Localization of an anti-CEA monoclonal antibody in colo-rectal carcinoma xenografts

Summary A mouse monoclonal anti-CEA antibody (11.285.14) has been examined for tumour localization potential by assessing its distribution in immunodeprived mice with xenografts of human colon carcinoma cell lines HCT-8, HRT-18, HT-29, and LS174T and a xenograft (HRVB) established from a primary rectal carcinoma. With four carcinomas (HCT-8, HT-29, LS174T, and HRVB) preferential tumour localization of ¹²⁵I anti-CEA was seen. Compared with ¹³¹I normal IgG₁ localization indices of up to 4.4:1 were achieved. Up to 10% of the injected dose of ¹²⁵I anti-CEA was present/g of tumour tissue and with the largest xenografts examined (3–4 g) up to 40% of the total body reactivity was localized in tumour tissue. The tumour localization of ¹³¹I labelled antibody was visualized by external gamma camera imaging. Overall antibody localization correlated with the CEA content of the xenografts and the fourth colon carcinoma xenograft (HRT-18) and an osteogenic sarcoma xenograft (791T), both with very low CEA levels, showed no localization of the monoclonal antibody.     

Localization of anti-osteogenic sarcoma monoclonal antibody 791T/36 in a primary human osteogenic sarcoma and its subsequent xenograft in immunodeprived mice

Summary A primary human osteogenic sarcoma was visualized in situ by external gamma-camera imaging following administration of ¹³¹I-labelled anti-osteogenic sarcoma monoclonal antibody 791T/36. A xenograft of the tumour established in immunodeprived mice also showed localization of ¹³¹I-791T/36 determined by both gamma-camera imaging and organ distribution studies. Radiolabelled normal immunoglobulin showed no tumour localization. Expression of the 791T/36-defined antigen on xenografted tissue was further confirmed by reaction of its in vitro-cultured cells with fluorescein-labelled 791T/36 antibody.     

Improved monoclonal antibody tumor/background ratios with exchange transfusions

Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both ¹³¹I-5G6.4 specific and ¹²⁵I-UPC-10 non-specific MoAb. Twenty-four hours later 65–80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P < 0.05) decreased normal tissue activities of ¹³¹I (except for muscle) by 63–85%. while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1–0.8 to 2.3–6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

Summary The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumourspecific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with¹²⁵iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b > IgG2a > IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab)₂ preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.     

The detection of virally induced tumors by 131I- and 125I-labeled syngeneic monoclonal antibodies

Summary The binding of the syngeneic monoclonal antibodies IC5F5 and 4D2B4 to Rauscher virus-induced myeloid leukemic (RMB-1) cells was analyzed in vivo in tumor-bearing BALB/c mice. To verify it these antibodies bind specifically to RMB-1 cells, purified antibodies were iodinated with the isotopes ¹²⁵I and ¹³¹I. Mice previously inoculated with tumor cells were injected with these labeled monoclonal antibodies and the plasma clearance and the tissue distribution were determined. The clearance in tumor-bearing animals was faster than in control mice. The tissue distribution was corrected for nonspecific accumulation by scoring for an unrelated antibody. Calculation of a localization index showed that IC5F5 binds at least 4.5 times more specifically to tumor cells than to other tissues. A preferential localization of radioactivity in s.c. tumor tissue was seen in the scanning of animals injected with ¹³¹I-labeled antibodies. The most direct proof of specific binding was observed in autoradiograms of animals treated with ¹²⁵I-labeled antibodies. Small islands of tumor cells in the livers of mice inoculated i.v. had a high density of grains compared to other tissues and also compared to tumor cells in mice treated with unrelated monoclonal antibodies. These results show efficient targeting of these monoclonal antibodies and make immunotherapy of these myeloid leukemic cells possible.     

Site-specifically modified 111In labelled antibodies give low liver backgrounds and improved radioimmunoscintigraphy

Site-specific modification of monoclonal antibodies at their oligosaccharide had previously been demonstrated to produce excellent ¹¹¹In imaging in a xenograft model using a Brown Norway (BN) rat lymphoma and a rat anti-BN MHC monoclonal antibody [Lee C. et al. Fed. Proc. Abstr. 43 3014 (1984)]. These results are due, in part, to lack of liver uptake, so we wanted to evaluate the extent of hepatic uptake observed with different monoclonal antibodies in normal mice. Biodistribution data were obtained for four monoclonal antibodies by first modifying each antibody at its carbohydrate with a diethylenetriaminepentaacetic acid (DTPA) derivative. The antibodies were then labelled with ¹¹¹In and injected into normal mice. Images were obtained 24 h post-injection, and at 48 h the mice were dissected and the tissue-to-blood (T:B) ratios determined. T:B ratios were approximately 1 (or less) for every organ evaluated, indicating minimal non-specific uptake into these organs. Data is also presented for the BN-rat system which shows excellent localization into the tumor xenograft and low non-specific organ uptake. These data indicate that modification of antibodies site-specifically at their oligosaccharide results in minimal non-specific uptake into non-target tissues and enhanced localization into the tumor target, and that this may represent a preferred method for production of ¹¹¹In labelled antibodies.     

