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Active Specific Vaccination Strategies

Engineering Customized Anti-Tumor Immune Response: Biology of MHC Class I Restricted TCR Engineered CD4 T Cells. T Cell Based Cancer Immunotherapy Approaches. A. B. Active Specific Vaccination Strategies. Adoptive Immunotherapy Approaches. Tumor Cells. CD8+ CTL Clone. TCR/CAR Engineered

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Active Specific Vaccination Strategies

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  1. Engineering Customized Anti-Tumor Immune Response: Biology of MHC Class I Restricted TCR Engineered CD4 T Cells

  2. T Cell Based Cancer Immunotherapy Approaches A. B. Active Specific Vaccination Strategies Adoptive Immunotherapy Approaches Tumor Cells CD8+ CTL Clone TCR/CAR Engineered T Cells DC DC LAK TIL Synthetic Peptide/ Tumor Lysate Pulsed DC Gene Modified DC Gene Modified Tumor Cells Ex-Vivo Generation of Large Scale Immune Effectors Synthetic Peptide

  3. TCR Engineering Approach CD4+ CD8+ CD8+ CD8+ CD4+ MHC II MHC I Antigen Presenting Cell CD8+ Engineered Anti-tumor T Cells Antigen Specific TCR

  4. Most active specific and adoptive immunotherapy approaches for cancer center around CD8+ cytolytic T lymphocyte (CTL)-based responses, generated through immunization or transferred adoptively with ex vivo generated CTL. Generating a robust and long-lived CTL response, however, needs “help” from CD4+ T helper cells. The task of simultaneously engaging CD4+ T helper cells and CD8+ CTL is quite difficult, especially in an antigen specific manner, since: (i). For most human tumor antigens HLA matched MHC class I and MHC class II antigenic epitopes are not characterized yet. (ii). In addition, even if HLA matched MHC class I and class II epitopes are available for a target antigen, since CD4 T cells recognize their target antigenic epitope in context to MHC class II molecules and most human tumors are MHC class II negative, engaging CD4 T cells in tumor immunity is technically a very challenging task.

  5. Creation of MHC Class I Restricted CD4 T Cells • Our working hypothesis was that the MHC class II-restricted natural CD4 T cells can be programmed to provide antigen specific “help” in tumor immunity by engrafting them with a MHC class I-restricted tumor epitope-specific T cell receptor (TCR), derived from the CD8+ anti-tumor CTL.

  6. Generation of MART-1 27-35 Epitope Specific Oligoclonal Line 6.9% 93.8% (i). MART-1 Tetramer CD8 (ii).

  7. Cloning and Characterization of MART-1 27-35 Epitope Specific TCR From M1 Specific CTL Line Vα2.1 Vβ14 TCR (M14.1 ) Vα2.2 Vβ14 TCR (M14.2 ) Control FUW-M14.2-TCR/sr39tk 0.9% 7.2% 12.7% Mart-1 Tetramer 5’ LTR TCR F2A TCR sr39tk WPRE P2A Ubi 3’ LTR CD3 50 MOI 100 MOI Control 10 MOI 49.8% 30.9% 32.5% 0.17% Mart-1 tetramer CD3 50.6% 68.6% 48% 0.07% V14 CD3 A. B. C.

  8. I- Biology of MHC Class I TCR Driven Human CD4 T Cells

  9. M1 Epitope Specific MHC Class I TCR Engineered CD4 T Cells Exhibit An Antigen Specific Effector Function JL-M PT-M A375 A375-M PT-M JL-M A375 A375-M T2 A. Control M14.2 TCR Transduced C. B. 0.66% 9.5% M1- Tetramer 97.3% 83.8% CD4

  10. Transgenic TCR Engineered CD4 T cells, Derived From Three Different Donors Exhibit A Th1 Biased Effector Function IL-2 IFN-γ IL-4 IL-10 IFN-γ IL-2 IL-4 IL-10 IL-2 IFN-γ IL-4 IL-10 Donor-2 Donor-3 Donor-1

  11. A. B. Transgenic TCR Engineered CD4 T cells Exhibit An Antigen Specific Th1 Biased Effector Response Against Human Melanoma Cells HLA-A2 MART-1 (i). A375 HLA-A2 Isotype HLA-A2 MART-1 A375-M A375-M A375-M A375 A375 A375 A375-MART-1 IFN-γ IL-2 IL-10 MART-1 (ii). HLA-A2 MART-1 PT-M (iii). IFN-γ IL-2 IL-10 IFN-γ IL-2 IL-10 HLA-A2 MART-1 JL-M PT-M JL-M

  12. Transgenic TCR Engineered CD4 T cells Display High Functional Avidity 14.8% Tetramer CD8 10μg 10ng 100ng 1μg 1ng 10μg 10ng 100ng 1μg 1ng 10ng 100ng 1μg 10μg 1ng A. B. C.

