Targeting of indium 111-labeled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: imaging of tumors hosted in nude mice

Antibody conjugates were prepared by coupling F(ab')2 or Fab' fragments of an antibody specific for the human high molecular weight-melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the antibody conjugate mediated binding of the 111In-labeled haptens to melanoma cells. In vivo, it allowed specific localization of the haptens in A375 tumors. The bivalent hapten exhibited much higher efficiency at targeting 111In onto cells, both in vitro and in vivo. Antibody conjugate and hapten doses (2 micrograms and 1 pmol, respectively) and the delay between antibody conjugate and tracer injections (24 h) were adjusted to maximize tumor uptake (4% injected dose/g) and tumor to normal tissue contrast (greater than 3) obtained 3 h after injection of the 111In-labeled bivalent hapten. This two-step technique, when compared to direct targeting of 111In-labeled F(ab')2 fragments, provided lower localization of injected activity into the tumor (x 0.25), but higher tumor/tissue ratios, especially with respect to liver (x 7), spleen (x 8), and kidneys (x 10). In addition, high contrast images were obtained within 3 hours, instead of days. Thus, antibody conjugate-mediated targeting of small bivalent haptens, labeled with short half-life isotopes, is proposed as a general method for improving tumor radioimmunolocalization.     

Tumor targeting properties of indium-111 labeled genetically engineered Fab' and F(ab')2 constructs of chimeric tumor necrosis treatment (chTNT)-3 antibody

Genetic engineering techniques have allowed the construction of Fab' and F(ab')2 constructs of chimeric tumor necrosis treatment antibody (chTNT-3), a chimeric monoclonal antibody (MAb) that targets necrotic regions of solid tumors. The purpose of this study is to evaluate the in vitro and in vivo properties of Fab' and F(ab')2 constructs radiolabeled with indium-111 (111In) using diethylentriamine pentaacetic acid (DTPA) conjugation to develop a clinically useful imaging agent for the detection of necrosis in solid tumors. Optimization of the MAb-to-DTPA ratio showed that a 1:2 ratio gave the best immunoreactivity while providing good radiolabeling efficiency and high specific activity for all three DPTA conjugates. In addition, 111In-labeled Fab' and F(ab')2 conjugates were found to have faster whole body clearance times and better biodistribution profiles compared to parental 111In-labeled chTNT-3 in tumor-bearing mice. Although radiolabeled Fab' and F(ab')2 constructs showed lower tumor uptake than radiolabeled chTNT-3, biodistribution results showed that these constructs had significantly lower uptake in liver, spleen, and other normal organs (except the kidney), and therefore had higher tumor-to-organ ratios. In addition, a comparison of all derivatives showed that the F(ab')2 reagent gave the best results in tumor imaging studies. These results demonstrate that stable, a genetically engineered F(ab')2 construct can be successfully radiolabeled with 111In to produce potential imaging reagents for the imaging and monitoring of tumor necrosis.     

Enhanced delivery of a monoclonal antibody F(ab')2 fragment to subcutaneous human glioma xenografts using local hyperthermia

The purpose of this study was to investigate the effects of tumor-localized hyperthermia at 42 degrees C on the tissue distribution of radioiodinated monoclonal antibody F(ab')2 fragments. Paired-label biodistribution measurements were performed in athymic mice bearing D-54 MG human glioma xenografts on one leg. Mice received both the 131I-labeled F(ab')2 fragment of Mel-14, reactive with human gliomas and melanomas, and nonspecific 125I-labeled RPC 5 F(ab')2. Tumor-bearing legs were placed in a 42 degrees C water bath or a 37 degrees C water bath (control) for 2 or 4 h. In mice sacrificed immediately after 2 h of heating, no hyperthermia-induced differences in the distribution of either fragment were observed. In the 4-h groups, tumor uptake of Mel-14 F(ab')2 increased from 7.04 +/- 1.59% injected dose (ID)/g at 37 degrees C to 20.65 +/- 4.53% ID/g at 42 degrees C (P less than 0.0001), and tumor localization of the control fragment rose from 5.23 +/- 1.35% ID/g to 14.51 +/- 1.37% ID/g (P less than 0.0001). In another experiment, F(ab')2 fragments were injected, tumors were heated for 4 h, and groups were sacrificed at 4, 8, and 16 h after injection. Statistically significant 2- to 3-fold higher uptake of both fragments in tumor were observed at all time points. Hyperthermic conditions also resulted in higher tumor:tissue ratios for both fragments. These results suggest that it may be possible to use tumor-localized hyperthermia to increase the therapeutic utility of radiolabeled monoclonal antibodies, particularly when labeled with short lived nuclides such as the 7.2-h alpha-emitter 211At.     

