67Ga-labeled antibodies for immunoscintigraphy and evaluation of tumor targeting of drug-antibody conjugates in mice

To assess the in vivo behavior of cytotoxic agents linked to antibodies, deferoxamine, known to form stable chelates with 67Ga, was conjugated with monoclonal antibodies using three different methods. One method used a homocoupling reagent, glutaraldehyde, whereas two other methods used heterocoupling reagents, N-succinimidyl-3-(2-pyridyldithio)propionate and succinimidyl-6-maleimidohexanoate, linking deferoxamine to antibodies through alkylamine, disulfide, and thioether bonds, respectively. Antibodies were efficiently labeled with 67Ga through chelation with deferoxamine without losing antigen-binding capability. 67Ga-labeled antibodies clearly visualized transplanted tumors in nude mice. However, the biodistribution of radioactivity was markedly different with the coupling methods used for the conjugation of deferoxamine and antibodies. High nonspecific uptake in the liver and spleen was observed with 67Ga-labeled antibodies prepared by the glutaraldehyde method. 67Ga-labeled antibodies linked by thioether bonds demonstrated in vivo stability and the highest tumor:liver ratio, whereas 67Ga-labeled antibodies conjugated with disulfide bonds were rapidly cleared from the circulation. These results indicate that antibody conjugates linked by thioether bonds are a better choice for drug targeting and that 67Ga-labeled antitumor monoclonal antibodies are useful not only for the immunoscintigraphy but also for the quantitative assessment and visualization of the biodistribution of drug-antibody conjugates.     

Labeling of sarcoma associated monoclonal antibody with 111In, 67Ga and 125I

Labeling of human sarcoma-associated murine monoclonal antibody (MAb) 23H7 with 67Ga and 111In by the bifunctional ligand method is reported. 67Ga was chelated to the MAb via desferrioxamine B and 111In via the cyclic anhydride of DTPA. Higher specific activity was obtained with 67Ga (4-5 microCi/micrograms) as compared with 111In (2 microCi/micrograms). The binding capacity of the MAb was confirmed by repeated indirect immuno-fluorescence assays performed before and after labeling. A fast blood clearance was observed: 33% recovered dose (R.D.) blood level 3 h post-injection as compared with 56% after injection of control polyclonal IgG. Preliminary results on chemically induced sarcoma bearing mice showed a relatively high tumor uptake of the labeled antibody.     

Stability, targeting, and biodistribution of scandium-46- and gallium-67-labeled monoclonal antibody in erythroleukemic mice

Using a monoclonal antibody specific for the Mr 70,000 glycoprotein of Rauscher erythroleukemia virus, we have determined the optimal conditions for conjugation with the cyclic anhydride of diethylene triamine pentaacetic acid and subsequent labeling with 46Sc. The conjugates were shown to retain their specificity and activity in vitro and to target specifically to virus-infected spleen cells in vivo. The stability of the 46Sc:diethylene triamine pentaacetic acid-antibody conjugates in vivo was studied using immunoaffinity chromatography; 25% of the isotope bound to transferrin, and 75% remained bound to the antibody conjugates. These results are discussed with respect to the potential for labeling antibodies with 47Sc for use in imaging and therapy. Studies with 111In-labeled antibody were used for comparison. Labeling with 67Ga was also performed; these labeled conjugates showed adventitious binding of isotope to the antibody and lack of stability of diethylene triamine pentaacetic acid-chelated gallium. Free EDTA was shown to stably incorporate 67Ga.     

