טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLeshem Jasmin
SubjectCombining Immunotoxin with Immune Check Point Inhibitors
DepartmentDepartment of Biology
Supervisors Professor Yoram Reiter
Professor Ira Harry Pastan
Full Thesis textFull thesis text - English Version


Abstract

Immune checkpoint blockade using antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) benefits a limited number of cancer patients. SS1P and LMB-100 are immunotoxins that target mesothelin. Atypical delayed responses were observed in mesothelioma patients that received SS1P suggesting that anti-tumor immunity was induced. Our goal was to develop a mouse model of mesothelin expressing cancer and use it to determine if combining SS1P or LMB-100 with immune check point inhibitors would stimulate anti-tumor immunity. We constructed a BALB/c breast cancer cell line expressing human mesothelin (66C14-M) and, in addition, used a murine mesothelioma cell line that was transfected with human mesothelin (AE17M). To evaluate in-vivo effects, tumors were implanted in one or two locations. SS1P or LMB-100 was injected directly into established tumors and anti-CTLA-4 was administered i.p. In the 66C14-M model, we analyzed tumors to determine changes in the immune-related gene transcription and immune cell composition. In the AE17-M model, we evaluated the ability of SS1P to induce ATP secretion and calreticulin expression on the cell surface. Both are associated with immunogenic cell death. We found that in mice bearing two 66C14-M tumors, immunotoxin injection into one tumor together with systemic (i.p.) anti-CTLA-4, caused complete tumor regressions in 86% of the injected tumors and 53% of un-injected tumors. No complete regressions occurred when drugs were given separately. In the combination treated tumors, a massive immune activation was demonstrated. Compared to vehicle treated tumors, 341 of 747 (45%) immune-related gene transcripts were induced. In addition, tumor regression was associated with increased numbers of tumor infiltrating CD8-T cells and blocked by administration of antibodies to CD8. In the AE17-M model, we found that exposing cells to SS1P induced calreticulin expression on their surface and fostered secretion of ATP. The combination of intra-tumoral immunotoxin together with i.p. anti-CTLA-4 significantly prolonged the survival of AE17M tumor bearing mice compared to control groups. In both models, surviving mice were protected from tumor re-challenge, indicating the development of anti-tumor immunity.

Altogether, our findings support developing therapy for patients composed of intra-tumoral or i.p.. immunotoxins and systemic CTLA-4 blockade.