Efficacy of Combinatorial anti-cancerchemotherapy and immunotherapy:    A central role of dendritic cells       Mohamed La...
The Beginning of Cancerous Growth:      A process that mimics the cancer grows in Egypt                             before...
EGYPT
Cancer Therapy             In clinic• Surgery:   Effective but doesn’t cure tumor  metastasis or prevent tumor  recurrence...
Cancer Therapy         In clinical trials• Immunotherapy:   can cure local and distant  tumors and prevent tumor  recurren...
Cancer Immunotherapy    Antibodies    Cytokines (IL-2, IFN-a)               (IL- IFN-    Adoptive T Cell Therapy    Va...
Activation of DCs is essential to prime T cells                    ?                                        ?             ...
Challenge to cancer Immunotherapy:          1- Tumor antigens are selfCancer cells originate from self cells (i.e. not for...
Challenge to cancer Immunotherapy:   2- Tumor cells escape from immune cells Down regulate the receptors required to inte...
Challenge to cancer Immunotherapy:                    3- High tumor cells/T cell ratio                 1:10,000 T cells   ...
Adoptive T cell transfer therapy3- Test T cell                                           4- Clonal   function             ...
Chemotherapy and Immunotherapy“Removal of weeds, and planting and watering good seeds”
Strategies to Improving Adoptive T Cell Therapy       Generation of      Chemotherapy    Vaccine       T cells in vitro   ...
Our Model of ChemotherapySystemic injection ofcyclophosphamide (CTX)                   (CTX)200 mg/kg (4 mg/mouse)        ...
Our models for adoptive T cell transfer              (Donor)                                     (Recipient)             T...
Chemotherapy and T cell Therapy          ProtocolPBS   CD8+ T cell Antigen                  AntigenCTX     1 x 106   primi...
CTX increased Ag-specific expansion of transferred     CD8 cells during effector and memory responses                     ...
How chemotherapy alters host    microenvironment                    10000          Blood                            8000  ...
Mechanisms of action of CTX:    1- Induces homeostatic expansion of transferred cells                                     ...
Mechanisms of action of CTX:                   2- A space “niche” is not a critical factor            CTX                 ...
Mechanisms of action of CTX:3- Decreases the numbers of regulatory T cells
Mechanisms of action of CTX:                     4- Type I IFNs are critical factors                                      ...
Mechanisms of action of CTX:              5- Inducing activation of dendritic cellsSalem et al. J Immunother 30(1): 40-53 ...
How Does Chemotherapy Benefit      Immunotherapy?              Creation of a space “niche”Elimination                     ...
Strategies to Improving Adoptive T Cell Therapy        Generation of      Chemotherapy    Vaccine        T cells in vitro ...
Focusing on Dendritic Cells   After Chemotherapy
Dendritic Cells are A Critical player in Anti-             Tumor Immunity   TLRLs                   No response
Mechanisms of action of CTX:    Inducing expansion of DCs during restoration phase                                        ...
CTX treatment induces an expansion of DCs         PBS               CTX
Post CTX expanded DCs express the phenotype of immature myeloid cells                                                Lymph...
How CTX Induces Dendritic cell               expansion? Is dose-dependent Induces proliferation of progenitors of  dendr...
Phases post CTX treatment relative to                      adoptive T cell therapy (Salem CII 2010)                       ...
Can post CTX expanded DCs be matured and benefit anti-tumor CD8+ T cell responses?
How to Mature Dendritic Cells Toll-Like  Toll-    Receptor (TLR) ligandsDendritic cells express TLRs Cytokines
Maturation of dendritic cells with TLRLs            TLR           agonistsBachmann et al. Nature Reviews Immunology 6: 159...
TLRs Alert Hosts From Invaders                                 NK cells                                 Macrophages       ...
TLRs and their specific TLR ligands         Microbial products        Dendritic cells
TL3 agonist (Poly (I:C))  • Poly (I:C) is a synthetic double stranded RNA    which mimics viral products  • It binds speci...
Can Poly(I:C)-activated dendritic cellsbenefit T cell responses to vaccination with a            self/tumor antigen?
Can Poly(I:C) Activate Dendritic Cells  Expanded after CTX Treatment?                   CTX                      CTX      ...