Human sarcoma-associated murine monoclonal antibody labeled with indium-111, gallium-67, and iodine-125

Monoclonal antibody (MAb) 23H7 is a hybridoma-derived IgG that is generated following fusion of mouse myeloma cell line P3U1 and spleen cells from BALB/c mice hyperimmunized with a human fibrosarcoma. It detects a mesenchymal antigen of 23KD expressed on human sarcoma tissues and other neoplasms, including myeloid leukemias, but it rarely binds to normal tissues. The MAb 23H7 was labeled with 67Ga and 111In using the bifunctional ligand method. The 67Ga was chelated to the MAb via desferrioxamine B, while 111In was chelated via the cyclic anhydride of diethylenetriamine pentaacetic acid. Higher specific activity was obtained with 67Ga than with 111In (4.5 and 2 muCi/microgram, respectively); both gave stable complexes. When 23H7 was labeled with 125I, considerable breakdown was observed. This, together with the physical shortcomings of this isotope, emphasizes the advantages of labeling with 111In and 67Ga. The rapid blood clearance of the labeled sarcoma-associated MAb may be beneficial for early tumor uptake and for imaging shortly after injection.     

Effect of unlabelled monoclonal antibodies on the biodistribution of 111In-labelled anti-prostate-specific acid phosphatase monoclonal antibodies in the mouse model

In order to optimize methods developed for the radioimaging of prostatic cancer, the effect of unlabelled monoclonal antibodies (MoAbs) on the biodistribution of 111In-labelled MoAbs was studied in nude mice carrying human prostatic cancer xenografts (PC-82). Since the intact IgG and its F(ab')2 fragment have different clearance patterns from the blood, we also studied which of these antibody forms is preferable for use as the unlabelled antibody, when injected prior to, simultaneously with, or following the injection of the labelled antibody. Due to their faster blood clearance, F(ab')2 fragments displayed higher tumour-to-blood ratios than the corresponding anti-prostate-specific acid phosphatase (anti-PAP) IgG1. However, tumour-to-blood ratios increased when the amount of the labelled anti-PAP-IgG1 was increased (from 10 micrograms to 25 micrograms) and the biodistribution measurements were carried out after a prolonged period (216 h). When a combination of labelled IgG1 (1, 10 micrograms) and unlabelled IgG1 (200 micrograms-300 micrograms) was used, tumour-to-blood ratios could not be improved. Contrary to what was observed with the intact IgG1, labelled F(ab')2 fragments did not display a dose-dependent accumulation in the tumour. A combination of labelled F(ab')2 fragments and unlabelled IgG1 resulted in a slight increase in tumour-to-blood ratios, but the administration of labelled F(ab')2 fragments with unlabelled F(ab')2 fragments resulted in a significant decrease (p = 0.04) in tumour-to-blood ratios. Liver-to-blood ratios could be decreased by using either a combination of unlabelled and labelled IgG1 (at ratios of 30:1, or 200:1), or a combination of unlabelled IgG1 and labelled F(ab')2 fragments (at ratios of 50:1, or 100:1), or a combination of the unlabelled and labelled F(ab')2 fragments (at a ratio of 50:1). The decrease of liver-to-blood ratios was independent of the mode of administration of the unlabelled substances. A "rinse" with an additional dose of unlabelled IgG1, 24 h before the sacrifice, resulted in even lower liver-to-blood ratios. The results obtained from this study suggest that, of the combinations investigated, to increase tumour-to-blood ratios and decrease liver-to-blood ratios thereby improving the radioimaging of prostatic cancer, the best one consists of 111In-labelled anti-PAP-F(ab')2 fragments and unlabelled anti-PAP-IgG1.     

Selective localisation of two radiolabelled anti-sarcoma monoclonal antibodies in human osteosarcoma xenografts

