Johanna Olweus, Dept Immunology, Institute for Cancer Research, Radiumshospitalet, Oslo, Norway, presents at the ProImmune Antigen Characterization and Biomarker Discovery Summit, January 2011. Cancer immunotherapy: finding allies among the "allos"

2. Johanna Olweus , Radiumhospitalet , Oslo University Hospital , Oslo , Norway

3. Differences between the British and the Norwegians British architecture Norwegian architecture

6. BERGEN

8. The main function of the immune system is to protect against INFECTIONS It protects by recognizing and attacking FOREIGN, DANGEROUS MATERIAL It does not attack self - TOLERANCE Important characteristics of the immune system

9. High affinity T cells reactive with self-antigens are negatively selected Negative selection of high affinity T cells reactive with self-antigens Ed Palmer (May 2003)

10. Most tumor associated antigens (TAA) are self antigens expressed on a variety of normal cells Autologous immune responses directed to TAA rarely give side effects, but also rarely give clinical responses High affinity TCRs yield more efficient anti-tumor responses Rosenberg S et al, J Immunol 2006 and Blood 2009 When TAA expressed on normal cells are targeted with high affinity T cells, toxicity and side effects occur Schendel et al, J Clin Invest 2010, Restifo et al PNAS 2008 Time to rethink cancer targets and identify high affinity T cells?

11. Survivin is widely expressed on normal healthy cells In Silico Transcriptomics online Rolf Skotheim

12. Immunotherapy of cancer - what are the options?

14. Graft-versus-leukemia Modified from Bleakley et al, Nat Rev Cancer 2004 Can graft-versus-host reactive T cells be separated from graft-versus-leukemia reactive T cells? Graft-versus-host disease Patient cured Leukemia patient Allogeneic bone marrow/ stem cell donor

15. Can graft-versus-host reactive T cells be separated from graft-versus-leukemia reactive T cells? Transplanted donor T cells Patient cells / organs GVL GVHD + -/+ - Goal: to generate allo-reactive T cells that specifically kill hematopoietic cells

16. Models explaining the high frequencies of T cells recognizing allogeneic (foreign) HLA Allogeneic HLA: Peptide- dominant Allogeneic HLA: HLA dominant Autologous HLA : Peptide- and HLA specific Minor HAg Major HAg Major HAg Foreign Foreign Patient cell Donor T cell

17. Targeting foreign versus normal cell-type specific peptides Foreign complex foreign self foreign self Vaccination targeting foreign peptide In vitro targeting Celltype specific peptides Peptides derived from cancer cells HLA antigen Peptide Danger signal

18. Protocol to generate allo-restricted T cells that kill specific cell types E Stronen et al, Scand J Immunol 2009; 69; 319-28 CD20 derived peptide

19. CD20/A2 pentamer+ T cells were generated from all A2 negative donors Donor T cells HLA-A*0201 neg Pentamer staining Cytotoxic T cells ” Patient APC” HLA-A*0201 Peptide specific for hematopoietic cells (CD20) HLA-A*0201neg. donors HLA-A*0201pos. donor I Abrahamsen et al, Leukemia, online Sept 16, 2010 CD8 CD20 pentamer 1.0% 2.8% 0.2% 0.1% 0.5% 0.6% CD8 Pentamer CD20 pentamer Control pentamer <0.01% <0.01%

21. Specificity: CD20-specific T cells are not activated by 4 different CD20neg target cells

22. CD20-specific T cells kill Chronic lymphocytic leukemia (CLL) cells CLL Cytotoxic T cell line I Abrahamsen et al, Leukemia, online Sept 16, 2010

23. CD20-specific T cells kill primary follicular lymphoma cells N=2 Ingerid Abrahamsen, Synneva Kjellevoll, submitted

24. The CD20-specific CTL line shows dependency on a large number of amino acids for peptide recognition CD20 WT peptide sequence: S L F L G I L S V Position: 1 2 3 4 5 6 7 8 9 I Abrahamsen et al, Leukemia, online Sept 16, 2010 Counts 100% 84.6% 155% 88.1% 35% 62.2% 42.9% 42.5% 102% 178% HLA-A*0201 multimer CD20 WT S188A L189A F190A L191A G192A I193A L194A S195A V196A 1 2 3 4 5 6 7 8 9

25. CD20-specific T cell clones are dependent on 6-9 amino acids for recognition of the CD20p I Abrahamsen et al, Leukemia, online Sept 16, 2010 HLA-A*0201 multimer Counts CD20 WT S188A L189A F190A L191A G192A I193A L194A S195A V196A 1 2 3 4 6 7 8 9 5 1 2 3 4 5 6 7 8 9

26. Specificity of the CD20-specific T cells can be explained by a striking homology in their peptide-binding domains (CDR3b) I Abrahamsen et al, Leukemia, online Sept 16, 2010

28. Allogeneic HLA: Peptide- and HLA specific Autologous HLA : Peptide- and HLA specific Minor HAg Major HAg Foreign HLA-A*0201 Foreign HLA-A*0201/CD20 can select highly peptide-specific T cells from donors with distinct MHC background CD20-derived peptide

29. T cell specific for cell-type restricted protein T cell receptor (TCR) + Patient T cells ” Re-directed” T cells killing leukemia cells HLA-A2 negative, T cell depleted bone marrow transplant to replace immune cells TCR transfer HLA-A2 positive leukemia/lymphoma patient Clone TCR peptide HLA-A2 Cell-type-specific T cell (from HLA-A2neg donor) leukemia Alternative 2 Alternative 1 Alt 1 Alt 2 Therapeutic strategies for adoptive T cell therapy

30. Predicting and screening candidate hematopoietic peptides Predicting peptides using computer algorithm from candidate proteins Peptide synthesis and biochemical validation Pentamer synthesis Validation of epitopes in T cell cultures Søren Buus’ lab NetMHC ProImmmune Ltd. Olweus lab 150 peptides 70 peptide/MHC pentamers

31. Responses to 18/26 hematopoietic peptides * response at d26

33. A polyclonal, multitargeted, high avidity attack! CANCER CELL

37. Allo Foreign HLA-A2 Self HLA Peptide x Peptide y Auto OUR STRATEGY 1 Target identification 2 Generation of T cells 4 TCR sequencing 3 a Epitope discovery 3 b Epitope selection 5 TCR expression/ modification 6 TCR selection 7 GMP development 8 Clinical trial 9 Immune monitoring