A potential new radiopharmaceutical for parathyroid imaging: Radiolabeled parathyroid-specific monoclonal antibody—I. Evaluation of 125I-labeled antibody in a nude mouse model system

Radioiodinated BB5-G1, a parathyroid-specific monoclonal antibody, and its F(ab′)₂ and Fab fragments were characterized using a nude mouse model system. Blood clearance studies indicated that the most slowly clearing species was the ¹²⁵I-BB5-G1 intact antibody, while the most rapidly clearing one was the ¹²⁵I-Fab fragment. ¹²⁵I-F(ab′)₂ retained its capacity to localize in the human parathyroid tissue implants with the uptake at 24 h being similar to that observed with the intact antibody. Poor localization was observed with the Fab fragment. These results suggest that BB5-G1 or its F(ab′)₂ fragment may be useful for parathyroid imaging.     

Comparison of the biodistribution of 75Se- and 131I-labelled monoclonal antibodies in nude mice

A monoclonal antibody, C-215, against colon cancer, was internally labelled with [⁷⁵Se]methionine. The biodistribution was studied in tumour-bearing nude mice and compared with the biodistribution of [¹³¹I]C-215. The tissue uptake was divided into three parts: antibody bound to the antigen, antibody in the extracellular space and uptake of the released radionuclide. [⁷⁵Se]C-215 showed a greater amount of antigen-bound antibody in the tumour, but also a greater unspecific uptake both in tumour and normal tissue.     

An antibody-lectin sandwich assay for the determination of CA125 antigen in ovarian cancer patients

A two-step forward sandwich assay was developed for the determination of the ovarian tumour associated glycoconjugate antigen CA125 with anti-CA125 Monoclonal antibody B27.1 on the solid phase and¹²⁵I-labelled wheat germ lectin as tracer in the solution phase. This Mab-lectin heterosandwich assay was optimized and the clinical utility was evaluated in sera from healthy volunteers and ovarian cancer patients. A correlation was established between Mab-lectin assay and the dual monoclonal antibody sandwich assay, TRUQUANT®OV2 RIA, that uses the same MAb B27.1 on the solid phase and a second¹²⁵I-labelled B43.13 MAb in the solution phase. A potentially improved clinical utility is suggested for the Mab-lectin assay. The unique format seems to identify novel isoforms of CA125 with different carbohydrate side chains that would otherwise be undetectable in the MAb-MAb sandwich assay wherein the paratopes are likely directed to protein determinants.     

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.     

Monoclonal antibodies against Mycoplasma hyorhinis: A secondary effect of immunization with cultured cells

Monoclonal antibodies were prepared against two different human tumour cell lines, the melanoma cell line SK-Mel-25 and the acute lymphoblastic leukemia T cell line CCRF-CEM. Presence of antibodies against human tumour cells in the supernatants of hybridoma cultures was tested by binding of ¹²⁵I-F(ab′)₂ anti-mouse IgG. On two occasions a hybridoma culture, initially selected for subsequent cloning as it seemingly produced antibodies against tumour cells, was later found to produce monoclonal antibodies specific for Mycoplasma hyorhinis. In immunofluorescent staining patchy structures were visible which seemed to be attached to the cell surface. By combined staining with FITC-conjugated anti-mouse immunoglobulin for monoclonal antibody, Evans blue for cytoplasm and Hoechst compound no. 33258 for DNA, the reaction against mycoplasma could be recognized. These results demonstrate that if cultured cells are used for preparation of monoclonal antibodies, there is a good chance that the selected hybridomas may produce antibodies against ‘culture artifacts’ such as mycoplasmas, in addition to the target antigens. Thus mycoplasma contamination of cell cultures poses a serious problem in the hybridoma research and the testing system for antibody specificity should be carefully monitored.     

Pharmacokinetics of a radioiodinated monoclonal antibody F(ab′)2 fragment in a xenograft model with circulating antigen

The pharmacokinetics of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment were investigated in athymic mice bearing OVCAR-3 ovarian carcinoma xenografts, a model in which the CA 125 antigen is present in serum. Nine antibody doses between 0.1 and 650 μg were studied. Optimal tumor to normal tissue ratios were obtained at 100–200 μg of F(ab′)₂. At most antibody doses, the pre-injection level of circulating CA 125 appeared to influence the localization of ¹³¹I activity in tumor, liver and spleen.     

Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fragment in mice with tumour xenografts revealed by the use of a residualizing radiolabel (dilactitol-125I-tyramine) and autoradiography

Summary Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (¹²⁵I) or by dilactitol-¹²⁵I-tyramine (¹²⁵I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with¹²⁵I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a binding site barrier effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.     

Unidirectional potentiation of binding between two anti-FBP MAbs: Evaluation of involved mechanisms

The monoclonal antibody MOv19 directed to a folate binding protein shows temperature-dependent potentiation of binding of the noncompeting monoclonal antibody MOv18 to the relevant antigen, but the mechanism involved in this phinomenon had remained unclear. Use of chimeric versions of both monoclonal antibodies and the F(ab′)₂ and fan fragments of MOv19 revealed an increment in MOv18 binding in all combinations irrespective of the orgin of the Fc portin of the monoclonal antibody. The potentiating effect of bivalent MOv19 fragments on ¹²⁵l-MOv18 binding was similar to that of the entire monoclonal antibody and occurred at saturating concentrations of both reagents at which monovalent binding prevails. Similarly, the monovalent fragment also induced a significant increase in MOv18 bunding. Howener, the potentiation sccurred only at very high concentrations of antibody fragment. Homologous inhibition was drastically reduced using MOv19 Fab fragment, suggesting a low binding stability of the monovalent reagent. Immunoblotting analysis and binding in the presence of exogenous purified folate binding protein indicated a cross-linking between soluble and cell surface molecules mediated by the bivalent monoclonal antibodies. The extentof the increase in MOv18 binging at O°C with high amounts of exogenous folate binding protein was lower than that obtained at 37⁰C in the absence of added molecule. Release of ¹²⁵l-MOv18 from the cell surface was significantly higher in the absence of MOv19 than in its presence. Affinity constant values of ¹²⁵l-MOv18 binding evaluated in the presence of MOv19 or control monoclonal antibody MINT5 were comparable, whereas the number of binding sites per cell detected by ¹²⁵l-MOv18 was significantly higher in the presence of MOv19 than MINT5. Together, the data suggest that monoclonal antibody MOv19 induces a conformational change of the molecule it binds that increases the number of antigenic sites anvailable for MOv18 binding and, in turn, the binding stability of the latter, MOv19 bivalency also contributes to the MOv18 binding increment by cross-linking released and cell surface–anchored folate binding protein molecules. © Wiley-Liss, Inc.     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

Drug localisation and growth inhibition studies of vindesine-monoclonal anti-CEA conjugates in a human tumour xenograft

Summary The distribution of tritiated vindesine (³H-VDS) was studied in the tissues and tumours of athymic mice bearing a human colorectal tumour xenograft. Selective tumour localisation was obtained when ³H-VDS was injected as a conjugate with a monoclonal anti-CEA antibody (11.285.14) but not as a conjugate with a non-binding monoclonal IgG1 (Ag8) or as free succinoyl-VDS. The amounts of VDS that localised in the tumour following injections of ³H-VDS-11.285.14 increased in proportion to the amount injected, over a wide dose range. Conjugates prepared using the Fab fragments of 11.285.14 showed no evidence of selective tumour uptake in comparison with normal tissues.Various dose levels of VDS-11.285.14 conjugate and free VDS were studied for effects on the growth of the tumour xenograft. A growth inhibition of 50% was obtained at 1.5 mg/kg with free VDS and at 2.5 mg/kg with conjugated VDS. The conjugate was, however, considerably less toxic.     

A monoclonal antibody (Po66) directed against human lung squamous cell carcinoma immunolocalization of tumour xenografts in nude mice

Summary Po66, a mouse IgG1 monoclonal antibody, was produced by immunization against a patient lung squamous cell carcinoma. The tissue reactivity of the antibody was measured by a radioimmunological assay with enzymatically dissociated cells, by an immunofluorescence test on frozen tissue sections and by peroxidase-staining of paraffin sections. The antibody bound to lung squamous cell carcinoma, oesophagous carcinoma and, inconsistantly to lung adenocarcinoma but not to the other tumours tested. Some normal tissues also reacted positively, in particular bronchial serous glands, oesophagus epithelium and renal distal and collecting tubules. In normal and malignant tissues showing epithelioid differentiation, Po66 bound to the intermediate maturation area. The antigen immunoprecipitated by Po66 from lung squamous cell carcinoma appeared as a single band with a molecular weight 47000 to 50000 daltons. Purified monoclonal antibody Po66 and an unrelated IgG1 immunoglobulin were labelled with radioactive iodine and injected i. v. into nude mice bearing subcutaneous xenografts of human lung squamous cell carcinoma. The localization index in the tumour was 3.3. Antibody labelled with ¹³¹I allowed gamma-scintigraphic imaging of the xenografts which were clearly outlined by days 9 to 11.This work was supported by Association pour la Recherche sur le Cancer     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA