טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKeren Sinik
SubjectBiological Properties of Recombinant TCR-like Antibodies
DepartmentDepartment of Biology
Supervisor Full Professor Reiter Yoram
Full Thesis textFull thesis text - English Version


Abstract

The advance made in understanding immune responses against cancer revealed the central role that HLA-peptide complexes play in these diseases. Our laboratory has isolated recombinant antibodies that recognize tumor-associated HLA-A2-peptide complexes with T-cell receptor like specificity. In the work presented herein, these antibodies were used to study the presentation of the melanoma differentiation antigens MART and gp100 on melanoma cells. Although HLA-peptide complexes are not thought to be involved in inducing changes in target cells, there are reports demonstrating that mAbs specific to HLA class-I can mediate apoptosis of hematopoietic target cells. Therefore we investigated the biological effects TCR-like antibodies may induce on melanoma cells by studying their ability to mediate protein synthesis inhibition or target cell death.

Our results demonstrate that TCR-like antibodies can specifically recognize MHC-peptide complexes on peptide-pulsed antigen presenting cells (APCs) and on melanoma cells. We showed that bivalent TCR-like IgGs exhibit improved binding properties toward peptide-pulsed APCs and tumor cells compared with scFv/Fab fragments. These IgGs were used to characterize primary melanoma cells from patients by specifically detecting the presentation of MART and gp100 derived epitopes on the surface of these cells.

Interestingly, the TCR-like monovalent scFv/Fab antibodies were able to induce a biological effect as indicated by their ability to inhibit 30% of protein synthesis in melanoma cells. The TCR-like bivalent IgG antibodies exhibited a specific dose and time dependent cytotoxic effect on peptide-pulsed APCs; this effect was not observed in melanoma cells. To increase HLA class-I expression and possible effective cross-linking in melanoma cells, IgG concentrations were decreased or cells were stimulated with INF-γ which induce MHC class-I expression. While stimulation with INF-γ had no effect on target cell death, decreasing antibody concentration resulted in specific dose dependent apoptosis.

Moreover, we present preliminary data on the effect of INF-γ on antigen presentation of melanoma differentiation antigens-derived epitopes. INF-γ affected differently the expression of MHC class-I, the HLA-A2 allele and epitopes derived from various melanoma antigens. This effect was cell line dependent.

In summary, the studies presented herein demonstrate the potential use of our TCR-like antibodies as a tool to study antigen presentation on APCs, tumor cells and more importantly on cells from patients. Further investigation is required to determine whether these antibodies can mediate target cell apoptosis. Our TCR-like antibodies enabled us to study the effects of INF-γ in a qualitative and quantitative manner to better understand the mechanisms involved in antigen presentation.