TLR3 agonist activates DCs and drives their   homing from blood to lymph nodes                          30                ...
Testing prime-boost vaccination strategy                        Prime          BoostB16      PBS T cells    Melanoma      ...
Pmel- Pmel-1 adoptive transfer mouse modelCD8 T cells react against gp100 melanoma peptide             Transgenic         ...
Stimulation of post CTX expanded DCs is essential for          augmenting CD8+ T cell responses                           ...
Boosting at the peak of post CTX DC expansion is essential to         establish therapeutic anti-tumor immunity           ...
Boosting with antigen/poly(I:C) at the peak of post CTX  DC expansion is effective against advanced tumor                 ...
Tripartite regimen generates memory T cells with robust                  anti-                  anti-tumor immunity       ...
Do DCs play a critical role in mediation of the beneficial                    effects of CTX?
Depletion of post CTX expanded DCs abrogates CTX effects                                            Spleen                ...
Poly(I:C) induces DCs to produce inflammatory                   cytokines                       18000                     ...
Treatment with G-CSF post CTX therapy increases                    the numbers of DCs                3.5                  ...
DCs number increased in breast cancer patients   treated with CTX-based chemotherapy                CTX- Patient    P14   ...
Conclusions   Chemotherapy with CTX induces marked    increases in the numbers of DCs in 12 days.   G-CSF corrects the l...
Strategies to Improving Adoptive T Cell Therapy    Generation of T   Chemotherapy    Vaccine     cells in vitro     Irradi...
Improving T cell functions in vitro before          their adoptive transfer in vivo      Donor     Tg Pmel                ...
Conditioning of pmel-1 cells with IL-12                pmel-             IL-    induces activation phenotype
Conditioning pmel-1 cells with IL-12 enhances              pmel-             IL- their function in vitro and expansion in ...
Conditioning pmel-1 cells with IL-12                pmel-            IL-enhances their systemic expansion in vivo         ...
Improving T cell functions in vitro before their adoptive           transfer in vivo increases anti-tumor responses       ...
IL-IL-12 induces pmel-1 cells to acquire              pmel-        stem cell phenotype
Current Preclinical Protocols developed at       National Cancer Institute, USA1.Total body irradiation2. Adoptive transfe...
Our treatment strategy1.Treatment with cyclophosphamide (CTX)2. Adoptive transfer of tumor-specific T cells3. Priming with...
Significance of our Findings   Replacement of ex vivo-based DC                        vivo-    vaccination with post chem...
Cancer immunotherapyCombination, Combination, Combination …..                         Nature 446, 964-966 (26 April 2007)
Future Studies   Does expansion of DCs occur in cancer    patients?   Can poly(I:C) mature DCs in cancer    patinets?   ...
ACKNOLWEDGEMENTSMedical University             University of Miamiof South Carolina              Alberto Montero, MDDavid ...
SPECIAL THANKS•    TWAS, The Academy of Sciences for    The Developing World (Third World    Academy of Sciences)•    CAS,...
THANKYOU
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Cas talk 9-2-2012 [compatibility mode]

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This talk on Cancer Immunotherapy was given by Prof. Mohamed Labib Salem
)ا.د. محمد لبيب سالم أستاذ المناعة بقسم علم الحيوان كلية العلوم جامعة طنطا مصر ومديروحدة المشروعات التنافسية بجامعة طنطا)
at the Chinese Academy of Sciences, Beijing, China on February 9, 2012 during his visit to CAS for a month supported by Third World Academy of Sciences (TWAS; Italy).

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Cas talk 9-2-2012 [compatibility mode]

  1. 1. Efficacy of Combinatorial anti-cancerchemotherapy and immunotherapy: A central role of dendritic cells Mohamed Labib Salem, PhD Prof. of Immunology Zoology Department, Faculty of Science Director, Competitive Project Unit Tanta University, Egypt Key Lab. Infection and Immunity Center for Infection and Immunity Institute of Biophysics Chinese Academy of Sciences
  2. 2. The Beginning of Cancerous Growth: A process that mimics the cancer grows in Egypt before January 25th, 2011 Underlying tissue
  3. 3. EGYPT
  4. 4. Cancer Therapy In clinic• Surgery: Effective but doesn’t cure tumor metastasis or prevent tumor recurrence• Radiotherapy/Chemotherapy: Effective but doesn’t prevent tumor recurrence besides its serous side effects
  5. 5. Cancer Therapy In clinical trials• Immunotherapy: can cure local and distant tumors and prevent tumor recurrence through developing tumor-specific memory T cell responses
  6. 6. Cancer Immunotherapy  Antibodies  Cytokines (IL-2, IFN-a) (IL- IFN-  Adoptive T Cell Therapy  Vaccines
  7. 7. Activation of DCs is essential to prime T cells ? ? ?? ?