Two mouse monoclonal antibodies (MoAbs), TP-1 and TP-3, previously shown in immunohistochemical studies to react with osteosarcomas, were labelled with 125I or 131I and evaluated for their ability to localise to human osteogenic sarcoma xenografts after intravenous injection. The radiolabelled TP-1 and TP-3 MoAbs had immunoreactive fractions of 70% and 67%, respectively, and bound to target cells with binding constants of 8.5 X 10(8) M-1 and 4.0 X 10(9) M-1, respectively. After injection of labelled TP-3 IgG, approximately 16% of the dose X g-1 tissue was found in the tumour after 24 hours. Maximum tumour/blood radioactivity ratios of 6-7 were achieved 3-4 days after antibody injection, while the ratios for the normal tissues were less than 1. The tumours could be clearly visualised by whole-body gamma scintigraphy without the need for subtraction techniques. The TP-1 IgG accumulated to a large extent also in the spleen. Hence, with this antibody the tumour was less well delineated from the adjacent normal tissues. However, the F(ab')2 fragments, derived from the TP-1 IgG, gave tumour/blood ratios up to approximately 40 after 3-4 days and yielded sharp gamma scintigrams of the tumour. Specificity of the antibody localisation was indicated by the lack of accumulation in a contralateral melanoma xenograft and the failure of 2 isotype-matched irrelevant MoAbs to localise to the sarcomas. With the F(ab')2 fragments satisfactory images could be obtained already after 16 hours. The results suggest that this preparation may be useful in clinical radioimmunodetection of osteogenic sarcomas.     

Uptake of gallium-67 by human leukemic cells: demonstration of transferrin receptor-dependent and transferrin-independent mechanisms

We have studied the role of transferrin and the transferrin receptor in the uptake of 67Ga by the human leukemic cell line HL60. In the absence of transferrin, HL60 cells incorporated about 1% of the 67Ga dose over 6 h. The presence of transferrin increased cellular 67Ga uptake approximately 10-fold. Transferrin-mediated uptake of 67Ga was blocked by an anti-transferrin receptor monoclonal antibody, and decreases in the density of cellular transferrin receptors led to corresponding decreases in the transferrin-dependent uptake of 67Ga. Changes in the cellular ferritin content did not significantly influence the uptake of 67Ga by either transferrin-independent or transferrin-dependent pathways. Regardless of the mechanism of uptake, a significant amount of intracellular 67Ga was found to be associated with immunoprecipitable ferritin as well as with a free pool. This free intracellular 67Ga appeared to be kinetically active since cells released 67Ga back to the media over time. Our results demonstrate the existence of a dual mechanism for the cellular uptake of 67Ga and suggest that the preferential uptake of 67Ga by lymphomas is related to the high density of transferrin receptors known to be expressed by these tumors in vivo.     

Gallium-67 radiotoxicity in human U937 lymphoma cells.

Promising clinical results have been obtained with radiolabeled antibodies in lymphoma patients. The higher uptake by lymphomas of 67Gallium (67Ga) compared with monoclonal antibodies makes selective radiotherapy by the widely available 67Ga appealing. However, the gamma radiation of 67Ga used in scintigraphy is considered to be almost non-toxic to lymphoma cells. However, in addition to photon radiation 67Ga emits low energy Auger electrons and 80-90 keV conversion electrons which could be cytotoxic. The objective of the present study was the assessment of radiotoxicity of 67Ga on a lymphoid cell line: U937. Proliferation (MTT-assay) and clonogenic capacity (CFU-assay) were measured after 3 and 6 days incubation with 10, 20 and 40 microCi ml-1 67Ga. Growth inhibition was 36% after 3 days incubation and 63% after 6 days incubation with 40 microCi 67Ga ml-1. Clonogenic capacity was reduced by 51% after 3 days and 72% after 6 days incubation with 40 microCi ml-1 67Ga. A survival curve showed an initial shoulder and became steeper beyond 200-250 pCi cell-1 (low linear energy transfer type). Iso-effect doses of 67Ga and 90Yttrium (90Y) were determined. The iso-effect dose of 40 microCi 67Ga ml-1 (cumulative dose of conversion electrons 306 cGy) was 2.5 microCi 90Y ml-1 (cumulative dose 494 cGy) and the iso-effect dose of 80 microCi 67Ga ml-1 was 5.0 microCi 90Y/ml. The main cytotoxic effect of 67Ga seems to be induced by the 80 keV conversion electrons. We conclude that the conversion electrons of 67Ga have a cytotoxic effect on U937 cells and that in our experiments a 16-fold higher microCi-dose of 67Ga than of 90Y was needed for the same cytotoxic effect. We believe that 67Ga holds promise for therapeutic use.     