Biodistribution of indium-111-labeled OC 125 monoclonal antibody intraperitoneally injected into patients operated on for ovarian carcinomas

The biodistribution of 111In-labeled monoclonal antibody (MAb) OC 125 was studied after i.p. injection in 28 patients who underwent surgery for ovarian carcinoma. Group I (eight patients) received intact 111In-labeled OC 125 MAb, Group II (three patients) intact 111In-labeled irrelevant NS, Group III (five patients) intact 111In-labeled OC 125 MAb associated with 20 mg of the same unlabeled MAb and Group IV (12 patients) F(ab')2 fragments of 111In-labeled OC 125 MAb. The patients were operated on 1 to 3 days after i.p. injection, and the surgeon removed large tumor fragments and/or small tumor nodules and, in some patients, collected the residual perfusion fluid from which malignant cell clusters were isolated. Uptake by large tumor fragments at 24 h was low: 0.0031 +/- 0.0032% injected dose per gram (%ID/g) for Group I and 0.0024 +/- 0.0022%ID/g for Group IV. It was moderately higher than that of Group II (0.0014 +/- 0.0006%ID/g) and Group III (0.0015 +/- 0.0007%ID/g). Uptake by small tumor nodules (0.1302 +/- 0.0802%ID/g at 72 h for Group I) and malignant cell clusters (median: 0.3322, with a maximum value of 4.1614%ID/g at 24 h for Group IV) was markedly higher. Tumor-to-normal tissue ratios with OC 125 MAb [intact or F(ab')2 fragments] ranged between 0.1 and 8.5 for large tumor fragments and 2 and 8,700 for small tumor nodules and malignant cell clusters. It would thus appear that RIT is feasible if an appropriate radionuclide can be selected for antibody labeling.     

Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma.

In clinical studies we have evaluated a unique monoclonal antibody-based drug delivery system, a bifunctional antibody designed to deliver imaging or therapeutic agents, such as radioisotopes, drugs, or biologics, to tumor cells, while minimizing the dose to normal tissue. The bifunctional antibody, with one specificity to a tumor-associated antigen (carcinoembryonic antigen) and another specificity to a hapten, is injected and allowed to localize at a tumor site for 4 days. A hapten, tagged with a radioisotope, is subsequently injected for delivery to and capture by the prelocalized antibody at the tumor site. In studies reported here, the sulfhydryl groups of Fab' fragments of ZCE-025 and CHA-255 were linked with bis-maleimidomethyl ether to form an F(ab')2 bifunctional antibody coupled by a stable thioether linkage. EOTUBE, a hydroxyethylthiourido derivative of benzyl EDTA, was used as the hapten carrier of 111In. Fourteen patients 62-82 years old with recurrent or metastatic adenocarcinoma of the colon were studied. Twenty of 21 known lesions were imaged, and eight of nine new lesions were confirmed. With this fundamentally new approach to drug delivery, clearance from normal tissue is rapid, and high tumor:normal tissue ratios are expeditiously achieved.     

Bispecific antibody pretargeting of tumor neovasculature for improved systemic radiotherapy of solid tumors.

