of the two conjugates, which were labeled with different metals via the same chelator, is different, especially regarding clearance of radioactivity from the kidneys.

Positron emission tomography imaging. PET imaging with intact 64Cu-labeled chCE7 antibody fragments permitted excellent visualization of two different L1-CAM-expressing tumor models. Both s.c. SK-N-BE2c tumor xenografts and peritoneal metastases from human ovarian carcinoma SKOV3 cells were visualized with high resolution 21 hours after i.v. injection of 15 MBq 64Cu-DOTA-triglycine-chCE7F(ab′)2 (Fig. 4). Images showed low background, thus corresponding to the low blood activity levels and high tumor/blood ratios obtained in the biodistribution experiments (Table 4; Fig. 3). PET imaging with 64Cu-DOTA-triglycine-chCE7F(ab′)2 also served to investigate regional distribution of radioactivity in tumor and normal tissues with greater detail compared with classic biodistribution studies. In particular, previous PET imaging studies with the intact 64Cu-chCE7 antibody revealed high accumulation of radioactivity in lymph nodes (33). In contrast, PET images of 64Cu-labeled antibody fragments showed a lack of radioactivity uptake in lymph nodes. As expected from the biodistribution studies, high activity concentrations were found in the kidneys, the ultrahigh resolution of the Nano-PET camera showing inhomogeneous kidney uptake localized in the kidney cortex (Fig. 4B). Prominent uptake of radioactivity was also apparent in the liver. Regions of interest analysis of PET images resulted in tumor/kidney ratios of 1.2 and tumor/liver ratios of 0.9, being in line with the values determined in the biodistribution studies (Table 4). Furthermore, high-resolution PET imaging of the SK-N-BE2c tumor mass allowed the delineation of heterogeneous distribution of radioactivity within the tumor. Figure 4C shows a series of coronal sections through the ovoid tumor, suggesting high vascularization and tracer perfusion even in the inner portions of the tumor. The images also suggest two areas within the tumor (arrows) with very low activity concentrations, probably representing necrotic tumor regions. Regions of interest analysis of these tumor subregions showed a 2.5-fold reduced tracer uptake in these putatively necrotic regions compared with average activity concentration in the residual tumor volume.

Fig. 4.

PET imaging of tumor-bearing mice 21 hours after injection of 64Cu-DOTA-triglycine-chCE7F(ab′)2. A, comparative PET imaging of a mouse with a small peritoneal SKOV metastasis (arrow) injected with 20 MBq (100 μg) 64Cu-chCE7 and 7 days later with 10 MBq (130 μg) 64Cu-chCE7F(ab′)2. Both whole body images show the same coronal plane through the tumor (slice thickness, 0.5 mm). B, PET imaging of a mouse with a SK-N-BE2c tumor (right) injected with 10 MBq (130 μg) 64Cu-chCE7F(ab′)2. The series of coronal whole body images show representative planes through the liver, kidneys, and tumor. The image to the right is the projection image. C, series of coronal slices with a more detailed view of tracer distribution within the tumor in B (arrows depicting putatively necrotic subregions).

Fig. 4.

PET imaging of tumor-bearing mice 21 hours after injection of 64Cu-DOTA-triglycine-chCE7F(ab′)2. A, comparative PET imaging of a mouse with a small peritoneal SKOV metastasis (arrow) injected with 20 MBq (100 μg) 64Cu-chCE7 and 7 days later with 10 MBq (130 μg) 64Cu-chCE7F(ab′)2. Both whole body images show the same coronal plane through the tumor (slice thickness, 0.5 mm). B, PET imaging of a mouse with a SK-N-BE2c tumor (right) injected with 10 MBq (130 μg) 64Cu-chCE7F(ab′)2. The series of coronal whole body images show representative planes through the liver, kidneys, and tumor. The image to the right is the projection image. C, series of coronal slices with a more detailed view of tracer distribution within the tumor in B (arrows depicting putatively necrotic subregions).

Close modal

Dosimetry.177Lu-labeled chCE7F(ab′)2 showed 2-fold higher radioactivity levels in the kidney and similar uptake of radioactivity in the liver compared with 67Cu-chCE7F(ab′)2 (Tables 3 and 4; Fig. 3). It was of interest to evaluate the effect of the different half-lives of the two nuclides on doses delivered to the kidneys and the liver. Dose calculations were done with pharmacokinetic data from Tables 3 and 4 using the OLINDA software (39). When taking the biological half-life of the conjugates into account, the calculated radiation burden of the (mouse) kidneys was 960 mGy/MBq for 67Cu-F(ab)2 and 2,000 mGy/MBq for 177Lu-F(ab)2. Doses for the liver were 850 and 690 mGy/MBq, respectively. The tumor was assumed to be a sphere of 0.5 g for which doses of 860 mGy/MBq 67Cu and 1,050 mGy/MBq 177Lu were obtained. If only the physical half-life of the radionuclides was considered for the time span 72 to 500 hours, a 20% higher dose was obtained for the 67Cu conjugate, whereas for the corresponding 177Lu compound the increase was 70%. The results show that due to the similar, rapid clearance of the 67Cu and 177Lu conjugates, the delivered dose is not significantly influenced by the longer physical half-life of 177Lu.