  13. Transgenic TCR Engineered CD4 T cells Display High Functional Avidity 1:100 1:50 1:10 1:1 10:1 1:100 1:50 1:10 1:1 10:1 E:T E:T

  14. Transgenic TCR Engineered CD4 T cells Exhibit Antigen Specific Cytolytic Effector Function A. B.

  15. Transgenic TCR Engineered CD4 T cells Kill Tumor Cells A. B.

  16. Cytolytic Machinery Of MHC Class I TCR Engineered CD4 T Cells Isotype Isotype Granzyme Perforin Isotype Isotype Granzyme Perforin Isotype T2 T2+M3 T2+M1 1.1 1.7.9 1.6 A. (ii). (i). CD4 CD107 B. C. T2 T2+M3 T2+M1 CD8 CD107

  17. Lytic Activity Exhibited by TCR Engineered CD4 T cells Is MHC class I Restricted And Granule Exocytosis Mediated Process Anti-MHCII Anti-MHCI EGTA Anti-MHCII Anti-MHCI EGTA A. B.

  18. MHC I restricted TCR transduced CD4+ T cells Exhibit An Epitope Specific • ``Helper Cytokine Response``. • Engineered CD4 T cells also exhibit an antigen specific, MHC class I restricted • and granule exocytosis-mediated cytolyticeffector response.

  19. Functional Profile of CD4 T Cells Engineered With A Different MHC Class I Restricted Transgenic TCR

  20. Transgenic TCR Engineered CD4 T cells Exhibit A Significant Epitope Specific Proliferation Potential Untransduced Baseline Transduced Stimulated A. 43.9% 85.1% 0.1% MART-1 CD4 B. c. a. b. d. e. MART-1 CFSE DC DC+M3 DC+M1

  21. Transgenic TCR Engineered CD4 T cells Undergo Epitope Specific Proliferation Against Human Melanoma Cells a. b. c. d. e. No Peptide MART-1 Tetramer +MART-1 Peptide CFSE A375-M1 A375 PT-M

  22. Transgenic TCR Engineered CD4 T cells Exhibit Polyfunctional Cytokine Profile

  23. Transgenic TCR Engineered CD4 T cells Facilitate ``Help`` For The Generation Of A Robust CD8+ CTL Response IVC IVC+CD4M1 IVC+ CD4 Control Fold Increase 2.7 28.7 1.4 1.3 Donor-1 25.3 97.3 11.1 58.0 92.7 3.5 2.5 6.0 26.8 Donor-2 9.3 Tetramer 88.8 83.5 18.5 49.0 2.2 1.6 34.2 2.3 Donor-3 2.7 85.0 95.6 16.7 46.9 CD8

  24. Transgenic TCR Engineered CD4 T cells Facilitate ``Help`` For The Generation Of A Robust CD8+ CTL Response IVC IVC+CD4M1 IVC IVC+CD4M1 1.0 0.45 2.2 1.0 +Cytokine -Cytokine

  25. In An In-Vitro CTL Generation Assay iTreg Mediate Immune Suppression CD8+ CD8+ CD8+ MHC Class I Restricted Interaction CD8+ CD8+ CD8+ CTL Response MHC I APC iTreg

  26. MHC Class I TCR Engineered CD4 T Cells Can Mitigate The iTreg Mediate Immune Suppression CD8+ CD8+ CD8+ MHC Class I Restricted Interaction CD8+ CD8+ CD8+ CTL Response MHC I APC CD4M1 iTreg