Polyclonal and monoclonal anti-CEA antibodies in immunolocalization of colorectal cancer

Antibodies to carcinoembryonic antigen (CEA) from sheep and monkey were immunoadsorbent purified. Mouse monoclonal antibody (MAb) anti-CEA I-38S1 and Fab fragments of this antibody were prepared from mouse ascitic fluid. The IgG preparations were labelled with 123I or 131I, the Fab fragments with 131I. The antibody reactivity was unchanged after labelling. Patients with advanced colorectal carcinomas received an intravenous injection of 50-200 MBq 123I or 30-160 MBq 131I coupled to 250-500 micrograms antibody or antibody fragment. Patient examinations were performed using emission tomography (SPECT) and/or conventional gamma camera scintigraphy. The specific localization of labelled anti-CEA to tumor was compared to known tumor localized by CAT-scan, other x-ray methods or laparotomy, 50% of known tumors were accurately localized with sheep anti-CEA. In contrast, 70-80% of known tumor sites were correctly localized with polyclonal monkey anti-CEA antibodies, with monoclonal anti-CEA antibodies or with Fab fragments of the latter. A few previously unknown tumors were detected.     

Epidermal growth factor receptor-reactive monoclonal antibodies: xenograft antitumor activity alone and as drug immunoconjugates

Antibodies reactive with human epidermal growth factor receptor (EGFr), such as 225 IgG1 (Masui et al., Cancer Res., 44: 1002-1007, 1984), are effective tumor-suppressive agents in xenograft models. In the present study an additional antibody reactive with EGFr was made and compared to 225 IgG1 for antitumor activity as an unmodified antibody or as a drug immunoconjugate. This IgG1 clone, designated EGFrL11, competed with EGF and immunoprecipitated a Mr 178,000 protein identical to that immunoprecipitated with 225 IgG1. Cross-competition and immunodepletion studies indicated that the two antibodies bound to distinct epitopes on the same molecule. Immunofluorescence studies confirmed that the EGFrL11 epitope was expressed on the surface of viable human squamous cell carcinoma lines including T222. Unmodified EGFrL11 and 225 IgG1 were tested for antitumor activity in T222 xenografts. At a dose of 81 mg/kg given twice weekly for 3 weeks, tumor suppression, but not regression, occurred with EGFrL11. A similar result was obtained with 225 IgG1. To gauge the potential of these antibodies as immunoconjugates, both were tested for antitumor activity in the T222 model after conjugation to the Vinca derivative 4-desacetylvinblastine-3-carboxhydrazide. Both immunoconjugates completely regressed established tumors. These data suggest that Vinca conjugates with EGFr-reactive monoclonal antibodies warrant further investigation as possible clinical candidates.     

Antibody targeting to the murine lymphoma ESb-MP: increased accumulation due to reduced internalization into lymphoma cells as compared to normal lymphoid cells

Using rat monoclonal antibody (MAb) 12-15A against the spontaneously metastasizing mouse lymphoma variant ESb-MP, we elaborated conditions for targeting. In vitro, high binding of labelled antibody to ESb-MP cells and low binding to lymphoid cells (e.g., spleen cells) was noted. In vivo, we observed pronounced accumulation in spleen, lymph nodes and bone marrow, the uptake kinetics indicating high accessibility of the target antigen in these tissues, and rapid clearance of radioactivity from blood and most normal tissues, indicating degradation of the antibody and excretion of the label. Binding to lymphoma tissue was slow but persistent, resulting in high tumor:tissue ratios only after 2-3 days. Biodistribution could be dramatically changed by pre-treatment of animals with excess cold antibody, which reduced trapping of labelled antibody in normal lymphatic tissue, leading to prolonged persistence in the blood and preferential uptake into tumor tissue. Monovalent 12-15A fragments showed less pronounced binding to lymphatic tissue, while being rapidly cleared from the circulation by virtue of their inherent tendency to bind to kidney tissue. Tumor:tissue ratios up to 56:1 were obtained by a combination of pre-treatment with unlabelled 12-15A IgG or Fab fragment, followed by injection of labelled fragment or IgG, respectively. This is interpreted on the basis of differences in the internalization and retention of antigen-antibody complexes. Pre-treatment obviously leads to a temporary blockade or removal of the target antigen, which is much more efficient with normal lymphoid cells than with tumor cells. Thus, it may become possible to target antibodies into the tumor despite concurrent antigen expression on normal tissue.     

