1. Not Only Chemotherapy Anti-Tumor Vaccines for Tumor Eradication
Irit Avivi
Department of Hematology and BMT
Rambam Medical Health Care Campus
Haifa
The papers presented by Drs. Mitchell, Kirkwood, weber and haluska all focus on the primary challenge confronting the field of melanoma cancer vaccines. Specifically how does one move from the potential promise of vaccine therapy towards the development of an effective treatment for patients with melanoma.

2. Talk Plan The immune system in the healthy person
Why does the immune system fail to eradicate cancer ?
Strategies to overcome this immunological impairment
Vaccine studies in Myeloma
Future directions

3. Current Approach to Cancer
Targeting tumor cells chemotherapy – attacking divided cells
The tumor is a foreign entity
Is their any evidence for anti-tumor response?

4. Evidence for the Role of the Immune System on Cancer Growth Allogeneic stem cell transplantation
recipient
donor

5. Evidence for the Role of the Immune System on Cancer Growth Allogeneic stem cell transplantation
recipient
donor
Graft-vs-host disease is associated with graft vs tumor effect

6. More Evidence for the Role of the Immune System on Cancer Growth
T cells & Antibodies directed against tumor cells
exist in the circulation
However
Immune responses are ineffective in
preventing disease establishment and progression

7. Why ?

8. The Immune Response T B T
FORIEGN
FORIEGN T T T T B DC

9. Dendritic Cells- The most important Antigen Presenting Cells
Monocytes
iDC
mDC
DC maturation

10. Brezofsky et. al., JCI 2004, 113(11): 1515-1525.
Berzofsky et al JCI 2004

11. Antigen Presentation
mDC T
MHC- peptide
CD40L
T cell activation

12. How does the Tumor Escape the Immune System?
Anergy
Activation DC
Tumor
Tumor
VEGF
IL6

13. Antigen Presentation
In patients with melanoma, the presence of tumor associated antigens and their recognition by the host T cell repertoire has, by definition, not led to an effective immune response. The primary explanation is thought to be due to ineffective presentation of antigen by tumor cells and compromised function of tumor specific effector cells in the cancer bearing patient. Vaccine development has sought to overcome these impediments. As outlined in this schema, presentation of antigen by tumor cells in the absence of costimulatory signals leads to anergy while presentation of tumor antigens by APC leads to a productive immune response

14. How does the Tumor Escape the Immune System?
CTLA-4 T T T T T T
Trg
PD1
Activation
PD1
Anergy
PDL1 DC
Tumor
Tumor
PDL1
VEGF
IL6

15. How Does the Tumor Escape the Immune System?
The tumor expresses Host Ags
It impairs DC maturation and function
Interferes with Antigen presentation
The tumor interacts with T cells Anergy
The tumor express inhibitory molecules
The tumor induces accumulation of regulatory T cells

16. Strategies to Overcome Tumor-Induced Immunological Defects
Tumor escape Overcoming
Host Ags Tumor specific Ags
Immunogenic stimulators
Impaired DCs Maturation Ex vivo DCs maturation
T cell Anergy Interfere with
mechanisms involved
in anergy

17. Results in Potent Mature DCs
Ex vivo DC Maturation
Results in Potent Mature DCs
Ex vivo
In Vivo= with Tumor T T T DC T DC
DC maturation

18. Strategies to Load Tumor Antigens onto DC
Individual Ag
Whole Cell
Loading of
Tumor peptides
Loading of Tumor lysate
Advantages
Tumor specificity
Feasibility
Monitoring of immunologic response against defined antigen
Disadvantages
Limited number of antigens
HLA restriction
Tumor evasion through down regulation of antigen expression
Tumor cell fusion

19. Vaccination with Individual Antigens
Advantages
Tumor specificity
Feasibility
Monitoring response against defined Ag
Lower risk for autoimmunity
Disadvantages
Limited number of antigens
HLA restriction
Tumor evasion through down regulation of antigen expression
Advantages
Tumor specificity
Feasibility
Monitoring of immunologic response against defined antigen
Disadvantages
Limited number of antigens
HLA restriction
Tumor evasion through down regulation of antigen expression