PURPOSE: Extra domain B (ED-B) fibronectin is a specific tumor matrix marker for targeting angiogenesis in solid tumors. In this study, the radiotherapeutic potential of the directly radioiodinated divalent anti-ED-B antibody fragment, L19 small immunoprotein (L19-SIP; 75,000 Da), was compared with a pretargeting approach using the bispecific antibody AP39xm679 (bsMAb; 75,000 Da). EXPERIMENTAL DESIGN: The bsMAb was prepared by coupling an anti-ED-B single-chain Fv (AP39) to the Fab' of the murine antibody m679, which binds to the small peptidic hapten histamine-succinyl-glycine (HSG). As an effector molecule for the pretargeting approach, the 111In-labeled HSG-DOTA complex was injected 25 or 41 hours after the bsMAb. The kinetics of both the iodinated bsMAb and the pretargeted 111In-labeled HSG hapten were investigated in mice bearing human glioblastoma xenografts (U251) and compared with the kinetics and tumor accumulation of radioiodinated L19-SIP. 111In and 125I were used as surrogate marker for the therapeutic radioisotopes 90Y/177Lu and 131I, respectively. RESULTS: Tumor uptake of the pretargeted 111In-labeled peptide was significantly higher than 125I-L19-SIP over 7 days. At the calculated maximally tolerated dose for each agent (with the kidney being the dose-limiting organ for pretargeting and the bone marrow for direct targeting), a mouse tumor dose of 146 Gy could be given by pretargeting versus 45 Gy delivered by the direct approach. CONCLUSIONS: These data suggest that pretargeting of ED-B with AP39xm679 and subsequent injection of the 90Y-hapten-peptide would improve the therapeutic efficacy in solid tumors by >3-fold compared with directly radiolabeled 131I-L19-SIP.     

Biodistribution of iodine-125 and indium-111 labeled OV-TL 3 intact antibodies and F(ab')2 fragments in tumor-bearing athymic mice.

The monoclonal antibody OV-TL 3, directed against an ovarian carcinoma-associated antigenic determinant, was tested as a vehicle for radioimmunolocalization of ovarian carcinomas in athymic mice bearing NIH:OVCAR-3 xenografts. The biodistribution of intact. OV-TL 3 was compared with the distribution of OC 125. Tumor uptake with OV-TL 3 was significantly higher than with OC 125, and almost 7 times higher than with a non-specific control antibody (OV-TL 19). Administration of a mixture of intact OV-TL 3 and OC 125 did not improve tumor uptake in comparison with OV-TL 3 alone. Subsequently, intact OV-TL 3 and its F(ab')2 fragments were labeled with either 111In or 125I. The highest tumor uptake was obtained with 111In-labeled intact OV-TL 3 (14.7% ID/g, 48 hr p.i.). For both antibody forms uptake of 111In in liver, spleen and kidneys was very high. Furthermore, 111In cleared more slowly from most tissues than 125I. As a result, tumor/tissue ratios with 111In-labeled OV-TL 3 were lower than with 125I-labeled OV-TL 3. The highest tumor/tissue ratios (6.9 to 53) were obtained with 125I-labeled OV-TL 3 F(ab')2 fragments, 48 hr post injection. 111In-labeled OV-TL 3 F(ab')2 has already been shown to be a clinically useful label for the detection of ovarian cancer. The results of our comparative animal study suggest that these clinical results may even be improved by using 123I-labeled OV-TL 3 F(ab')2.     

Biodistribution properties of (111)indium-labeled C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid humanized 3S193 diabody and F(ab')(2) constructs in a breast carcinoma xenograft model.

The humanized complementarity determining region-grafted anti-Lewis Y (Le(y)) monoclonal antibody [humanized 3S193 (hu3S193)] was developed for targeting Le(y)-expressing epithelial tumors such as breast, colon, lung, prostate, and ovarian carcinoma. We are exploring the potential use of smaller molecular size, bivalent analogues of hu3S193, because the faster blood clearance of M(r) approximately 54,000 diabody and M(r) approximately 110,000 F(ab')(2) molecules may be advantageous in achieving optimal and rapid tumor uptake for diagnostic and potential therapeutic applications. The single-chain variable fragment-5 residue linker construct (diabody) was expressed using the bacterial secretion vector pPOW3, and soluble product was purified without refolding processes. The F(ab')(2) fragment was obtained by pepsin digest of parental hu3S193. To facilitate evaluations, the radiometal (111)In was used to label C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid chelated diabody and F(ab')(2). The immunoreactivity of the radiolabeled constructs was 41.3 and 58.6%, and the K(a) was 1.68 x 10(6) M(-1) and 5.33 x 10(6) M(-1) for the diabody and F(ab')(2), respectively. Radioconjugates were injected into mice bearing Le(y)-positive MCF-7 tumors, and biodistribution properties were determined at various time points after injection. The uptake of radiolabeled diabody in xenografts was maximal at 1 h after injection (4.7 +/- 0.6% injected dose/g), whereas the F(ab')(2) peaked at 8 h after injection (14.2 +/- 2.4% injected dose/g). The tumor:blood ratio at 4 h for the diabody and F(ab')(2) was 5:1 and 2:1, which increased to 20:1 and 5:1, respectively, at 8 h and increased further to 40:1 and 130:1, respectively, at 48 h. These results demonstrate that the diabody construct may have applications as a diagnostic imaging reagent, whereas F(ab')(2) displayed effective tumor targeting and may have potential as a therapeutic molecule in patients with Le(y)-expressing tumors.     