An idiotypic replica of carcinoembryonic antigen inducing cellular and humoral responses directed against human colorectal tumours.

A monoclonal anti-idiotypic antibody (anti-Id MAb) 708 (IgG2b), which inhibited the binding of NCRC23 (IgG1) MAb to CEA and prevented radiolabelled CEA from binding to MAb NCRC23, was produced. No recognition of 3 other anti-CEA antibodies, 3 other IgG1 or 2 IgM MAbs was observed with this anti-idiotypic antibody. When an immunoblotting technique was used, 708 anti-Id MAb failed to bind to isolated heavy or light chains of MAb NCRC23, whereas binding was observed with intact antibody. Mouse, rat and human lymphocytes (in vitro) were immunized with 708 anti-Id MAb and the resultant Ab3 antibodies all inhibited binding of labelled 708 anti-Id MAb to MAb NCRC23 and also reacted with CEA, showing that 708 anti-Id MAb induced anti-CEA antibody responses. Similarly, mice immunized with 708 anti-Id MAb could be restimulated in vitro with either CEA or tumour cells expressing CEA which induced specific T-cell proliferative responses. Human tumour-infiltrating lymphocytes isolated from colorectal tumours or peripheral blood T cells from cancer patients were stimulated in vitro with 708 anti-Id MAb or an irrelevant IgG2b antibody. Six days later both sets of lymphocytes were restimulated with CEA, and lymphocytes primed to 708 anti-Id MAb proliferated in response to CEA. These results suggest that 708 anti-Id MAb can act as an idiotypic replica of CEA and stimulate cellular and humoral anti-CEA immune responses. It is therefore of great interest as an idiotypic vaccine against colorectal cancer.     

Localisation and toxicity study of a vindesine-anti-CEA conjugate in patients with advanced cancer.

Safety of administration of a vindesine (VDS)-anti-CEA conjugate and its ability to localise after radiolabelling were investigated in patients with advanced metastatic carcinoma (4 colorectal and 4 ovarian). For imaging, patients received between 230 and 520 micrograms of 131I labelled antibody. In 5, localisation of conjugate was demonstrated, in another it was equivocal and in 2 patients, undetectable. For assessment of safety each patient also received a single dose of conjugate increasing from 1.2 to 42 mg antibody linked to 24 to 1800 micrograms VDS. The in vitro activity of the anti-CEA antibody and its ability to localise in vivo were preserved after conjugation. There was no obvious toxicity or hypersensitivity attributable to either the radiolocalisation or escalated doses of conjugate in any of the patients. The feasibility of the preparation and administration to patients of a vindesine-antibody conjugate has been demonstrated.     

Biodistribution of antibodies after intraperitoneal or intravenous injection and effect of carbohydrate modifications

These studies were designed to improve the strategy for intraperitoneal immunotherapy of human ovarian carcinoma with monoclonal antibodies (MAbs). Since ovarian tumor cells generally appear to be confined to the peritoneal cavity, regional therapy is appropriate and can reduce the need for strictly tumor-specific MAbs. In normal mice, with the use of radioiodine-labeled MAbs, transfer from peritoneal cavity to blood was found to be very rapid, within hours, and this transfer was delayed slightly by increasing the volume injected. The presence of ascitic fluid in mice greatly delayed the rate of transfer. For reduction of possible toxicity for normal cells outside the peritoneal cavity, the hepatic receptor for desialylated serum glycoproteins was used. Neuraminidase treatment of all major mouse immunoglobulin classes and subclasses, including IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA, did not cause their rapid blood clearance, although similar treatment of fetuin was effective. Conjugation of IgG with galactose, with use of the cyanomethyl derivative, did result in very rapid blood clearance via the hepatic lectin; within 3 minutes clearance was essentially complete. The specificity of uptake was demonstrated by inhibition with desialylated fetuin. Degradation within the liver, release of the radioiodine, and excretion from the animal were also quite rapid, within hours. This conjugation procedure had no effect on the antibody activity of the two MAbs tested. Such modified MAbs, therefore, are degraded almost immediately after entering the blood and would be advantageous in intraperitoneal therapy and in other situations in which regional immunotherapy is appropriate.     

