| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 88 === English Abstract
Tumor formation is a multi-step process involving the accumulation of genetic mutations. Tumor cells often express abnormal gene products due to their transformation, producing tumor specific antigens that may be used for vaccination to induce immunity against parental or metastastic tumor cells. Malignant tumors, however, have a high rate of TAP-1, TAP-2 and HLA class I loss. Activation of T-cells requires both signal 1 (interaction of TCR and peptide-MHC) and signal 2 (interaction of costimulatory molecules). If a tumor cell down regulates its antigen-presenting molecules, an active antitumor response may not be generated.
We designed transgenes to produce cell membrane-bound proteins which contain the transmembrane region of murine B7-1 fused to a single chain antibody fragment (scFv) of anti-CD3. A human IgG1 Fc domain (from hinge to CH3 domain, g1) was inserted between these two domains. The B7 transmembrane domain was designed to allow the scFv to be anchored on the cell surface. The scFv can bind to CD3 and directly activate T cells. The g1 domain can allow the chimeric protein to form disulfide-linked dimers. The plasmid was transfected into tumor cells to generate ''immunostimulatory tumor cells''. The genetically engineered tumor cells should stimulate and activate the naive T-cells.
Anti-CD3 scFv dimers expressed on the cell surface induced CD25 (IL-2 receptor a-chain) expression and proliferation of splenocytes. CT26 tumor cells engineered to express surface scFv dimers (CT26/2C11) also induced potent lymphocyte cytotoxicity with or without addition of exogenous IL-2. Splenocytes activated by CT26/2C11 cells also killed wild-type CT26 cells, indicating that activated splenocytes could kill bystander tumor cells. Immunization of BALB/c mice with irradiated CT26/2C11 cells did not protect against a lethal challenge of CT26 cells, suggesting that systemic immunity was not induced. However, the growth of CT26 tumors containing 50% CT26/2C11 cells was significantly retarded compared to CT26 tumors whereas CT26/2C11 tumors did not grow in syngeneic mice. These results suggest that expression of anti-CD3 scFv dimers on tumors may form the basis for a novel therapeutic strategy for tumors that exhibit defects in antigen processing or presentation.
Coexpression of anti-CD3 scFv and costimulatory molecules CD80 or CD86 on tumor cells promoted more efficient activation of T cells. We show that coexpression of CD80 or CD86 with scFv enhanced T-cell proliferation, reduced the stimulation threshold, increased IL-2 production, prolonged T-cell viability and generated more potent T-cell cytotoxicity compared with scFv alone. Coexpression of scFv with CD80 or CD86 allowed activation of both CD8+ and CD4+ T-cells whereas scFv alone only activated CD8+ T-cells. The potent activation of naive splenocytes by scFv dimers and B7 molecules expressed on tumor cells indicates that this strategy may be more useful for the gene-mediated therapy of tumors that display defects in antigen processing and presentation.
Expression of chimeric proteins on the plasma membrane has some obstacles. Several membrane-bound protein transgenes were constructed to investigate the surface display of chimeric proteins. We found that the extracellular domain, TM and spacer are all important for surface display. The membrane-bound proteins also appeared to possess an universal proteolytic cleavage site near the plasma membrane. If surface proteins can be designed to prevent this proteolytic degradation, surface display may be greatly improved. According our investigation, the design of membrane-bound proteins should include a proper TM which contains an intact cytoplasmic domain and a long length spacer which can allow the chimeric proteins to form dimers and protect the chimeric proteins from proteolytic degradation.
Chinese abstract ..................................................................... 13
English abstract ..................................................................... 15
Chapter 1 Background and Strategy
1.1. Tumor antigens .............................................................. 18
1.2. Activation of T-cells ..................................................... 19
1.3. Tumor vaccines .............................................................. 21
1.4. Bispecific antibodies ..................................................... 24
1.5. Strategy ............................................................................ 25
Chapter 2 The expression of chimeric scFv on the cell surface
2.1 Abstract ............................................................................. 35
2.2 Introduction ....................................................................... 36
scFv
1. Anti-tumor antigen scFv-TM chimeric receptors ........................... 37
2. ScFv-TM chimeric receptors for activating T-cells ......................... 39
3. Anti-phOx scFv-TM chimeric receptors ......................................... 40
4. Other functional surface scFv ........................................................ 42
Other novel MBPs ................................................................... 43
1. Membrane-bound Enzymes ........................................................... 43
2. Membrane-bound Cytokines ........................................................ 44
3. Membrane-bound Fc Chimeric proteins ........................................ 44
4. Membrane-anchored reporter chimeric proteins ............................. 45
2.3 Factors that affect surface expression ......................... 46
1. Transmembrane domains .............................................................. 46
2. Spacer ........................................................................................ 47
3. Protein stability ........................................................................... 48
4. Extracellular domains .................................................................. 49
2.4 Conclusions ....................................................................... 50
Chapter 3 Active anti-CD3 single-chain antibody dimers expressed on the plasma membrane of tumor cells to induce cytotoxicity
3.1 Summary .............................................................................. 62
3.2 Introduction ......................................................................... 63
3.3 Materials and methods ...................................................... 65
3.4 Results .................................................................................. 72
3.5 Discussion ............................................................................ 77
Chapter 4 Enhanced cytotoxicity of lymphocytes activated by anti-CD3 single-chain antibody dimers and B7 molecules expressed on the plasma membrane of tumor cells
4.1 Summary .............................................................................. 99
4.2 Introduction ......................................................................... 100
4.3 Materials and methods .................................................... 102
4.4 Results ................................................................................ 108
4.5 Discussion ......................................................................... 113
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