- Defined as approaches that enhance, suppress or modify the immune system to treat or cure diseases.

Transcription

1 암의면역치료 권병세

2 Immunotheraphy - Defined as approaches that enhance, suppress or modify the immune system to treat or cure diseases. - Applied for the treatment of cancer, autoimmune diseases, transplant rejection, and chronic infection. - Little or no side effects. - Take advantage of inherent properties of immune system. - Selectivity.

3 Cancer Immunotherapy 1. Active a. Vaccine Tumor cells DNA b. Idiotype antibodies 2. Passive a. mab b. Cells

4 mab therapeutics 1. Direct killing of Cancer cells - ADCC and CDC Ex) anti-cd20, anti-her2 2. Immunomodulation - Enhance effector mechanisms Ex) anti-ctla4, anti-4-1bb (CD137)

5 Antibody Therapeutics

6 Immunomodulatory mab therapeutics 1. Ipilimumab (anti-ctla4) 2. Anti-4-1BB 3. Anti-PD1 4. Anti-PDL1 5. Anti-AITR

7 Immunomodulatory mab Therapeutics Anti-4-1BB Anti-CTLA4 Anti-4-1BB Anti-CTLA4 4-1BB Lymph node T cells Cancer -specific T APC cancer Cancer antigen 7

8 ICAM-1 CD48/58 ICOS-L B7-1 LFA-1 CD2 ICOS CD28 Positive costimulation B7-2 CTLA-4 PD-L1 PD-L2 PD-1? Negative costimulation HVEM BTLA-4 APC MHC TCR T cell LIGHT HVEM GITR-L GITR IgG superfamily CD40 CD40L TNF superfamily CD70 CD27 Positive costimulation 4-1BB-L 4-1BB CD30-L CD30 OX40-L OX40

9 Adoptive Immune Cell Therapeutics

10 I-1. Adoptive Immunotherapy Adoptive Immunotherapy DCs NKs CTLs Tumor killing mechanism CTL Induction Direct killing Direct killing Immunological Memory + -/+ +++ Intra-subset regulation >7 subsets yes >2 subsets yes ~2 subsets no Ag specificity Specific Non-specific Specific Proliferation in vivo Peripheral % < 1% 1-5% 10-30% Patient preconditioning No Yes Yes Production procedure Simple Simple Ag-specific T cell selection 10

11 Adoptive T cell therapy of NCI Gattinoni L. Nat Rev Imm 2006 Steve Rosenberg & Nick Restifo NCI, NIH, USA

12 Lymphodepletion 1. Homeostatic lympho-proliferation (Tan et al., JEM 195:1523, 2002; Gattinoni et al., JEM 202:907, 2005) 2. Preconditioning of cancer patient (Rosenberg et al., Clin Cancer Res 17:4550, 2011)

13 Survival of patients with metastatic melanoma treated with autologous tumor-infiltrating lymphocytes and IL-2 : Cross-clonal targeting and modification of microenvironment No TBI (43 pts) OR: 49% (CR:13%) Clin Cancer Res 17:4550, 2011 TBI (200cGy) (13 pts) OR: 52% (CR:20%) TBI (1200cGy) (18 pts) OR: 72% (CR:56%)

14 1. To aim cross-clonal common targets to overcome intratumoral clonal heterogeneity. 2. To modify immunosuppressive tumor microenvironment.

15 CTL therapeutics (Clinical Experience) Polyclonal CTLs Hurdle 1 PBMC OKT3 Polyclonal CTLs including some Agspecific CTLs Antigen-specific CTLs PBMC Lymphoblastoid Cell Lines (LCL) Antigen-specific CTLs PBMC Ag-pulsed DCs Antigen-specific CTLs PBMC Ag-pulsed Allogeneic APCs Antigen-specific CTLs

16 Solution Mixed Lymphocyte Peptide Culture (MLPC)

17 Problems with Purification of tumor-specific T cells Hurdle 2 Tumor T cell repertoire a b c d e.. 1. pmhc multimers - Lack of available reagents: peptide and MHC - TCR down regulation 2. Functional separation - cytokines or degranulation - Detect only subsets of T cells