  8. 8. Challenge to cancer Immunotherapy: 1- Tumor antigens are selfCancer cells originate from self cells (i.e. not foreign)• Poorly immunogenic• Induce very weak (undetectable) immune responseMicrobes are foreign• Immunogenic• Induce strong immune responseCancer cells can be immunogenic if a danger signal induced by microbial products
  9. 9. Challenge to cancer Immunotherapy: 2- Tumor cells escape from immune cells Down regulate the receptors required to interact with immune cells Upregulate receptors that suppress immune cells upon interaction Produce factors that suppress immune cell functions Favors expansion of suppressor immune cells
  10. 10. Challenge to cancer Immunotherapy: 3- High tumor cells/T cell ratio 1:10,000 T cells 1:50 T cells 1:2 T cellsDisease Burden Clinically Localized Advanced Un-detectable Disease Disease
  11. 11. Adoptive T cell transfer therapy3- Test T cell 4- Clonal function expansion of T cells with IL-2 & CD3 Ab2- Grow T cells With IL-2 5- Infusion of T cells + vaccine 1- Tumor excision 6- Chemo or Irradiation Before T cell transfer
  12. 12. Chemotherapy and Immunotherapy“Removal of weeds, and planting and watering good seeds”
  13. 13. Strategies to Improving Adoptive T Cell Therapy Generation of Chemotherapy Vaccine T cells in vitro Irradiation adjuvant In vitro In vivo In vivo
  14. 14. Our Model of ChemotherapySystemic injection ofcyclophosphamide (CTX) (CTX)200 mg/kg (4 mg/mouse) (4
  15. 15. Our models for adoptive T cell transfer (Donor) (Recipient) Transgenic Naive Ly 5.1 mouse Ly 5.2 mouse CD8+ T cellsCD8 T cells are engineered to 1 x 106recognize tumor antigens I.V. injection Donor CD8+ T cells (0.2 %) are clearly visualized & quantified CD49low CD44low CD69low CD62Lhigh
  16. 16. Chemotherapy and T cell Therapy ProtocolPBS CD8+ T cell Antigen AntigenCTX 1 x 106 priming boosting d0 d1 d2 d3 d5 d7 d70 Readout (Number of Donor Cells)
  17. 17. CTX increased Ag-specific expansion of transferred CD8 cells during effector and memory responses 25 PBS % OT-1 cells in PBL 20 CTX 1 mg CTX 4 mg 15 10 5 0 3 6 7 8 14 21 Days post vaccination Of priming Re-vaccination Day 70 Day 7 Day 14 4 12 12 3.5 PBS % OT-1 cells in PBL % OT-1 cells in PBL 10 10 3 CTX % OT-1 cells 2.5 8 8 2 6 6 1.5 4 4 1 0.5 2 2 0 0 0 PBS CTX PBL Spleen PBL SpleenSalem et al. J Immunother 30(1): 40-53 (2007)
  18. 18. How chemotherapy alters host microenvironment 10000 Blood 8000 # PBL (10 /L) 6000 6 4000 2000 0 0 3 6 9 12 15 18 100 Spleen and Bone marrow Spleen # Leucocytes (10)/organ BM 80 6 60 40 20 0 0 3 6 9 12 15 18 Days post CTX treatment
  19. 19. Mechanisms of action of CTX: 1- Induces homeostatic expansion of transferred cells 80 Before vaccination Total number (106) per spleen 70 1.2 60 % OT-1 cells in PBL 1 50 40 ** 0.8 30 0.6 20 0.4 10 ** 0.2 0 0 6 24 72 0 Hr after CTX PBS CTX -6 hr CTX -24 hr CTX -48hr After vaccination 70 % OT-1 cells in PBL on day 7 60 50 40 30 20 10 0 PBS CTX -6 hr CTX -24 hr CTX -48hrSalem et al. J Immunother 30(1): 40-53 (2007)