  27. A. B. Donor-1 Donor-2

  28. A. B. (i). (ii).

  29. A. CD86 CD80 MHCII MHCI CD40 (i). DC Isotype DC Isotype DC DC+CD4-C DC DC+CD4-C DC+CD4-C DC DC+CD4-M1 DC+CD4-M1 Total Gated Population DC+CD4-C DC+CD4-M1 DC+CD4-C DC DC+CD4-M1 DC+CD4-M1 DC+CD4-C DC+CD4-M1 Isotype Isotype Isotype (ii). DC DC+CD4-C DC DC CD11c+ve Gated Population DC+CD4-C DC+CD4-M1 DC+CD4-C DC DC+CD4-M1 DC+CD4-M1 DC+CD4-C DC+CD4-M1 C. B. CD86 CD80 MHCII MHCI CD40 iDC iDC mDC iDC mDC iDC Total Gated Population iDC mDC mDC mDC iDC iDC mDC CD11c+ve Gated Population mDC iDC iDC iDC mDC mDC mDC

  30. SUMMARY • MHC I restricted TCR transduced CD4+ T cells Exhibit An Epitope Specific • ``Helper Cytokine Response``. • These MHC class I-restricted tumor epitope specific TCR engineered CD4+ T • cells can facilitate the generation of a robust CD8+ CTL response. • Engineered CD4 T cells also exhibit an antigen specific, MHC class I restricted • and granule exocytosis-mediated cytolyticeffector response.

  31. (PCD/AICD) Magnitude of response contraction expansion memory Time Time

  32. II. Are MHC Class I TCR Engineered CD4 and CD8 T Cells Susceptible to Epitope Specific AICD? & Can Long Lasting Anti-Tumor T Cells Be Generated By Interfering With The Premature AICD ?

  33. MHC Class I Restricted CD4 T Cells Undergo Epitope Specific AICD Just Like the TCR Engineered CD8+ T Cells T2 T2+M3 T2+M1 (B). (A). T2 T2+M3 T2+M1 56.3 5.0 56.7 5.1 15.0 33.2 9.7 27.3 9.1 58.0 30.9 59.3 M1 Tet. M1-Tet. Annexin (CD4) Annexin (CD8) 1.9 66.0 3.9 66.5 18.1 44.3 51.5 73.7 2.0 21.2 7.5 M1 Tet. M1-Tet. 79.6 DiOC6 DiOC6 DiOC6 12.5 10.6 34.9 3.1 40.9 9.5 DiOC6 DiOC6

  34. MHC Class I Restricted CD4 T Cells Undergo Epitope Specific AICD In A Dose Dependent Manner T2+M1 25ug/ml 10ug/ml 1ug/ml 0.1ug/ml 0.01ug/ml T2 T2+M3 62.3% 21.4% 10.7% 69.0% 70.1% 24.2% 9.2±0.7 14.4±0.1 24.6±0.4 6.7±0.3 22.4±0.6 7.3±0.2 31.5±0.9 3.3±0.5 30.6±0.6 6.6±0.7 14.7±0.8 6.9±0.1 16.2±0.1 3.7±0.5 9.9 14.5 24.8 6.6 23.2 7.1 31.3 3.0 31.3 3.4 6.7 13.8 7.1 16.3 M1 Tet. Annexin 8.9 11.0 43.6 38.9 43.5 35.5 24.5 DiOC6

  35. Comparative AICD Susceptibility in MHC Class I TCR Engineered CD4 & CD8 T Cells Antigen Experienced Freshly Transduced CD8M1 Antigen Experienced Freshly Transduced CD4M1

  36. AICD In MHC Class I TCR Driven CD4 T Cells Is DR-Independent 13.2% 12.9% 64.5% 62.2% 65.2% 62.7% 62.6% 58.9% T2 Fas/Fc TNF-sRI/Fc T2+M3 T2+M1 IFN-g RI TRAIL-RI TRAIL-RII 8.9 14.0 14.7 8.2 15.4 8.8 14.8 9.0 15.1 9.8 14.1 35.0 7.7 34.0 5.2 5.2 M1 Tet. Annexin (CD4 T Cells) 56.6 55.0 50.5 19.5 33.8 56.1 51.9 55.8 DiOC6