18 T 세포의다양성 T cell diversity 유전자조합가능한 TCR: ~10 15 TCR 종류 : 총 T 세포수 : ~10 12

19 I-4. Cacer Immunotherapy at National Cancer Center 4-1BB (CD137) 4-1BB Cancer cells APC MHC I TCR Anti-tumor CD8 T cells Anti-apoptosis Proliferation htert WT1 NY-ESO1 Memory formation Longevity 19

20 pcmv pp65 Cell % 4-1BB pcmv pp65 4-1BB expression and Expansion of CMV + CD8 + T cells A #1 #2 #3 #4 # CD8 B 1 day 3 day 5 day 7 day 14 day C 100 pcmv + CD8 + T BB + CD8 + T CD CD Incubation time (day)

21 pcmv pcmv 4-1BB 4-1BB 4-1BB pcmv 4-1BB 4-1BB expression upon restimulation on Day 14 A Before 1 After restimulation Exp #1 Exp #2 Exp # B R1 Exp #1 Exp # R1 CD8 54 CD R2 FSC R2 CD CD8 CD8 CD8

22 pcmv pcmv + CD8 + T (%) Recovery rate (%) 4-1BB pcmv 4-1BB Use of 4-1BB for selection of CMV + CD8 + T cells A R1 Exp #1 Exp # R1 R2 FSC R2 CD CD8 B Panning CD8 Bead Exp #1 Exp #2 C Bead Panning D Bead Panning

23 I-4. Cancer Immunotherapy at National Cancer Center Adoptive 4-1BBCTL Therapy 암항원 (TAAs) Peptide(s) PBMC 에암항원을첨가하여배양 4-1BB 암항원에반응하는 T 세포의 4-1BB 발현 항 -4-1BB 항체를이용한암항원특이적 T 세포의분리 분리된 T 세포의대량배양 말초혈액 PBMC 분리 암환자투여 Pre-conditioning 23

24 I-4. Cancer Immunotherapy at National Cancer Center Clinical Trials at NCC ( 국립암센터 ) Name: EBViNT Cell; EBV-induced NCC T Cell Indications: Standard therapy-failed EBV-positive Cancers; Lymphomas, Gastric Cancers, Nasopharyngeal Carcinomas Date of Approval:

25 I-4. Cancer Immunotherapy at National Cancer Center EBViNT Cell Dose II (1/2 bag; cells, NCC-02 HCS) NCC-02 HCS 62 year-old, male ECOG PS 1 EBViNT Cells Lot # Ag HCS-CT HLA-A*24-restricted EBV/LMP2A Hodgkin's disease Prior chemotherapy regimen: #2 25

26 I-4. Cancer Immunotherapy at National Cancer Center EBViNT Cell Dose II (1 bag; cells, NCC-03 KMS) Rt lower leg lesion NCC-03 KMS 63 year-old, female 4 weeks after EBViNT Therapy ECOG PS 1 Extranodal NK/T cell lymphoma nasal cavity: EBV-ISH negative Rt lower leg mass: EBV-ISH positive r/o renal abscess, r/o tumor 6 months after EBViNT Therapy Prior chemotherapy regimen: #1 EBViNT Cells Lot # Ag KMS-CT HLA-A*24-restricted EBV/LMP2A 9 months after EBViNT Therapy

27 Benefits Complete durable regression can be induced. No/minimum toxicity. Epitope Spreading at a full dose of CTL. High rate of manufacturing success (7/8). 27

28 I-5. Lessons from the NCC Cancer Immunotherapy Strong 2 nd immune responses are found in the patient with complete regression Weak 2 nd response Strong 2 nd response No 2 nd response 28

29 I-5. Lessons from the NCC Cancer Immunotherapy Epitope spreading is required for the complete regression of tumor Tumor cells Cancer Heterogeneity IFN-g Driver CTLs kill target tumor cells Release of new tumor Ags Suppressive tumor microenvironment Epitope Spreading IFN-g-mediated Driver CTL-dependent Strong 2 nd immune response Intra- & inter-molecular epitope spreading Overcome Ag loss & MHC downregulation 29

30 Measures to improve NCC CTL Therapy 1. Effective in vivo expansion and Memory - CTL Subset and Homeostatic lymphoproliferation. 2. Immunosuppressive tumor microenvironment. 3. Immune cell suppressors in patient serum. 4. Manufacturing period of NCC CTL. 5. Immunosuppressive NK, myeloid cells and RBC during induction phase. 6. Why PD in NPC?