  20. 20. Mechanisms of action of CTX: 2- A space “niche” is not a critical factor CTX ACT Vac i.p. i.v. s.c. Measuring Ag-specific expansion of adoptively d-2 d0 d1 transferred CD8+ T cells Filling the space 20 % OT-1 cells in PBL on day 3 18 16 14 12 10 8 6 4 2 0 PBS CTX CTX CTX CTX +50m +100m +150mSalem et al. J Immunother 30(1): 40-53 (2007)
  21. 21. Mechanisms of action of CTX:3- Decreases the numbers of regulatory T cells
  22. 22. Mechanisms of action of CTX: 4- Type I IFNs are critical factors 100 90 80 CTX IFN- (pg/ml) 70 60 50 40 30 20 PBS 10 0 1 4 8 24 48 72 96 Hours post CTX (4 mg) treatment 12 % CD8 T cells in PBL 10 CTX/Wild Type 8 6 4 CTX/IFNaKO 2 PBS 0 3 5 7 45 Days post vaccinationSalem et al. J Immunother 30(1): 40-53 (2007)
  23. 23. Mechanisms of action of CTX: 5- Inducing activation of dendritic cellsSalem et al. J Immunother 30(1): 40-53 (2007)
  24. 24. How Does Chemotherapy Benefit Immunotherapy? Creation of a space “niche”Elimination Induction of of activation of dendritic cellssuppressive cells Induction of survival cytokines
  25. 25. Strategies to Improving Adoptive T Cell Therapy Generation of Chemotherapy Vaccine T cells in vitro Irradiation adjuvant In vitro In vivo In vivo
  26. 26. Focusing on Dendritic Cells After Chemotherapy
  27. 27. Dendritic Cells are A Critical player in Anti- Tumor Immunity TLRLs No response
  28. 28. Mechanisms of action of CTX: Inducing expansion of DCs during restoration phase Lymphopenic phase Restoration phase Day 0 Day 2 Day 6 Day 9 Day 12 5.6 ± 1.1 3.5 ± 0.9 6.4 ± 0.8 25.2 ± 1.1 18.7 ± 4.1CD11c CD11b # CD11c CD11b cells (10 /L) 1400 6 1200 1000 800 + 600 400 + 200 0 0 2 6 10 14 Days post CTX treatment
  29. 29. CTX treatment induces an expansion of DCs PBS CTX
  30. 30. Post CTX expanded DCs express the phenotype of immature myeloid cells Lymphopenic phase Restoration phase d0 d2 d4 d10 d14 4.5% 25% 35% 7.2% 2.1% CD80 35 PBS 30 Day 12 post treatment CTX % CD11c cells 25 20 + 15 10 5 0 mDCs pDCs Myeloid DCs (mDCs): CD11chighCD11bhighLy6GlowB220low Plasmacytoid DCs (pDCs): CD11chighCD11blowLy6GhighB220high
  31. 31. How CTX Induces Dendritic cell expansion? Is dose-dependent Induces proliferation of progenitors of dendritic cells in bone marrow Induces production of stem cell mobilizing factors  Flt3 and CCR2 signaling is criticalSalem et al. 2009 J ImmunologySalem et al., 2010 J ImmunologySalem et al., 2010 Cell Immunology
  32. 32. Phases post CTX treatment relative to adoptive T cell therapy (Salem CII 2010) 2010) Lymphopenic phase Recovery phase Number of dendritic cells • Creation of a space niche due to • Cellular recovery from lymphopenia the induced lymphopenia • Less of lymphopenia • Homeostatic expansion of T cells • Less homeostatic proliferation of T cells • Elimination of regulatory cells • Expansion of immature dendritic cells • Elimination of cytokine competition • Microbial translocation • Activation of dendritic cells Donor T cells DCs -1 0 3 6 9 12 15 18 20 Days after CTX treatmentACT Antigen priming Antigen boosting + TLR agonists + TLR agonists
  33. 33. Can post CTX expanded DCs be matured and benefit anti-tumor CD8+ T cell responses?