Tumour and cellular localization by use of monoclonal and polyclonal antibodies to placental alkaline phosphatase

Monoclonal and polyclonal antibodies against placental alkaline phosphatase (PLAP) were evaluated for tumour immunolocalization of human PLAP-producing Hep 2 tumours in nude mice. The antibodies were labelled with 125I and injected i.p. in mice with developing Hep 2 tumours. The distribution of 125I-anti PLAP in various tissues showed that the labelled antibody was enriched in the tumour, the mean concentration ratio being 7.1 and 6.8 for polyclonal and monoclonal antibodies, respectively. A PLAP negative tumour (RD) showed a mean ratio of 1.2. There was a positive correlation between PLAP content and uptake of labelled antibody in the tumours. Hep 2 tumour cells in tissue culture showed 100% positivity for PLAP, while imprints of the tumour after passage in nude mice showed 40-50% positivity. PLAP offers potential as a useful marker for localizing tumours in humans.     

Immunotargeting with Monoclonal Cytokeratin 8 Antibodies of Human Urothelial Cancer Transplanted to Nude Mice

The possibility of using cytokeratin antibodies for the radioimmunolocalization of urinary bladder cancer was studied. A monoclonal murine IgG antibody was raised against cytokeratin 8 and labelled with iodine-125; normal murine IgG was used for control purposes. The urothelial cancer cell line RT4 was transplanted into immunodeficient nude mice. The anti-cytokeratin 8 antibody was administered intraperitoneally and its uptake in the tumour and other organs was analyzed with a computerized gamma camera. Optimal scintigraphic visualization occurred 11 days after antibody administration. The tumour/blood ratio of the specific antibody was 5.64 (±5.01 SD) on day 11, compared with 0.73 (±0.35 SD) in the control. Autoradiography demonstrated antibody uptake preferentially in viable sections of the tumour. The antibody uptake is presumed to be the result mainly of binding to the released cytokeratin in and around cells lysed during natural cellular death. The monoclonal murine anti-cytokeratin antibody is of potential interest in studies aimed at improving the clinical staging of urinary bladder cancer.     

Use of simultaneous double-tracer SPECT with 99m-Tc-technetril and 67-Ga-citrate for follow-up of patients with non-small cell lung cancer

In 20 primary patients with focal abnormalities on conventional CT we evaluated diagnostic properties of simultaneous double-tracer SPECT. Scintigraphy was performed as a single examination with simultaneous registration of 67Ga and 99mTc-MIBI. Image acquisition was started 48-74 hours after IV injection of 130-175 MBq 67Ga-citrate and immediately after IV injection of 500-740 MBq of 99mTc-MIBI. All images for each agent were classified as positive and negative for primary tumor, N1 and N2 lymph-nodes (LN). According to histology 18 of 20 evaluated patients had non-small cell lung cancer (NSCLC), the other two patients had tuberculosis and nonspecific inflammation. SPECT with 99mTc-MIBI correctly visualized tumor in 18, 67Ga allowed correct visualization in 16 cases. Both tracers were truly negative in a patient with tuberculosis and false positive in a patient with nonspecific inflammation. Double-tracer SPECT was slightly more specific than CT in primary lesions. In 18 patients histological verification of LN status was obtained: NO was revealed in 9 cases, N1 in 4 and N2 in 5 cases. Both tracers correctly discriminated LN-positive and LN-negative cases with 94% specificity. On the contrary, CT was false-positive in 3 and false-negative in another 5 patients. Differentiation between N1 and N2 LN involvement is crucial for therapy planning. 99mTc MIBI and 67Ga revealed N1 in 2 cases and N2 in 4 cases, the diagnosis was later verified by postoperative morphology. In 2 patients SPECT overestimated extent of LN involvement and LN status was changed after surgery from N2 to N1. In 18 patients results of 99mTc-MIBI and 67Ga augmented each other. Accuracy of LN staging by SPECT with 99mTc-MIBI and 67Ga was 83%. CT accurately determined LN stage only in 7 patients, it was overestimated in 7 and underestimated in 4 cases. SPECT with 99mTc-MIBI and 67Ga demonstrated high overall accuracy in diagnostics of regional LN invasion for patients with NSCLC. Diagnostic value of conventional CT was significantly lower. Correct level of LN involvement was determined by SPECT in 83% of cases.     

