A method of membrane preparation for immunoassay.

Membranes prepared from a variety of solid tissues were used as solid-phase antigens for ELISA or RIA after fixation onto polylysine-primed 96-well plates. The preservation of antigens in these membrane preparations was tested by reactivity in ELISA using 2 monoclonal antibodies: W6/32, which recognizes an HLA framework antigen (a protein antigen) and anti-SSEA-1, directed to a carbohydrate antigen carried on glycoproteins. Levels of antigen deposition and usefulness as solid-phase antigens were assessed for ELISA as compared to RIA. Coated plates may be frozen for many months with preservation of antigenic activity. This method is relatively simple, rapid, and is useful for preparation of tissue antigens for immunoassay, especially for screening monoclonal antibodies.

Radioimmunolocation of a heterotransplanted human choriocarcinoma (BeWo) using monoclonal anti-SSEA-1: pharmacokinetics.

Stage-Specific Embryonic Antigen-1 (SSEA-1), originally discovered on mouse teratocarcinomas, has since been found on some human non-seminomatous germ-cell tumors and adenocarcinomas, as well as on some adult mouse and human tissues. A monoclonal antibody to this antigen (anti-SSEA-1; IgM, kappa) was used for radioimmunolocation. Nude mice bearing the human choriocarcinoma BeWo, which is SSEA-1 positive, were injected using a mixture of [131I]anti-SSEA-1 and [125I]MOPC 104E, an unselected myeloma protein of the same heavy-chain isotype. Animals were sacrificed at 24 hour intervals; the radioactive deposition due to both antibodies was determined for both tumors and normal organs. Accumulation of anti-SSEA-1 in the tumor was consistantly rapid and specific, while little accumulation of the unselected myeloma protein occurred. At five days after injection, an average of 3% of the initial dose of specific antibody was retained per gram of tumor; the tumor/blood ratio was 11, tumor/muscle was 80. Gamma-camera imaging allowed ready location of the tumors. Tumors could also be imaged using F(ab')2 antibody fragments.

In vivo antitumor effect of methotrexate conjugated to a monoclonal IgM antibody specific for stage-specific embryonic antigen-1, on MH-15 mouse teratocarcinoma.

Methotrexate (MTX) was coupled to an IgM monoclonal antibody specific for stage-specific embryonic antigen-1 (SSEA-1), and the resulting immunoconjugate (MTX-anti-SSEA-1) was used for in vivo drug targeting in mice bearing MH-15 teratocarcinoma. Immunoconjugates having an average of 65 mol MTX/mol antibody retained full antigen-binding capacity. Mice bearing well-established tumors (approx. 1 g) were treated i.v. using the immunoconjugate. MTX-anti-SSEA-1 at 15 mg/kg of drug had significant antitumor activity with no significant systemic toxicity. Neither an irrelevant isotype-matched conjugate, MTX-MOPC-104E, prepared from the MOPC 104E myeloma protein, nor free MTX injected alone or with either antibody had any significant antitumor effect. These results indicate that IgMs can be effective drug carriers for tumor targeting in spite of their high molecular mass, and that antigens that are selectively accessible in tumors, even though present in normal tissues, can be suitable targets for in vivo chemoimmunotherapy.

Tumor location and drug targeting using a monoclonal antibody (anti-SSEA-1) and antigen-binding fragments.

Both murine and heterotransplanted human nonseminomatous germ-cell tumors have been successfully located by external scintigraphy using radioiodinated anti-SSEA-1, a monoclonal IgM, and its pepsin-derived antigen-binding fragment, F(ab')2 mu. Antibody localization in the tumor is mainly due to antigenic specificity, rather than to nonspecific trapping, and depends strongly on the amount of time after injection. The antibody has been used for drug targeting in vitro and in vivo.

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies.

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, kappa) at ratios of 1.2-35 mol dye/mol antibody and 9.2.27 (IgG2a, kappa) at 0.6-6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.

Toxicity of intravesical recombinant human tumor necrosis factor in cynomolgus monkeys.

Six groups of two cynomolgus monkeys were treated with escalating intravesicular doses of recombinant human tumor necrosis factor (rHuTNF) for a 6 week interval. The doses of rHuTNF ranged from 10 ng to 1 mg and were instilled weekly. Two monkeys had instillation of saline only and served as controls. The monkeys were weighed and temperatures determined before, immediately after, and 2 days following each treatment. Cystoscopic examination was performed 2 days after each treatment and blood samples were obtained. At the conclusion of the study, animals were killed and necropsy was performed. There was no observable toxicity from treatment with rHuTNF. There was no difference between treated and control monkeys with respect to temperature, weight, or blood measurements. No drug-induced alteration in bladder morphology was found by either cystoscopic or microscopic pathologic examination.