Harness the immune system in the treatment of cancer: A model

1. Harness the immune system in the treatment of cancer: A model Kirk R. Schultz, MD

2. Goals Of Presentation • A clinical model to understand immune responses to cancer in children • ALL • GVL • Components of GVL • Approaches to develop anti-ALL immune responses

3. Goals Of Presentation • A clinical model to understand immune responses to cancer in children • ALL • GVL • Components of GVL • Approaches to develop anti-ALL immune responses

4. Acute Lymphoblastic Leukemia In Children/adolescents • One of 4 children and adolescents diagnosed with cancer have ALL • ALL is the most common leukemia • Relapsed ALL is the sixth most common diagnosis in children with cancer

5. Life Years Saved In Children And Adults With Acute Leukemia In BC • Children/Adolescents vs. Adults Life Years Saved • 88% of life years ALL • 51% of life years saved total leukemias Life years saved

6. Characteristics of ALL therapy • One of the few malignancies that has an optimal response with a prolonged (2 - 3 years) low dose chemotherapy • Relapses can occur up to 7 to 10 years off of therapy

7. Biology of ALL • About 60-70% of diagnosed childhood ALL had the clone at birth • Approximately 1% of all umbilical cord blood studied have ETV6-AML1 (correlate with ALL) • Two-hit hypotheses (virus?): • Second hit results in second molecular event-transformation • Second hit results in loss of immune surveillance

8. Graft-Versus-Leukemia (ALL) • GVL first described in ALL • CML > AML > ALL • GVL is a well established form of immune therapy for childhood cancer • Understanding the mechanisms involved in GVL is an important tool to: • Improve GVL • Develop anti-leukemia vaccines

9. Graft-Versus-Leukemia • Primarily associated with chronic GVHD • Evidence lower relapse • URD vs. Auto BMT • Sib vs. Twin BMT

10. Goals Of Presentation • A clinical model to understand immune responses to cancer in children • ALL • GVL • Components of GVL • Approaches to develop anti-ALL immune responses

11. Immune Populations Involved in the GVL Response • T cells • TcRb • TcR • NK cells • Antigen presenting cells • DCs, B cells, and macrophages • Target cells - ALL

12. Components of the anti-ALL response we need to better understand • Effector cells • NK cells • T cells • Antigens recognized in an anti-ALL response • Role of APCs • ALL as antigen presenting cells

13. Effector cells in an anti-ALL response

14. T cells in the anti-ALL response • Much of the anti-ALL response in GVL appears to be T cell mediated • In most responses the antigen is poorly characterized • Alpha/beta T cells appear to be the major population

15. ALL-reactive T cells post BMT ALL specific response NK Activity

16. NK cells and ALL • NK cells may be important effectors in reactivity with pre-B ALL • NK-92ci is a IL-2 transfected Non-Hodgkin's cell line with activated NK cell activity • NK-92ci may be used as therapy for ALL after BMT or chemotherapy

17. TNF-a Treatment of ALL cells Increases NK-92ci Killing of ALL untreated 60 wortmannin 50 40 30 % lysis 20 10 0 untreated TNF-

18. NK-92ci Killing of ALL: Conclusions (Reid G et. al. Clin Exp Immunol. 2002) • TNF-a is able to augment ALL cell susceptibility to killing by NK-92ci • The activation incorporates both PI3 kinase dependent and independent • This support s further development of NK-92ci as a therapy for ALL post BMT and chemotherapy.

19. Antigens in the anti-ALL response

20. Antigens recognized in anti-ALL response • Fusion gene products • TEL-AL1 • BCR-ABL • Lack of GVL in MLL-AF4 • Overexpressed oncogenes • WT1

21. Antigen presenting cells in the anti-ALL response

22. Professional Antigen Presenting Cells • Dendritic cells • Most efficient • Primary response • B cells • Specific • Can be as efficient as DCs • Maintenance of response • Macrophages • All purpose

23. T cell - APC Interaction T cell IL-2R CD3 CD40L CD28 CTLA4 OX40 CD4 TCR CD40 TNF- CD80/86 IL-2 Ag OX40L MHC ALL cell

24. Antigen presentation in an anti-ALL response • DCs are important in anti-All response • We have previously shown that B cells are important for in vivo T cell priming to leukemia • Little is understood regarding the role of APCs in generation or maintenance of the anti-ALL response