Antibody guided targeting of non-small cell lung cancer using 111In-labeled HMFG1 F(ab')2 fragments

Immunoscintigraphy using F(ab')2 fragments of tumor-associated monoclonal antibody HMFG1 was performed in 14 patients with primary and metastatic non-small cell carcinoma of lung cancer. The antibody was conjugated with diethylenetriamine pentaacetic acid and labeled with 111In. Quality control studies showed efficient incorporation of 111In onto antibody (5 mCi/mg), no significant loss of immunoreactivity, and in vitro and in vivo stability. The optimal time for imaging was between 48 and 72 h. Following i.v. administration, serum activity fell rapidly (t1/2a = 2.5 +/- 1.3 (SD) h; t1/2b = 42 +/- 4.5 h). The majority of the radioactivity was associated with the plasma and not with the blood cells. All patients had a significant concentration of 111In in the liver (approximately 20% of the injected dose, 48 h postadministration). No toxicity was encountered. No human antimurine-IgG antibody was detected in any of the patients within 4 months of follow-up, even in patients receiving two administrations of F(ab')2 fragments. Localization of all primary lesions and the majority (80%) of metastatic lesions was achieved. Seven of 14 patients were also studied using a 111In-labeled nonspecific antibody (Fab')2 fragment (4C4). In three patients the specificity index was higher than the other four (P less than 0.05). We conclude that although successful targeting of 111In-labeled (Fab')2 fragments of HMFG1 can be achieved in patients with non-small cell carcinoma of lung, observable tumor localization can also be achieved using a nonspecific antibody. Based on these findings, we recommend that in order to demonstrate specific radioimmunolocalization, patients with lung and possibly other tumor types should be studied using both specific and nonspecific antibodies.     

Specific localization, gamma camera imaging, and intracellular trafficking of radiolabelled chimeric anti-G(D3) ganglioside monoclonal antibody KM871 in SK-MEL-28 melanoma xenografts

The chimeric monoclonal antibody KM871, directed against the G(D3) antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with (125)I via tyrosine residues and with (111)In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A"-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with (111)In or (125)I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G(D3)-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed. (111)In-labeled CHX-A"-DTPA-KM871 showed a maximum tumor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of (111)In-labeled CHX-A"-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G(D3) monoclonal antibody to G(D3)-expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.     

Effect of tumor mass and antigenic nature on the biodistribution of labeled monoclonal antibodies in mice

The effect of tumor mass and antigenic nature on the biodistribution of 111In- and 125I-labeled monoclonal antibodies (MoAbs) was studied using F(ab')2 fragments of three representative anti-tumor MoAbs and SW1116 human colorectal carcinoma grown in nude mice. The 19-9, F33-104 anti-CEA, and 17-1A MoAbs showed specific binding to SW1116 cells. The former two MoAbs recognize circulating CA 19-9 with molecular weights of more than 5,000,000 and CEA of Mr 170,000-180,000, respectively, whereas 17-1A reacts with a nonshedding antigen. Both percentage injected dose per gram tumor and tumor-to-blood ratios were inversely proportional to the tumor mass in nude mice administered 111In- and 125I-labeled 19-9, but liver uptake increased as tumor size increased. Analysis of serum samples and tumor homogenates demonstrated the presence of a high-molecular-weight species, probably due to the antibody binding to CA 19-9. In the case of 111In-labeled anti-CEA MoAb, tumor uptake also decreased and liver uptake increased with tumor size, but this effect was less obvious than that of 19-9. In contrast, tumor and liver uptake of 125I-labeled anti-CEA MoAb, 111In- and 125I-labeled 17-1A and control antibodies were independent of tumor mass. The absolute tumor uptake and tumor-to-blood ratios of all 125I-labeled antibodies were lower than those of the 111In-labeled ones. And the effect of tumor mass was also weaker with 125I-labeled antibodies, probably due to in vivo dehalogenation. These results indicate that the effect of tumor size on the incorporation of labeled MoAb into tumors is dependent on the antigenic nature to be targeted and/or radionuclides used for labeling and that high concentrations of circulating high molecular weight antigens may limit in vivo use of MoAb conjugates.     

