Three separate techniques were used to demonstrate B and T cells forming rosettes in unimmunized and immunized mice: a) anti-@ serum and brain-absorbed anti@ serum as control, b) anti-H-2 serum in neonatally thymectomized mice reconstituted with semiallogeneic thymus cells, c) radioiodinated anti-immunoglobulin serum to demonstrate surface immunoglobulins o n rosette-forming cells (RFC).The three methods gave very c.onsistent results. More than 80 5% of background RFC were B cells; after priming with sheep red blood cells the proportion of T RFC rose t o 40 -50 % by days 6 --10, and then declined.

Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme-antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies on this unique approach to ADEPT.

Sensitivity to allogeneic cell surface antigens was transferred to naive mice by lymphoid cells from sensitized mice. Sensitivity was detected 24–48 h after antigen challenge and was associated with a mononuclear cell infiltration. The cells responsible for transfer were T lymphocytes as demonstrated by successful transfer with cell suspensions enriched for T lymphocytes, and by abrogation of transfer following treatment with anti-Thy-1.2 serum and complement. Both Ly1⁺ and Ly2⁺3⁺ bearing T cells were involved. Removal of Ia⁺ cells had no effect on the ability of sensitized lymphoid cells to transfer the reaction to naive mice.

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