Clinical studies with the high-energy β particle–emitting nuclide 90Y linked to anti-CD20 antibody ibritumomab showed that radioimmunotherapy with this radiopharmaceutical (“Zevalin”) is a safe and effective treatment for patients with relapsed or refractory non-Hodgkin's lymphoma (40). For treatment of small metastases, medium-energy β particle emitters, such as 177Lu or 67Cu, may be more favorable due to their shorter effective path length and consequent sparing of normal tissues. Data from first patient studies with 177Lu-labeled Rituximab (27) and preclinical studies with 177Lu-labeled peptides (41) support this hypothesis. A further step toward optimizing the treatment of small metastases could be the introduction of antibody fragments labeled with 177Lu or 67Cu. To this purpose, we have engineered divalent fragments of the internalizing anti-L1-CAM antibody chCE7 [F(ab′)2 110 kDa]. A comparison between 67Cu- and 177Lu-labeled fragments was done, because development of radiolabeled antibody fragments has to take into account availability of the chosen radionuclides in addition to good protein production yields and convenience of labeling protocols. Now, 177Lu is more widely available than 67Cu and has been successfully used for labeling of antibody fragments (9, 42) and peptides (29). In this study, almost identical procedures were used for radiolabeling with the two nuclides and fully immunoreactive fragments with similar specific activities were obtained. Maximal activity levels in tumors reached 12%ID/g to 14%ID/g at 24 hours for the 67Cu- and 177Lu-labeled antibody fragments. The kinetics of tumor uptake and clearance of radioactivity from the blood (Fig. 3) were slower than those observed for divalent diabodies (55 kDa). Pharmacokinetics were comparable with single-chain constructs engineered to dimerize via constant domain sequences, such as single-chain Fv-CH3 T84.66 (80 kDa; ref. 43) or single-chain Fv-CH4 L19 SIP (80 kDa; ref. 10) and also similar to CH2 domain–deleted antibodies CC49δCH2 (120 kDa; ref. 44). The tumor/blood ratios, which were reached at 24 hours, were 14.4 for the 177Lu compound and 5.2 for the 67Cu compound, which is lower than in the case of single-chain Fv–derived constructs labeled with radiometals.

Recombinant 67Cu-F(ab′)2 and 177Lu-F(ab′)2 conjugates were stable for at least 48 hours in human plasma, without evidence of degradation or aggregation (data not shown), indicating that the lower tumor/blood ratios are not due to circulating aggregates or metabolites. The clear PET images of both a large s.c. tumor and small-sized peritoneal metastases with 64Cu-DOTA-triglycine-chCE7F(ab′)2 (Fig. 4) indicate that tumor/blood ratios are sufficient for good imaging. Figure 4C shows heterogeneous uptake of radioactivity in the tumor xenograft and illustrates the value of PET imaging for exact dosimetry as opposed to measuring uptake values from overall tumor (or other organs, such as the kidney). The possibility to perform PET imaging may well represent an additional advantage of the radiocopper immunoconjugate as opposed to the lutetium conjugate.

Nontarget tissue uptake of radiometal-labeled immunoconjugates depends on the size of the constructs and the clearance properties of the labeled metabolites, which usually consist of the metal complexes linked to amino acid residues used for ligand coupling. It seems that single-chain constructs, such as diabodies, which are devoid of constant region sequences, show generally excessively high levels of radioactivity in the kidneys, similar to radiometal-labeled peptides. In contrast, some of the larger-sized immunoconjugates, which were tested in radiometal-labeled forms, showed better tumor/kidney ratios and sometimes considerable uptake of radioactivity in the liver and the spleen. In radiocopper-labeled F(ab′)2 fragments, such as 64Cu-CPTA-1A3F(ab′)2 (31) and 67Cu-CPTA-chCE7F(ab′)2 (35), it was found that fragments labeled with positively charged copper complexes showed higher kidney uptake than fragments labeled with negatively charged copper complexes. A similar effect of charge on kidney uptake was observed in an octreotate conjugate labeled with 64Cu using a cross-bridged macrocyclic chelator (45). For comparing 67Cu and 177Lu fragments, a DOTA-triglycine–linked chelator, which has been found previously to reduce kidney uptake of 67Cu-F(ab′)2 (32), was used and conjugation conditions were optimized. The main difference found between the conjugates consisted in 2-fold increased tumor/kidney ratios of the 67Cu-DOTA-triglycine-F(ab′)2. The negative charge of the Cu-DOTA complex may be responsible for reduced kidney uptake of the radiocopper-labeled conjugate as opposed to the neutral charge of the lutetium complex in the 177Lu-labeled fragment. Apart from charge effects, our results show that biodistributions are also strongly influenced by the number of ligands attached to antibody fragments as indicated by the data shown in Fig. 2, where protein substitution with increased molar excess of the DOTA ligand lead to 177Lu immunoconjugates exhibiting loss of tumor uptake, a 7-fold reduction in kidney accumulation, and a 3.5-fold increase of liver uptake. Compared with an 111In-DOTA-labeled T84.66 minibody with excellent tumor-targeting properties (43), liver uptake is lower with both 67Cu- and 177Lu-labeled chCE7F(ab′)2. The strong uptake of radioactivity in lymph nodes we observed with 64Cu-labeled chCE7 antibody (33) may be due to accumulation of radiolabeled metabolites in the reticuloendothelial system of the lymph nodes or to binding of the Fc region to activated lymphocytes and is not observed with 64Cu-labeled chCE7 antibody fragments. We conclude that, with the exception of tumor/blood ratios, the tumor/tissue levels of the 67Cu conjugate seem more favorable than those of the 177Lu compound. Recently, we increased 67Cu production to 15 GBq to provide therapeutic doses of 67Cu-labeled intact antibodies. The results of this study suggest that the development of radiocopper-labeled antibody fragments is worthwhile both for PET imaging in 64Cu-labeled form and for therapy in 67Cu-labeled form.

Grant support: Swiss National Science Foundation grant 3100A0-100200.

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.

We thank Susan Cohrs, Christine DePasquale, and Yvonne Eichholzer for excellent technical assistance.

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