20. Vaccination with Whole Cell Derived Antigens
Advantages
Broad response limits risk of tumor evasion
Presentation of unidentified & patient’s -tumor specific Ags
Presence of helper (DC4) and CTL (CD8) response crucial for maintenance of long term immune response
Disadvantages
Technical challenge of manipulating whole cells for multi-center setting
Risk of auto-immunity
Advantages
Broad response elicits helper and CTL immunity limits risk of evasion Presence of helper and CTL response crucial for the maintenance of long term immune response
Presentation of unidentified and patient specific antigens, allows host immunity to “choose” immunogenic epitopes
Disadvantages
Technical challenge of manipulating whole cells for multi-center setting
Risk of auto-immunity

21. Fusion Vaccines

22. Why did we Choose Myeloma?
Relapse rate
7 year survival
Khaled Y et al, BMT 44:325, 2009

23. Myeloma- Immunomodulated Disease

24. “Vaccination” with DC/MM Fusions

25. Vaccination with DC/MM Fusions: Trial Design
Phase I dose escalation trial
Eligibility
Patients with advanced stage myeloma
Presence of measurable disease
> 20% plasma cells in bone marrow for vaccine generation
Vaccination Schedule:
3 subcutaneous vaccinations in conjunction with GM-CSF administered every 3 weeks

26. n=18 Monocytes GM-CSF 100ug at vaccine site for 4 days Leukapharesis
Adherent PBMCs cultured for 5-7 days with GM-CSF & IL-4; TNF-a added for hours
Bone marrow aspiration
Doses prepared & frozen microbiology testing sent
Myeloma cells isolated
Fusion cells quantified by measuring dual expression of unique DC & tumor markers
CD38
CD138
CD86
Myeloma cells assessed for tumor & DC specific markers
MUC1
DC & myeloma fused with 50% PEG at DC: tumor, 3:1 to 10:1
CD138
CD38
DCs assessed for DC & tumor specific markers DR
CD40
CD80
CD86
CD83

27. Vaccine Characterization
Myeloma Cells CD-38
Dendritic Cells CD86
Fusion cell

28. Adverse Events Treatment associated events, transient grade I-II:
Injection site reactions 37
Edema 6
Muscle Aches 5
Fatigue 2
Fever 1
Chills/sweats 2
Diarrhea 1
Pruritis 1
Rash 2
Anorexia 1
No Autoimmune disorders
Episode of DVT/PE with antecedent history of DVT

29. Vaccine site reaction: Skin Biopsy

30. Immunological Response:
Vaccine Site Reaction
Recruitment of “ attacking” CD8 T cells
Recruitment of antigen- presenting DCs (CD1a)

31. Fusion Cells Stimulate
Allo – CD8 +T cell
Stimulation Index (SI)
(n=16)

32. Tumor Lysate Induced IFNγ Expression by CD8+ T cells

33. Vaccination Induced Immune Response
Pre-
vaccination#3
55.8
10.8
1 month post
vaccination 57
4.5
mobilization 51
1.78
Post-
transplantation 72
4.1
IFN
CD8 FITC 33

34. Persistent Response against Tumor Specific Antigen
% MUC1
CD8
Pre vaccine
1 month post
3 months post
Immunologic response associated with disease stabilization

35. Vaccination with DC/Myeloma Fusions: Conclusions
Vaccination is feasible and well tolerated
Majority of patients show immunologic response
Disease stabilization in a majority of patients

36. How to Improve the Efficacy of Fusion Vaccines?
Administer to patients with
minimal residual disease
Administer after eliminating Regulatory T cells

37. High Dose Therapy (HDT)
Provides minimal residual disease
Induces a significant reduction in
suppressive regulatory T cells
“ A vaccine supportive environment ”

38. Inclusion: ASCT ; Exclusion: Autoimmunity
Cohort 2
Induction
Therapy
Cohort 1 and 2 38

39. Study Update 41 patients enrolled to date
All underwent successful vaccine generation
21 - completed vaccinations, Med FU
22 - ongoing study
Vaccine details:
Mean percent of fusion: 40%; Fusion viability: 78%
Mean dose: 3.9 x 106 fusion cells
Safety: unchanged. 2 ANA without symptoms 39

40. Percentage of CD4+ T cells Percentage of CD8+ T cells
Immunological Response to Vaccine
Mean Percentage of Tumor Reactive T Cells
CD4
CD8
Percentage of CD4+ T cells
expressing IFN
Percentage of CD8+ T cells
expressing IFN
Pre Tx
Post Tx
Peak Post
Vaccine
Pre Tx
Post Tx
Peak Post
Vaccine 40

