ACTIVE SPECIFIC IMMUNOTHERAPY IN MAN--A MURINE MODEL
Principal Investigator: Barry Kahan
Abstract: Tumor-specific transplantation antigens (TSTA), extracted and purified from methylcholanthrene (MCA)-induced C3H/HeJ murine sarcomas offer unique reagents to dissect and augment syngeneic host resistance and to analyze weak peptide determinants. During the previous grant period TSTA, extracted with 1-butanol and partially purified by preparative isoelectric focusing, was demonstrated to potentiate the therapeutic effects of a variety of immunostimulants: (1) cyclophosphamide (20 mg/kg IP once), (2) continuous intrasplenic (IV x3) infusion of Interleukin-2 (120 U/day x7 days), (3) liposome-encapsulated 10 mug muramyl-tripeptide (IV x3), and/or (4) adoptive IV systemic transfer of 107 cloned cytotoxic T lymphocytes. The multi-modality TSTA regimens overcame the outgrowth of 3 (<1 mm), 7 (2.6 mm) or 14 (10 mm) day established MCA-F subcutaneous neoplasms, and almost vitiated spontaneous pulmonary metastases following amputation of a limb bearing the MCA-F-4 (lung avid) tumor. The studies proposed herein represent extensions to models of poorly- or non-immunogenic tumors, such as autochthonous MCA-induced, non-immunogenic MCA-T, and spontaneous teratocarcinoma ASKN neoplasms, and to the di- methylhydrazine-induced C57BL MC-38 colonic adenocarcinoma, as well as to almost subliminal (102) or massive (107) initial MCA-F cell inoculations. A second series of studies strives to improve the immunogenicity of extracted TSTA by a) purification using preparative isotachophoresis and/or high performance gel permeation chromatography; b) ex vivo incorporation of antigen into liposomes or incubation with resting or gamma- interferon/bacteria lipopolysaccharide activated, butanol-treated or untreated macrophages; c) autoaggregation, homologous cross-linkage, or keyhole limpet hemocyanin hetero-conjugation; and d) bead particle exposure. The contribution of antigen processing will be evaluated using paraformaldehyde-fixed macrophages or their plasma membranes, as well as by exposure to lysosomotropic and other agents to disrupt antigen digestion and cytoplasmic association with Ia determinants. A third series of studies uses already produced T- cell clones, which were isolated from an in vivo-sensitized, host spleen cell population specifically re-stimulated in vitro with extracted TSTA. Clones fused with BW5147 to obtain T-cell hybridomas will provide indicator cells for antigenic epitopes on purified TSTA molecules and their tryptic peptides, as well as a source of specific T-cell receptors for immunization to produce anti-clonotypic antibodies, possibly suitable as therapeutic immunogens (with or without linkage to TSTA or to anti-CD3 antibodies). The proposed studies thus proffer a molecular approach to dissect potential antigens augmenting host resistance for active specific immunotherapy.
Funding Period: 1981-03-15 - 1993-06-30
more information: NIH RePORT