  37. AICD In MHC Class I TCR Engineered CD8 T Cells Is Also DR-Independent 19.5 33.8 56.1 51.9 55.8 56.6 55.0 50.5 T2 Fas/Fc TNF-sRI/Fc T2+M3 T2+M1 IFN-g RI TRAIL-RI TRAIL-RII M1-Tet. Annexin 14.0 7.7 34.0 5.2 35.0 8.9 14.7 8.2 15.4 8.8 14.8 9.0 15.1 9.8 14.1 5.2 DiOC6

  38. AICD In MHC Class I TCR Driven CD4 T Cells Involves JNK T2 M3 M1 SP PD 90.8% 47.1% 61.3% 51.0% 91.6% p-cJun Actin A. T2 T2+M3 T2+M1 SP PD M1-Tet. 19.9±0.8 52.8±0.8 5.3±0.3 23.3±0.6 20.8±0.5 31.6±1.4 22.2±0.8 53.7±0.8 5.4±0.3 21.2±0.6 5.3 23.9 19.0 53.7 5.0 54.7 21.1 32.8 21.7 21.7 Annexin (CD4) 7.3±0.9 9.8±4.0 25.6±2.0 21.9±3.3 30.1±1.8 6.3 7.1 18.9 29.3 27.2 C. B. (i). (ii). DiOC6

  39. AICD In MHC Class I TCR Eng. CD8 T Cells Also Involves JNK T2 T2+M3 T2+M1 SP PD 33% 33% 44% 89% 91% A. M1-Tet. 32.0 2.7 31.7 3.0 6.5 16.8 12.2 15.0 6.4 12.9 Annexin (CD8) 5.0 3.1 50.7 32.3 44.5 DiOC6 C. B. (i). (ii). T2 M3 M1 SP PD p-cJun SP PD cJun Actin SP PD

  40. AICD In Human Tumor Antigen Specific MHC Class I TCR Engineered CD4 & CD8 T Cells Is a caspase-independent, DR-independent, JNK Activation Driven Process.

  41. Intrinsic Death Signal JNK ? Caspase independent death (via AIF, EndoG etc.) Caspase dependent death (via cytochrome-c)

  42. AICD In MART-1 Epitope Specific CTL Involves Selective Release Of Mitochondria Resident Death Executioner, AIF HM S-100 C M1 C M1 AIF Cyto-C Endo-G Smac Htr-A2 Hsp-60 Actin

  43. Control AIF Cytox green Overlay AICD Cytox green Overlay AIF SP Cytox green Overlay AIF Cytox green Overlay AIF PD Mitochondrio-nuclear Translocation of AIF during AICD and blockade of this translocation by SP600125

  44. Mitochondrio-nuclear Translocation of AIF during AICD

  45. AICD In MART-1 Epitope Specific CTL Involves Large Scale DNA Fragmentation, Characteristic of AIF Mediated Death C M1 C M1 C Stau C-ssDNA C-DAPI C-ssDNA+DAPI 4.0Kb 4.0Kb 2.3Kb 2.3Kb 50 Kb 2.0Kb 2.0Kb AICD-ssDNA AICD- DAPI AICD-ssDNA+DAPI PFGE 0.5Kb 0.5Kb 24 hr 16.1 9.1 6.1 Control CTL Control CTL 26.4 19.5 12.9 CTL+ Control Peptide CTL+ Control Peptide 39.3 38.2 20.4 CTL+ M1 Peptide CTL+ M1 Peptide ss-DNA PI A. B. C. 4 hr 24 hr

  46. AIF Is Localized At The ssDNA Breaks In CTL Undergoing AICD ssDNA AIF AIF+ssDNA AIF+ssDNA+DAPI

  47. AIF release in TCR driven Death of Jurkat cells AIF Hsp60 Cyt-C Actin AIF Hsp-60 Cyt-C Actin S-100 Fractions-CD3 HM Fractions-CD3 C 30` 1hr 4hr 8hr 24hr 48hr C 30` 1hr 4hr 8hr 24hr 48hr

  48. CD3 CD3+SP CD3+PD CD3 CD3+SP CD3+PD HM Fractions-CD3 S-100 Fractions-CD3 C 4h 24h 4h 24h 4h 24h C 4h 24h 4h 24h 4h 24h AIF Cyto-C Actin 4h 24h Effect of SP600125 on AIF Release in TCR Driven Death of Jurkat

  49. How Does JNK activation regulates Mitochondria-Centric AICD In MHC Class I TCR Driven Human Primary Anti-Tumor T Cells?

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