  34. 34. How to Mature Dendritic Cells Toll-Like Toll- Receptor (TLR) ligandsDendritic cells express TLRs Cytokines
  35. 35. Maturation of dendritic cells with TLRLs TLR agonistsBachmann et al. Nature Reviews Immunology 6: 159–164 (2006)
  36. 36. TLRs Alert Hosts From Invaders NK cells Macrophages Dendritic cells
  37. 37. TLRs and their specific TLR ligands Microbial products Dendritic cells
  38. 38. TL3 agonist (Poly (I:C)) • Poly (I:C) is a synthetic double stranded RNA which mimics viral products • It binds specifically to TLR3 expressed in DCs. • TLR3/TLRL triggers inflammatory mediators and activate DCs. • We and others have established that poly(I:C) is a potent adjuvant for CD8+ T cell responses in different vaccination settings.Salem et al. J Immunother 28:220-228 (2005)Salem et al. Vaccine 24:5119-5132 (2006)
  39. 39. Can Poly(I:C)-activated dendritic cellsbenefit T cell responses to vaccination with a self/tumor antigen?
  40. 40. Can Poly(I:C) Activate Dendritic Cells Expanded after CTX Treatment? CTX CTX PBS None Poly PBS None PolyBlood 2.36 1.35 36.3 1.8 1.6 4.8Lymph 40.7 28.2 83.3 34.7 49.5 85%Nodes CD80 CCR7
  41. 41. TLR3 agonist activates DCs and drives their homing from blood to lymph nodes 30 25 % CD11c+CD11b+ DCs 20 15 10 5 0 CTX CTX CTX CTX PBS Poly PBS Poly Blood Lymph nodes
  42. 42. Testing prime-boost vaccination strategy Prime BoostB16 PBS T cells Melanoma Melanomas.c. CTX 106 antigen antigen ± poly(I:C) ± poly(I:C)d-12 d0 d1 d2 d12 d15 Readout
  43. 43. Pmel- Pmel-1 adoptive transfer mouse modelCD8 T cells react against gp100 melanoma peptide Transgenic mice Naïve spleen CD8+ T cells Tumor Vaccine+ Vaccine+ (500,000) cells Poly(I:C) Poly(I:C) D -10 D-2 D -1 D0 D 12 CTX T cells can grow DCs are expanded
  44. 44. Stimulation of post CTX expanded DCs is essential for augmenting CD8+ T cell responses 12.5% 2500 0.25 39.4% # pmel-1 in DLNs (106) 2000 0.2 # pmel-1 in PBL (106/L) 1500 0.15 1000 0.1 0.05% 500 3.1% 0.6% 0.05 0.1% 0.1% 4.5% 2.1% 0.02% 0.04% 0.2% 0 0 1500 0.18 9.1% 18.2% 1250 0.15 # DCs in PBL (106/L) # DCs in DLNs (106) 1000 0.12 14.9% 14.6% 750 6.0% 0.09 6.5% 7.6% 500 0.06 3.6% 1.9% 1.7% 2.2% 4.6% 250 0.03 0 0 B16 - - + + + + - - + + + + CTX - + - - + + - + - - + + gp100 - - + + + + - - + + + + Poly I:C - - - + - + - - - + - +
  45. 45. Boosting at the peak of post CTX DC expansion is essential to establish therapeutic anti-tumor immunity 450 400 350 Tumor area (mm ) 2 300 250 200 150 Single vaccination 100 50 Two vaccinations 0 9 12 15 18 21 24 27 30 33 Days post tumor inoculation 100 PBS (No pmel-1) CTX (No pmel-1) 80 PBS/gp100 + poly I:C d1 PBS/gp100 + poly I:C d1 and d12 % Survival 60 CTX/gp100 + poly I:C d1 40 CTX/gp100 + poly I:C d1 and d12 20 0 0 10 20 30 40 Days post tumor inoculation
  46. 46. Boosting with antigen/poly(I:C) at the peak of post CTX DC expansion is effective against advanced tumor CTX 450 (No T cells) PBS (No T cells) 400 PBS/Vac + poly(I:C) Tumor area (mm2) 350 300 CTX/Vac + IL-2 250 200 150 100 CTX/Vac+ poly(I:C) 50 0 12 14 17 20 22 24 26 28 32 Days post tumor inoculation
  47. 47. Tripartite regimen generates memory T cells with robust anti- anti-tumor immunity 450 400 CTX (no 2nd ACT) Tumor area (mm2) PBS (no 2nd ACT) 350 300 250 CTX/hgp100 (2nd ACT) 200 150 100 CTX/hgp100+poly(I:C) (2nd ACT) 50 0 11 14 16 18 22 26 28 Days post tumor inoculation
  48. 48. Do DCs play a critical role in mediation of the beneficial effects of CTX?