Multicenter study of immunoscintigraphy with radiolabeled monoclonal antibodies in patients with melanoma

A multicenter study was performed to analyze the efficacy of 99mTc- and 111In-labeled F(ab')2 fragments of monoclonal antibody (MoAb) 225.28S (reactive with a high molecular weight melanoma associated antigen) to radioimage malignant lesions in patients with melanoma. A total of 254 melanoma patients, carrying 412 documented melanoma lesions, were studied in 10 nuclear medicine departments. A total of 377 lesions were visualized in 206 patients; in particular (a) 250 of 412 known lesions were visualized in 159 of 191 patients known to carry melanoma lesions; (b) 95 occult lesions were visualized in 61 patients of the same group; and (c) 32 lesions were visualized in 15 of 63 patients without diagnosed lesions. The melanomic nature of 101 of 127 radioimaged occult lesions was confirmed by clinical criteria and/or by additional laboratory investigations. These results indicate that immunoscintigraphy with radiolabeled F(ab')2 fragments of MoAb 225.28S can provide clinically useful information. Analysis of the variables influencing the outcome of immunoscintigraphy with 99mTc- and 111In-labeled F(ab')2 fragments of MoAb 225.28S confirmed the role of size, anatomic site, and level of high molecular weight melanoma associated antigen in melanoma lesions. Such analysis also showed, for the first time, the influence (a) of the isotope used to radiolabel the antibody fragments and (b) of the clinical stage of the patients. The present study has shown good agreement in the results obtained by the 10 nuclear medicine departments, suggesting that immunoscintigraphy with radiolabeled F(ab')2 fragments of MoAb 225.28S is a reliable procedure.     

Biodistribution of indium-111-labeled OC 125 monoclonal antibody after intraperitoneal injection in nude mice intraperitoneally grafted with ovarian carcinoma

The purpose of this work was to study the biodistribution of 111In-labeled OC 125 monoclonal antibody (MAb) with known affinity for ovarian carcinomas in a nude mouse model grafted i.p. with a human ovarian cancer (NIH:OVCAR-3). Tumor uptake 24 h after i.p. injection was higher with intact 111In-labeled OC 125 MAb (28 +/- 7.44%ID/g) than with 111In-nonspecific immunoglobulin (6.86 +/- 1.35%ID/g). The kinetics of tumor uptake also differed, showing a plateau followed by a drop at Day 7 with 111In-OC 125 MAb and a decrease beginning at 24 h with 111In-nonspecific immunoglobulin. Tumor-to-normal tissue ratios ranged between 29.91 +/- 11.85 and 0.68 +/- 0.15 with 111In-OC 125 MAb and between 4.50 +/- 1.06 and 0.53 +/- 0.04 with 111In-nonspecific immunoglobulin according to the normal tissues and the time points considered. Tumor uptake 2 h after injection was the same for F(ab')2 fragments as for intact MAb, whereas maximum uptake at 24 h (18.76 +/- 4.62%ID/g) was lower and was followed by a decrease at Day 4. Tumor-to-normal tissue ratios were in the same range, except for the tumor to blood ratio which was higher and the tumor to kidney ratio which was lower at 24 and 96 h. Maximum tumor uptake was higher after i.p. (30.77 +/- 4.76%ID/g) than i.v. (14.59 +/- 2.70%ID/g) injection. Instead of attaining the plateau noted after i.p. injection, tumor uptake after i.v. injection remained low at 2 h (2.11 +/- 1.66%ID/g), reaching its peak only after 96 h. 131I-OC 125 injected i.p., which reached maximum tumor uptake at 2 h (13.53 +/- 4.25%ID/g), showed tumor-to-tissue ratios ranging between 15.98 +/- 2.63 and 0.96 +/- 0.86, i.e., not very different from those with 111In. After i.p. injection of a radiolabeled colloid solution, maximum tumor uptake was reached at 96 h (20.22 +/- 5.35%ID/g), but with very high nonspecific uptake in liver (31.06 +/- 6.22%ID/g) and spleen (55.23 +/- 14.11%ID/g). These results indicate high, selective tumor uptake of 111In-OC 125 after i.p. injection and demonstrate the feasibility of i.p. radioimmunotherapy of ovarian carcinomas.     

