Six cobalamin-biotin conjugates have been prepared. The cobalamin-biotin conjugates were prepared to evaluate the effect that the location of attachment had on the binding with transcobalamin II (TCII), the cobalamin binding protein in plasma, and to evaluate their potential use for in vitro and in vivo applications. This study focused only on the effect of binding with TCII. To decrease the possibility of steric problems in binding of the cobalamin conjugates with TCII, and biotin's binding with streptavidin or avidin, moieties of 11-18 atoms in length were used as linkers. Four biotin conjugates were prepared which were attached to the corrin ring of the cobalamin molecule (on b-, c-, d-, and e-side chains). One conjugate was attached to the 5'-OH of the ribose moiety, and another conjugate was attached at the cobalt metal (in place of the cyanide moiety of cyanocobalamin). Competitive binding studies were conducted where various amounts of the cobalamin-biotin conjugates and their precursor cobalamin derivatives competed with [57Co]cyanocobalamin for binding of recombinant human TCII (rhTCII). Evaluation of cobalamin derivatives which were conjugated at the 5'-OH of ribose or the cobalt metal center indicated that conjugation at either of these positions had little effect on binding with rhTCII. However, conjugates where the attachment was made on the corrin ring substituents had a large variation in binding with rhTCII. Conjugates on the e-propionamide side chain had little effect (relative affinity was equal to or decreased less than a factor of 3) on binding with rhTCII, conjugates of the b-isomer had decreased binding (relative affinity decreased less than a factor of 10), conjugates of the d-propionamide had further decreased binding (relative affinity decreased between 44 and 69 times), and conjugates on the c-acetamide group had poor binding to rhTCII (relative affinity decreased between 295 and 1160 times). The significance of the side chains on the corrin ring in providing specificity and high-affinity binding with rhTCII is discussed.

Technetium-99m labeling of antibodies has been suboptimal because of low affinity adventitious binding, nonspecific labeling, and loss of immunoreactivity. The diamide dithiolate ligand system (N2S2) forms highly stable, well-defined tetradentate complexes with Tc(V). Antibodies and their fragments have been labeled by conjugation of preformed "mTc4,5-bis(thioacetamido)pentanoate active ester to protein amine groups to give a chemically known 99"Tc-N2S2 complex covalently linked to antibody. Evaluations of the "'Tc-N2S2-bound antibodies and their fragments have shown high stability and retained immunoreactivity.Successful targeting ofdiagnostic radionuclides to tumors not only provides a tool to diagnose and stage cancer but also demonstrates feasibility for therapy where ligand systems can be applied to therapy radionuclides. Early studies with radiolabeled antibodies utilized radioiodine (1231/1311) because of extensive experience in protein radioiodination, covalent attachment, and ready availability of the radionuclide (1,2). Improved tumor-to-nontumor ratios were achieved with 1"'In compared to 131I by using diethylenetriaminepentaacetate (DTPA) bifunctional chelating agent technology (3)(4)(5) MATERIALS AND METHODS Preparation of 99mTc-4,5-bis(thioacetamido)pentanoyl (N2S2)-Conjugated Anti-Melanoma 9.2.27 F(ab')2 Fragment. To a mixture of 25 1.l of 4,5-bis(benzoylthioacetamido)pentanoic acid (1.0 mg/ml solution in 90% CH3CN) and 100 ,ul of 1 M NaOH was added 100 mCi of sodium [99mTc]pertechnetate in 1.0 ml of saline (0.9% NaCl). Then 1.0 mg of sodium dithionite (0.10 ml of a freshly prepared 10 mg/ml solution) was added, and the mixture was heated at 750C for 15 min.The pH was brought to about 6 with 0.10 ml of 1 M HCI and 0.30 ml of 0.2 M sodium phosphate buffer (pH 6.0). Then 10.0 mg of 2,3,5,6-tetrafluorophenol (0.10 ml of a 100 mg/ml solution in 90% CH3CN) and 12.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.10 ml of a 125.0 mg/ml solution in 90% CH3CN) were added, and the solution was heated at 75°C for 30 min. The resulting tetrafluorophenyl active ester derivative of "mTc-4,5-bis(thioacetamido)pentanoate was purified by loading the reaction mixture on a conditioned C18 cartridge (J. T. Baker), washing with 2.0 ml of 20% (vol/vol) ethyl alcohol/0.01 M sodium phosphate, pH 7.0, eight times, and eluting with 100% CH3CN. The solvent was evaporated under a stream of N2. Then 0.5 ml of the 9.2.27 F(ab')2 fragment (16) at 2.5 mg/ml and 0.50 ml of 0.2 M sodium phosphate (pH 9.0) were added for conjugation. After 15 min at room temperature, 25 mg of lysine (0.25 ml of a 250-mg/ml solution at pH 9.0) was added to quench unreacted ester. The 99mTc-N2S2-9.2.27 F(ab')2 was purified by passage through a G-25 Sephadex column (Pharmacia) equilibrated with phosphate-buffered saline.Abbreviations: N2S2, diamide dithiolate chelating system; 99M Tc-N2S2-9.2.27 F(ab')2, 99mTc-4,5-bis(thioacetamido)pentanoyl-9.2.27 F(ab')2 fragments; DPTA, diethylenetriaminepentaacetate.

An immunogen consisting of a 4M urea extract derived from human melanoma cells (M14), that was devoid of HLA-A,B,C, HLA-DR antigens and fibronectin was adsorbed to lens culinaris lectin-Sepharose 4B and used to immunize mice for production of monoclonal antibody to a melanoma-specific glycoprotein. Screening for hybridomas secreting antibodies to melanoma associated antigens was facilitated by use of a solid phase target antigen of chemically defined medium of melanoma cells (CDM). Use of these procedures allowed us to select 40 hybridomas secreting antibody which recognized determinants on melanoma cells not found on lymphoid cells. Further characterization of one of these hybridomas, 9.2.27, indicated that the antibody it secreted recognized a 240K dalton glycoprotein found on all melanoma cell lines tested but not on carcinoma, lymphoid, or fibroblastoid cultures. These results demonstrate the utility of soluble antigen preparations devoid of strongly immunogenic non tumor-specific molecules in the elicitation of tumor specific antibody. Preliminary results suggest that immunogens of this kind are superior to intact melanoma cells for production of tumor specific hybridomas.

The murine antimelanoma monoclonal antibody, 9.2.27, was administered intravenously to eight patients with metastatic malignant melanoma. Biopsies of metastatic nodules clearly demonstrate the selective localization of this antibody on the melanoma cell surface with a dose-response relationship to the quantity of administered antibody. The antibody infusions were clinically well tolerated and the pharmacokinetics of the antibody and the antiglobulin responses are described. This study indicates that murine monoclonal antibodies have potential as selective targeting agents in the design of future therapeutic trials using monoclonal antibodies or conjugates thereof in the treatment of cancer.

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