  49. 49. Depletion of post CTX expanded DCs abrogates CTX effects Spleen LNs 5 0.8 25.2% # pmel-1 in DLNs (106) 11.6% # pmel-1 in spleen (106) 4 0.6 3 1.5% 4.5% 0.4 2 2.2% 0.2 1 0.8% 0.03% 2.4% 0 0 PBS CTX PBS CTX PBS CTX PBS CTX WT DTR Tg WT DTR Tg
  50. 50. Poly(I:C) induces DCs to produce inflammatory cytokines 18000 1800 16000 1600 TNFα (pg/ml serum) IL-6 (pg/ml serum) 14000 1400 12000 1200 10000 1000 8000 800 6000 600 4000 400 2000 200 0 0 350 16000 MCP1 (pg/ml serum) 300 14000 IFN-γ (pg/ml serum) 250 12000 10000 200 8000 150 6000 100 4000 50 2000 0 0 CTX/poly DTR/CTX Poly Poly PBS PBS DTR/CTX CTX/poly + poly + poly
  51. 51. Treatment with G-CSF post CTX therapy increases the numbers of DCs 3.5 PBS 3 CTX 2.5# Dendritic cells CTX + G-CSF 2 (10^6/ml) 1.5 1 0.5 0 Day 7 Day 9 Day12 Days post CTX treatment
  52. 52. DCs number increased in breast cancer patients treated with CTX-based chemotherapy CTX- Patient P14 P17 P26 P29 P36 P40 P45 P47 P54 P56 P66 P74Baseline 175.1 264.6 696.6 810.3 591.6 660.9 883.8 141.0 489.3 253.3 395.6 252.0 AC 216.5 701.5 872.9 1750.7 865.8 921.0 854.8 144.6 592.8 881.4 938.9 561.4% Change 20 160 20 115 50 40 -3 2.5 21.1 240 137 123Blood were collected from newly diagnosed breast cancer patients (n=12; stage I-IV) who hadreceived four courses of standard ddAC: doxorubicin 60 mg/m2 plus CT 600 mg/m2 on day 1,followed by paclitaxel 175 mg/m2 (ddT) on day 1 for four cycles. The numbers of HLA-DR+ cells wereanalyzed by flow cytometry and expressed as the changes relative to the healthy donor.