Pharmacokinetics of the monoclonal antibody B72.3 and its fragments labeled with either 125I or 111In

A comparison of the pharmacokinetics of intact B72.3 (a murine monoclonal antibody specific for human breast and colon carcinoma) with F(ab')2 and Fab fragments labeled with 111In and 125I was done in athymic mice bearing target (LS174T) and non-target (HCT-15) tumors. IgG B72.3 labeled with either isotype imaged LS174T. Biodistributions of both labels were similar in all organs except liver. F(ab')2 also imaged the LS174T tumor, while Fab bearing either isotype did not. The blood clearance was Fab greater than F(ab')2 greater than immunoglobulin G B72.3 for both isotopes. 111In-labeled fragments yielded large accumulations in the kidneys which persisted for 2 days. The different patterns of biodistribution for the various forms of B72.3 labeled with the two isotopes suggest that the most desirable combination of fragment and isotope will depend on the intended use.     

Tumor targeting by a multivalent single-chain Fv (scFv) anti-Lewis Y antibody construct

The use of single-chain variable fragment (scFv) constructs has been investigated in cancer radioimmunotherapy (RIT) and radioimmunodetection, as these molecules permit rapid tumor penetration and clearance from the serum relative to whole IgG. Multimerization of scFv constructs has demonstrated improvements in functional affinity (i.e., avidity) and maximal tumor uptake. In this paper, we report the first biodistribution and pharmacokinetics studies of a noncovalent, direct-linked scFv (V(L)-0-V(H)) trimeric/tetrameric "multimer" of the anti-Lewis Y monoclonal antibody, hu3S193. The in vitro binding and in vivo biodistribution of the hu3S193 multimer was characterized alongside the hu3S193 F(ab')(2) following radiolabeling with the Indium-111 ((111)In) radioisotope. Immunoreactivities of the radiolabeled multimer and F(ab')(2) were 73% and 53.2%, and binding affinities (K(a)) were 1.58 x 10(7) M(1) and 4.31 x 10(6) M (1) for the multimer and F(ab')(2), respectively. Maximal tumor uptake in Le(y)-positive MCF-7 breast cancer xenografted BALB/c nude mice was 12.6 +/- 2.5 percent injected dose/per gram (%ID/g) at 6 hours postinjection for the multimer and 15.7 +/- 2.1 %ID/g at 24 hours postinjection for the F(ab')(2). However, limited in vitro stability and high renal localization of radiolabeled constructs were observed, which, despite the observed tumor targeting of the hu3S193 multimer, most likely preclude its use in RIT and imaging modalities.     

Radioimmunotherapy of intracerebral human glioma xenografts with 131I-labeled F(ab')2 fragments of monoclonal antibody Mel-14