25. CpG ODNs and Antigen Presentation • CpG ODNs are unmethylated bacterial DNA with immune stimulatory sequences • Effects include: • Effect B cells, DCs, NK cells through TLR9 • Increased co-stimulatory molecules • Increased T cell responses in vivo • We hypothesized that CpG ODN would alter immunogenicity of • pre-B ALL • Normal microenvironmental APCs (B cells, DCs)

26. 50 ALL 40 mature B 30 20 increase in % positive cells 10 0 -10 CD40 CD80 CD86 CpG ODN Treatment and Costimulatory Molecules on Patient ALL and Normal B Cells

27. CpG ODNs and ALL: Conclusions • CpG ODNs did not alter the immunogenicity of pre-B ALL • CpG ODNs do increase co-stimulatory molecule expression by resident antigen presenting cells in the bone marrow.

28. ALL as an antigen presenting cell

29. TEL-AML1 Acute Lymphoblastic Leukemia • Good prognosis • Late relapse off chemotherapy • Does a host response impact on recurrence of TEL-AML1 ALL?

30. TEL-AML1 ALL CO-STIMULATORY MOLECULES • Higher in TEL-AML1 ALL • CD40 • Lower in TEL-AML1 • CD86 % Positivity

31. TEL-AML1 ALLMHC MOLECULES (MCF) • Higher in TEL-AML1 ALL • HLA-DR MCF

32. Does Immune selection play a role in Relapse of pre-B ALL • We obtained ALL samples treated in selected therapy protocols for initial and relapsed ALL samples • Some paired samples

33. 1000 initial mean Relapsed mean 750 500 250 0 HLA-ABC HLA-DR Initial Diagnosis vs. Relapsed ALL HLA Class I and II Mean Channel Fluorescence • No change in • MHC class I • MHC class II

34. Allo-reactive T cell response - Cytokines • Initial • Th1 and Th2 • Relapsed • Th2 (IL-5) only

35. Initial Diagnosis vs. Relapsed pre-B ALL in Children 60 initial 50 * Relapsed 40 30 % positive cells 20 10 0 CD40 CD80 CD86 CD137L CLIP CD74 CD48

36. Changes in Immunogenicity of ALL Relapse after Chemotherapy • Differences initial diagnosis vs. relapsed ALL • No major differences • Costimulatory, MHC, adhesion molecules • Major difference • CD137L, a co-stimulatory molecule in T cell responses • Other evaluations are being performed • Paired samples • Other APC functions

37. Goals Of Presentation • A clinical model to understand immune responses to cancer in children • ALL • GVL • Components of GVL • Approaches to develop anti-ALL immune responses

38. Methylation and Acetylation’s Impact on Immunogenicity • Methylation of DNA and actelyation of histones both regulate the function of promoter genes • Inhibition of deacetylation can affect CD40 and CD86 expression • Inhibition of methylation can alter tumor suppressor and oncogenes

39. Methylation (AzaC) and Deacetylation (TSA) Inhibitors on Costimulatory Molecules 40 40 AzaC AzaC 30 TSA 30 TSA 20 increase in % positive cells 20 10 10 0 0 -10 -10 ALL1 REH RS4:11 ALL1 REH RS4:11 CD86 CD40

40. COG Relapsed ALL Study • Patients will receive 3 courses of intensive therapy followed by BMT for early relapse • Three hypothesized reasons that ALL will relapse after BMT • Drug resistance • Acquired ability of ALL to escape surveillance • Inability of donor to generate anti-ALL response

41. COG Relapsed ALL Study • Evaluation of ALL blasts at first and second relapse • Surrogate marker for relapse - MRD (Willman) • Evaluation for mechanism associated with drug resistance - microarray (Carroll) • Evaluation for loss of ALL immunogenicity - Schultz • Evaluation for ineffective donor anti-ALL response (measured 3, 6 and 12 months post BMT) • Cytokine polymorphisms - Goyal • Immune reconstitution - Schultz

42. ACKNOWLEDGEMENTS • Vancouver • Gregor Reid • Angela Alessandri • Rusung Tan • Jan Dutz • Sharon Bader • C-C Hsiao • Winnipeg • Kent HayGlass • Kansas City • Andy Gilman • Iowa City • Donald MacFarlane • COG • Steve Hunger • Paul Gaynon • Bill Carroll