Susceptibility to and escape from complement-mediated lysis of guinea-pig hepatoma line-10.

Rabbit xenoantisera produced against diethyl-nitrosamine-induced strain-2 guinea-pig hepatoma line-10 cells (L-10) according to various immunization schedules were compared for their cytotoxicity on L-10 cells in the presence of guinea-pig complement. The highest activity was obtained with antisera (Cx-1) produced by repeated intravenous injections of living L-10 cells at high cell dosage, whereas intramuscular injections of living or glutaraldehyde-treated L-10 cells at similar frequency and cell dosage were less effective for the production of cytotoxic antibodies against L-10 cells. Intravenous injections of smaller cell doses were less effective. The cytotoxic antibody in Cx-1 antiserum was shown to be IgG by various methods including gel filtration on Sephadex G-200, ion exchange chromatography and immunoelectrophoresis of purified tumor-specific antibodies. It was concluded that L-10 cells can be lysed by guinea-pig complement and tumor-specific antibodies (IgG). Some antisera contained IgM antibodies which were not cytotoxic. A decrease in susceptibility of L-10 cells to complement-mediated lysis was observed when the cells were maintained in vivo for a long period (more than 20 passage generations). This was due to a lower density of tumor antigens on the cell surface. When tumor cells were treated with a second antibody directed against rabbit IgG or F(ab')2, cytotoxicity of Cx-1 antisera was completely abolished.     

Tumor targeting potential of liposomes encapsulating Ga-67 and antibody to Dalton's lymphoma associated antigen (anti-DLAA)

Dalton's lymphoma (solid tumor) was induced in Swiss mice by an 'Air-pouch' technique using ascites cells. A liposomal technique for radioimmunodetection and localization using Ga-67 and antibody has been developed. Dalton's lymphoma associated antigen (DLAA) was isolated and purified by ammonium sulfate fractionation and chromatography. The antibody to this antigen, anti-DLAA, was prepared by immunization of rabbits and purified by CNBr activated chromatography. The specificity of this antibody to the solid tumor antigen was ascertained by agar gel diffusion. Scintiscan studies were carried out using controls and Dalton's lymphoma bearing mice at various time intervals after i.p. administration of 10-20 mu ci of Ga-67, Ga-67 Ga-67 as liposomes and Ga-67 incorporated with antibody to DLAA as liposomes. Extensive studies carried out using liposomes of different sizes and charges revealed that anti-DLAA significantly improved the tumor uptake of Ga-67, thereby facilitating better visualization of the tumor. Negative and neutral liposomes delivered maximum radioactivity at the target tissue, whereas positive liposomes did not cause significant tumor accumulation of 67Ga. Biodistribution studies showed maximum tumor-associated activity that is 15.43% of the injected dose per gram tumor tissue was achieved with Ga-67 anti-DLAA liposomes compared to 6.6% with Ga-67 administered as such. Injection of Ga-67 liposome resulted in minimum tumor-associated activity that is 5.33%, with maximum uptake by liver and spleen. This might be caused by accumulation of liposomes in these organs. Incorporation of anti-DLAA might significantly improve uptake of Ga-67 by target tissue, due to the specificity of the antigen antibody reaction.     

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.