The administration of radiolabeled monoclonal antibodies to improve the treatment of malignant gliomas is dependent upon achieving effective tumor radiation dose while sparing normal tissues. We have evaluated the efficacy of 131I-labeled F(ab')2 fragment of monoclonal antibody Mel-14, an IgG2a reactive with the chondroitin sulfate proteoglycan antigen of gliomas, melanomas, and other neoplasms, in prolonging survival of athymic mice transplanted intracerebrally with D-54 MG human glioma xenografts. Studies indicated that in vitro immunoreactivity, affinity, and tumor localization in vivo of radiolabeled Mel-14 F(ab')2 were maintained at specific activities of 10-13 microCi/micrograms. Intravenous injection of 1500 microCi/115 micrograms or 2000 microCi/154 micrograms 131I-labeled Mel-14 F(ab')2 into mice 6-7 days after xenograft implantation resulted in significant survival prolongation over control animals (P = 0.009 using Wilcoxon rank sum analysis). In another experiment, 1500 microCi/126 micrograms 131I-labeled Mel-14 F(ab')2 improved survival significantly over controls (P = 0.006), while 1500 microCi/220 micrograms 131I-labeled nonspecific antibody did not (P = 0.2). Increasing the injected radiation dose to 3000 microCi 131I-labeled Mel-14 F(ab')2 did not significantly increase survival in tumor-bearing mice, because of supervening radiation toxicity. However, giving 3000 microCi 131I-labeled Mel-14 F(ab')2 in two doses of 1500 microCi, 48 h apart, did significantly prolong animal survival over controls (P = 0.001). Estimated radiation dose to tumor was 915 rad after injection of 3000 microCi 131I-labeled Mel-14 F(ab')2 in two doses, a dose higher than that delivered to normal tissues. The results of this study suggest that radiolabeled Mel-14 F(ab')2 be evaluated as an agent for radioimmunotherapy trials.     

In vivo biodistribution of a humanized anti-Lewis Y monoclonal antibody (hu3S193) in MCF-7 xenografted BALB/c nude mice

The biodistribution characteristics of a humanized anti-Lewis(y) antibody (hu3S193) radiolabeled to three radioisotopes, 125I, 111In, and 90Y, were examined in a BALB/c nude mouse xenograft model of breast cancer. The immunoreactivity of both 125I- and 111In-bound hu3S193 exceeded 50% and was 20% for 90Y. In vivo, labeled antibody was shown by gamma camera imaging and immunohistochemical and autoradiographic techniques to localize to Lewis(y)-expressing breast xenografts with minimal normal tissue uptake. Maximal radioisotope uptake peaked at 48 h for all three isotopes; however, the percentage of injected dose/gram and tumor retention were greater for 111In- and 90Y-bound antibody than for 125I-bound antibody. Although immunoreactivity of 111In- and 125I-labeled hu3S193 in serum was stable over a 5-day period, the amount of unlabeled 111In in serum was lower than 125I, which together with higher tumor uptake indicates better retention of 111In-labeled hu3S193 and catabolites within the tumor cells. Superior tumor uptake and retention of 111In-labeled hu3S193 and similar blood clearance compared with 125I-labeled hu3S193, suggest that radiometals are the preferred radioisotope for this antibody-antigen system. Humanized 3S193 is a promising new construct for the targeting and potential therapy of Lewis(y)-expressing tumors.     

Enhanced clearance of radiolabeled murine monoclonal antibody by a syngeneic anti-idiotype antibody in tumor-bearing nude mice.

A syngeneic anti-idiotype monoclonal antibody (MAb) (CM-11) directed against an anti-carcinoembryonic antigen (CEA) murine MAb (NP-4) was evaluated as a second antibody (SA) to promote the rapid clearance of radiolabeled NP-4 from the blood. Initial studies confirmed that CM-11 IgG removed 131I-NP-4 IgG from the blood as effectively as a polyclonal donkey anti-goat IgG removed 131I-goat IgG. However, use of an F(ab')2 in place of either the NP-4 or CM-11 IgG was not as effective in removing primary radiolabeled antibody, despite the formation of high-molecular-weight complexes. In accordance with previous results, the timing and dose of the SA injection was critical for optimizing tumor uptake and improving tumor/non-tumor ratios. In nude mice bearing GW-39 human colonic tumor xenografts, a delay in the injection of CM-11 by 48 hr after injection of radiolabeled NP-4 was optimal, since this allowed maximum tumor accretion. At a 200:1 CM-11:NP-4 ratio, tumor uptake was reduced, suggesting inhibition of NP-4 binding to CEA within the tumor. Despite optimizing tumor uptake by delaying SA injection and adjusting its dose, the percentage of 131I-NP-4 in the tumor decreased 2- to 3-fold within 2 days after CM-11 injection. A similar effect was seen for 111In-labeled NP-4 IgG with CM-11. Injection of excess unlabeled NP-4 given to block CM-11 shortly after its injection failed to curtail the loss of NP-4 from the tumor. Our results suggest that high blood levels of MAb are important for sustaining NP-4 in the tumor. Radiation-dose predictions derived from biodistribution studies indicate that a higher tumor dose may be delivered using the SA method than with either 131I-NP-4 IgG or F(ab')2 alone. Use of the SA method with 90Y-labeled NP-4 IgG, as modeled from biodistribution studies with 111In-NP-4 IgG, would likely be limited by liver toxicity.     

