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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA029592-09
Application #
3482155
Study Section
Experimental Immunology Study Section (EI)
Project Start
1981-03-15
Project End
1993-02-28
Budget Start
1989-03-01
Budget End
1990-02-28
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
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