T Cell Receptor Cdr3¦Ä Transplant Antibody (ot3) - Immunotoxin Anti-tumor Research And Cdr3¦Ä Peptide Conformational Studies,

γδT cells have a broad anti-tumor spectrum with distinct antigen recognition mechanism fromαβT cells and immunoglobulins.γδTCR recognizes antigens in a way like pattern recognition receptors.The specificity and affinity ofγδT cell receptor(γδTCR) lies in its CDR3δ,the Complementary Determinant Region 3 ofδchain.In our previous studies,the OT3 peptide,whose sequence was from CDR3δof ovarian cancer infiltratedγδT cells,could bind with many kinds of tumor cell lines and tissues.In this experiment,application of a novel antibody in experimental oncology was evaluated,whose CDR3H is replaced by sequence of OT3.First,this antibody named OT3Ab was expressed by murine J558L myeloma.The heavy chain with CDR3δcame from human IgG1 expression vector PNγ1-V_H62-CDR38 and light chain was from J558L cells.After reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting confirmation,OT3Ab exhibited specific binding with ovarian cancer cell line SKOV3 by flow cytomtry.In vitro experiment also demonstrated OT3Ab could mediate antibody-dependent cell-mediated cytotoxicity against SKOV3 cell line.In vivo radioisptope imaging in nude mice bearing SKOV3 tumors showed targeting of ^99mTc labeled OT3Ab to tumor tissue.Then an immunotoxin OT3AbDT was constructed by chemically conjugation of OT3Ab and diphtheria toxin(DT).After purification through high performance liquid chromatography(HPLC) on a size exclusion column,non-reducing SDS-PAGE and Western blotting were carried out to confirm the successful conjugation.The binding ability of OT3AbDT with SKOV3 cells was not affected,as shown by immunofluorescence under light microscope.³H-Leucine incorporation inhibition assay showed the 50%inhibition concentration of OT3AbDT for SKOV3 tumor cells was between 1 - 10μg/ml. Then OT3AbDT was used for experimental immunotherapy in subcutaneous tumor bearing nude mice models.Four intratumoral injections of OT3AbDT(13.8μg each time) greatly decreased the sizes of SKOV3 tumors,significantly different from PBS control. The OT3AbDT was also injected intravenously to tumor-bearing nude mice subcutaneously inoculated with SKOV3,HepG2(hepatocellular cancer cell line),Hela (cervical cancer cell line) and HR8348(rectal cancer cell line) cells.The growth of tumors with different origins exhibited slowing down to different extent after 4 times administration,too.In the second part of the dissertation,the CDR3δpeptides OT3 and ch1 that was from chronic hepatitis B circulatingγδT cells,undertook molecular dynamics simulation to study their structure in solution.The conformations of OT3 and chl peptides after clustering analysis were both "loop"-like.The V,D,and J segment random mutations in chl peptide,led to conformational distortions to different extent;but the changes in conformation did not correlate with the decrease of binding to hepatitis B virus(HBV) genome transfected HepG2 2.2.15 cells.It suggested the CDR3δpeptides bind with HBV infected cells in a conformation-independent way.