Improved delivery of radiolabeled anti-B1 monoclonal antibody to Raji lymphoma xenografts by predosing with unlabeled anti-B1 monoclonal antibody.

A human B-cell lymphoma xenograft model was used to test whether the administration of unlabeled MoAb prior to injection of radiolabeled monoclonal antibody (MoAb) improves delivery of the radiolabeled MoAb to tumor prior to testing in clinical radioimmunotherapy trials. The anti-B1/CD20 pan-B-cell MoAb reactive with human B-cell lymphomas and leukemias but not reactive with mouse B-cells was used in this study. Athymic nude mice bearing human Raji Burkitt lymphoma xenografts were given injections of 2.5 muCi (0.3 microgram) 131I-labeled anti-B1 with or without a 2-h prior single injection of 100 micrograms of unlabeled anti-B1 antibody. Four days later the animals given injections of 131I-labeled anti-B1 and the unlabeled anti-B1 predose had a tumor uptake of 12.72 +/- 1.17% (SEM) of injected dose/g which was 44% greater than the animals receiving the 131I-labeled anti-B1 alone (P = 0.014). The uptake in most normal tissues was unchanged, although the blood level of 131I-labeled anti-B1 appeared to be greater following unlabeled anti-B1 predosing (P = 0.067). Predosing with isotype matched irrelevant MoAb did not result in a greater tumor uptake or blood concentration of 131I-labeled anti-B1 compared to the administration of 131I-labeled anti-B1 alone. In studies using 111In-labeled anti-B1, the effect of unlabeled antibody predosing was more pronounced. For animals given injections of 4.5 muCi (0.4 microgram) 111In-labeled anti-B1 and the unlabeled anti-B1 predose, the uptake in tumor was 12.37 +/- 2.07% of injected dose/g which was 162% greater than the animals receiving the 111In-labeled anti-B1 alone (P = 0.009). Predosing decreased 111In-labeled anti-B1 uptake in spleen, while the blood level was significantly greater. Predosing was more effective than simultaneous injection in improving tumor delivery. When tumor-bearing mice were either simultaneously given injections of 36 micrograms of unlabeled anti-B1 and 4 micrograms 111In-labeled anti-B1 or were given preinjections of 36 micrograms unlabeled anti-B1 3 h prior to injection of 4 micrograms 111In-labeled anti-B1, tumor uptake 3 days later was 1.3-fold higher in the animals which received the preinjection of unlabeled antibody (P = 0.011). As the quantity of unlabeled anti-B1 was increased (36, 96, 996 micrograms) in the predose, significantly greater uptake in tumor was observed, although this uptake appeared to plateau at the highest predoses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radioimmunoimaging of human bladder tumor xenografts in nude mice by using monoclonal antibodies

Monoclonal antibodies HBJ127 and HBJ8, raised against T24 human bladder cancer cells, predominantly react with the cells in proliferating stages and with a portion of epithelial tumor cells, respectively. To investigate the in vivo localization of these monoclonal antibodies, the antibodies were labeled with radioiodine and indium-111 (111In) and injected into nude mice transplanted with human bladder tumors. The BT-11 bladder tumor had the highest concentration of radioiodinated HBJ127 and HBJ8 monoclonal antibodies, with 11.6 and 14.3% of the injected dose per gram and with a tumor-to-blood ratio of 2.6 and 1.6, respectively, at 4 days after the administration. An irrelevant monoclonal antibody did not show any specific accumulation in the BT-11 tumor. The 111In-labeled HBJ127 antibody was also localized in the tumor with a higher tumor-to-blood ratio than the radioiodinated antibody. The xenografted BT-11 tumor was successfully visualized with the radiolabeled HBJ127 and HBJ8 antibodies by scintigraphy. These monoclonal antibodies and the human bladder tumor xenografts may provide a good model for radioimmunoimaging and possibly therapy.