41. Clinical Response CR/nCR 67% Vaccination Post-tx Post-tx Transplant
Post-tx 1 month
Transplant
Post-tx
4 months
Post-tx
9 months 41

42. Response Rate Percentage of Patients Achieving CR Over Time
68% -complete response or very good partial response
>>>> 40% with HDT only 42

43. Vaccination with DC/MM Fusions Following SCT Conclusions
Feasible & well tolerated
Induces a significant anti-myeloma
immune response
Results in a high response rate
Strong correlation between immunological and clinical responses 43

44. How to improve?

45. How does the Tumor Escape the Immune System?
CTLA-4 T T T T T T
Trg
PD1
Activation
PD1
Anergy
PDL1 DC
Tumor
Tumor
PDL1
VEGF
IL6

46. PD-L1/PD-1 Pathway PD-L1 -an inhibitory ligand expressed by APCs
Signals via the costimulatory
complex
promotes
Inhibits T cell activation
Induces apoptosis of memory effector
cells & disrupts CTL mediated lysis

47. PDL-1 Expressed by Tumor is Associated with Tolerance
Shin & Rehermann Nature Medicine 2006

48. PDL-1 is Expressed by DCs and Myeloma Cells
IgG
PDL1 DC MM

49. PD-1 Expression on T cells
Control
Myeloma Patients
IgG
IgG
4.8%
17.3%
PD-1
PD-1
CD 4

50. Mean PD-1 Expression on CD4 T cells : Increase with Advanced Disease
% Expression (CD 4 T Cells)
p=0.004

51. CD4 T cell expressing PD-1 Increase Following Fusion Stimulation
% Expression of PD-1 (CD 4 T cells)

52. PDL-1 Expression on DC/Myeloma Fusions
CD 86
CD 38
PDL-1

53. PD-1 Blockade CT-011: clinical humanized PD1 antibody
Binds effector memory Tcs, resulting in their activation and inhibition of apoptosis
In animal models, administration induced anti-tumor immune responses & tumor regression
Clinical Trials:
Phase I trial : tolerated with evidence of clinical benefit
Administration post ASCT results in increased CD4+ effector memory T cells (Rotem-Yehudar et al., A1216; Ash 2009)

54. PD-1 Blockade Results in Downregulation of PD-1
Expression on CD 4 T Cells following Fusion Stimulation
% Expression of PD-1 (CD 4 T cells)

55. PD-1 Blockade Results in Increased
Interferon-gamma Following Fusion Stimulation
% Expression of IFNγ (CD4 T cells)
p=0.048

56. In Vivo Vaccine Primed Tumor Reactive T cells
are Increased in the Presence of Ex-vivo PD-1 Blockade )
CD CD8 INFG CD8INFG + AntiPD1
CD8/IgG
CD8/IFNg
CD8/IFNg 10 1 2 3 4
FL1-H
0.03
0.21
0.55
Pre-Vaccine #1
0.03
0.21
0.55
1 month
post vaccines
0.01
0.9
2.51

57. PD-1 Blockade Results in Increased Lysis of Autologous Tumor by Fusion Stimulated T Cells
Control
+ Anti-PD-1
23%
32%
Tumor
Granzyme

58. PD1-PDL1 pathway in Myeloma
Strong in vitro data supporting the potential role of PD1-PDL1 pathway in myeloma
- High expression of PDL1 on DCs & MM cells
- High expression of PDL on T cells
PD1-PDL1 blockade improved anti-tumor response in vitro
The in vitro data provide strong platform for combining anti PD1-PDL1 hrapy with

59. Directions Choose the right tumor: Immunogenic vs “Non Immunogenic?
Improve the immunogenic target:
specific peptides vs whole tumor ?
Control the inhibitory environment

60. Potential Approaches T T T T T T Trg Activation PD1 Anergy PDL1 CTLA-4DC
Tumor
Tumor
PDL1
VEGF
IL6

61. The Rambam- Harvard Vaccine Program
DC/MM – anti PD1L Myeloma vaccine study
DC/MM- immuno-mudulatory drug study
DC/AML- anti PD1L Myeloma vaccine study
DC/RCC – Sunitinib Study
Sunitinib -kinase inhibitor of Flt3, Kit, VEGF
and PDGF receptors
Pre-clinical study for vaccination for CLL

62. Acknowledgments
Hematology Department, RMC Tami Katz, Lina Bisharat, Jacob M Rowe Hematology Department, BIDMC Jacalyn Rosenblat David Avigan

63. Thanks