  53. 53. Conclusions Chemotherapy with CTX induces marked increases in the numbers of DCs in 12 days. G-CSF corrects the lymphopenia induced by CTX but increases the expansion of DCs. CTX- CTX-expanded DCs express immature phenotype Poly(I:C) can mature CTX-expanded DCs in CTX- vivo Poly(I:C)- Poly(I:C)-matured DCs induce therapeutic CD8 CD8+ mediated anti-tumor immunity anti-
  54. 54. Strategies to Improving Adoptive T Cell Therapy Generation of T Chemotherapy Vaccine cells in vitro Irradiation adjuvant In vivo In vivo In vitro
  55. 55. Improving T cell functions in vitro before their adoptive transfer in vivo Donor Tg Pmel Culture T cells and stimulate with + gp10025-33 peptide ± IL12 for 3~5 days CD8+ T cells arespecific to the MHC-1gp10025-33 melanoma Pept alone Pept + IL12 peptide CD62L (pmelsham ) (pmelIL12 ) CD44 PBS PBS Vaccination + Poly (I:C) CTX CTX
  56. 56. Conditioning of pmel-1 cells with IL-12 pmel- IL- induces activation phenotype
  57. 57. Conditioning pmel-1 cells with IL-12 enhances pmel- IL- their function in vitro and expansion in vivo In press
  58. 58. Conditioning pmel-1 cells with IL-12 pmel- IL-enhances their systemic expansion in vivo 90 80 % Pmel cells in PBL 70 60 50 40 30 20 10 0 PBS CTX PBS CTX CD8sham CD8IL12
  59. 59. Improving T cell functions in vitro before their adoptive transfer in vivo increases anti-tumor responses 400 None 350 None 350 Pmelsham 300 Pmelsham (1 x 106) Pmelsham+gp100 PmelIL12 (1 x 106) 300 Tumor area (mm2) PmelIL12 250Tumor area (mm2) Pmelsham (5 x 106) 250 PmelIL12+gp100 PmelIL12 (5 x 106) 200 200 150 150 100 100 50 50 0 0 9 12 15 18 21 24 9 12 15 18 21 24 27 Days post pmel cell transfer Days post pmel-1 cell transfer 450 No CTX, no pmel 100 400 CTX Pmelsham 350 80 Pmelsham+gp100 Percent survivalTumor area (mm2) 300 PmelIL12 PmelIL12+gp100 60 250 200 40 150 100 20 50 0 0 4 8 12 16 20 24 28 32 36 40 44 0 5 10 15 20 25 30 35 40 Days post pmel-1 cell transfer Days post pmel cell adoptive transfer
  60. 60. IL-IL-12 induces pmel-1 cells to acquire pmel- stem cell phenotype
  61. 61. Current Preclinical Protocols developed at National Cancer Institute, USA1.Total body irradiation2. Adoptive transfer of tumor-specific T cells3. Vaccination with Viral vector encoding tumor antigen or dendritic cells loaded with antigen4. Treatment with high dose of IL-2 for 5 days This strategy is significantly effective in melanoma setting but very toxic
  62. 62. Our treatment strategy1.Treatment with cyclophosphamide (CTX)2. Adoptive transfer of tumor-specific T cells3. Priming with tumor antigen during the lymphopenic phase post CTX therapy + Poly(I:C)4. Boosting with tumor antigen at the peak of dendritic cell expansion + Poly(I:C) This strategy is significantly effective in melanoma setting but non toxic and less expensive
  63. 63. Significance of our Findings Replacement of ex vivo-based DC vivo- vaccination with post chemotherapy expanded DCs Replacement of highly toxic IL-2 with IL- effective and non-toxic poly(I:C) non- Non- Non-viral delivery of antigen for vaccination Adoptive transfer of IL-12 activated T IL- cells can further augments the anti- anti- tumor immunity.
  64. 64. Cancer immunotherapyCombination, Combination, Combination ….. Nature 446, 964-966 (26 April 2007)
  65. 65. Future Studies Does expansion of DCs occur in cancer patients? Can poly(I:C) mature DCs in cancer patinets? patinets? Is vaccination/poly(I:C) of cancer patients sufficient to generate anti-tumor responses? anti- What cancer patients and what cancer population can benefit from post CTX expansion of DCs?
  66. 66. ACKNOLWEDGEMENTSMedical University University of Miamiof South Carolina Alberto Montero, MDDavid J. Cole, MD Diaz Montero, MDMike Nishimura, PhDMark Rubstein, PhD Rubstein, Oncovir, Oncovir, Inc, WashingtonAmir A Alkhami, PhD Alkhami, Andreas Salazar, MDMarcela Montero, PhDAndre Kadima, MD Kadima, Tanta University, EgyptYian Chen, PhD Sabry EL-Naggar, PhD EL-Naggar,Osama Naga, DDS Randa Al-Naggar, Phd Al-Naggar,Elizabeth Little, BSc Wael Y. Attia, PhD Attia,Rick Peppler, MS Peppler,Narender Nath, PhD Nath,Suez Canal University, EgyptAhmed Khafagy, PhD Khafagy,
  67. 67. SPECIAL THANKS• TWAS, The Academy of Sciences for The Developing World (Third World Academy of Sciences)• CAS, Chinese Academy of Sciences• Prof. Shengedian Wang, MD/PhD Director, Key Lab. Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing
  68. 68. THANKYOU

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