31 The inverse relationship of in vitro and in vivo effector functions of adoptively transferred T cell subsets

32 % Tcm CD8 T (CD45RO + CD62L + ) # CD8 T cells Production of Tcm NCC CTL IL-2+PBS IL-7+PBS IL-15+PBS IL-21+PBS IL-2+IL-7 IL-2+IL-15 IL-2+IL-21 IL-7+IL-15 IL-7+IL-21 IL-15+IL IL-2+PBS IL-7+PBS IL-15+PBS IL-21+PBS IL-2+IL-7 IL-2+IL-15 IL-2+IL-21 IL-7+IL-15 IL-7+IL-21 IL-15+IL-21

33 CD8 CD8 In vivo effect of cytokines on adoptively-transferred CD8 + T cells gc cytokine-induced CMV + CD8 T ( cells/ i.v./ mice) Analysis Rag2gc DKO mice IL-2 (IL-7 or IL-21) (50,000IU/i.p./mice) IL-2+PBS IL-7+PBS IL-21+PBS IL-2+IL-7 IL-2+IL-21 IL-7+IL-21 IL-2 CD4 + IL-2 IL-2+PBS IL-7+PBS IL-21+PBS IL-2+IL-7 IL-2+IL-21 IL-7+IL CD4

34 Modification of Cancer Microenvironment Cancer Cells CTL Lymphodepletion Immune Sppressors Cancer CTL

35 Shortening of production period of NCC CTL Modification 1 (shortening of restimulaton and rapid expansion periods) A. Original protocol (50cc) (50cc) (100cc) (300cc) (500cc) culture medium days Induction phase Restimulation Isolation Rapid expansion B. Test protocol (-2 days) (-4 days) (200cc) (300cc) (500cc) days Induction phase Restimulation Isolation Rapid expansion Modification 2 (increase of number of seed cells) - CD8 T cell number: current (5 x 10 5 cells per bag) - suggested ( 1 x 10 6 cells per bag) - Culture bag number: current (1 ~ 2 bags) - suggested ( 3 bags) A. Original protocol (50cc) (50cc) (100cc) (300cc) (500cc) culture medium days Induction phase Restimulation Isolation Rapid expansion B. Test protocol (-2 days) (-7 days) (200cc) (300cc) (500cc) days Induction phase Restimulation Isolation Rapid expansion

36 IV-1. 제 1 세부과제 - 선행연구 Epitope Screening for 4-1BBCTLs Cancer Antigens Response rate (%) EBV + cancers Hematologic Oncology Clinic EBV LMP2a (18) ~ 81% Glioblastomas Neuro-Oncology Clinic WT1 (20) ~ 68% Pancreatic cancers Center for Liver Cancer htert (25) ~ 55% Lung cancers Sarcomas Center for Lung Cancer Orthopedic Oncology Clinic htert (25) WT1 (20) MAGE-A3 (24) NY-ESO-1 (20) MAGE-A3 (24) ~ 60% ~ 70% ~ 70% ~ 53% ~ 58% NY-ESO-1 (20) ~ 70% Ovarian cancers OY-TES-1 (10) ~ 44% Center for Uterine Cancer Cervical cancers HPV (23) ~ 40% Gastric cancers Center for Gastric Cancer htert (25) ~ 70% Prostate Cancers Center for Prostate Cancer PSMA TARP Planned Breast Cancers Center for Breast Cancer NY-ESO-1 Planned Solid tumors Mutated tumor Ags Planned 36

37 Clinical Trial with WTiNT Cell Name WTiNT Cell; WT1-induced NCC T Cell Indications Standard therapy-failed glioblastoma pateints Data of Approval

38 Clinical Trial with TERTiNT Cell Name TERTiNT Cell; htert-induced NCC T Cell Indications Standard therapy-failed solid cancers; Lung cancers, Gastric cancers, Pancreatic cancers & Melanomas Data of Approval Under IND filing

39 Plans for future clinical trials Antigen Cell type Product Name KFDA approval Condition for Phase I clinical trial Indication EBV/LMP2A CD8 T EBViNT Cell EBViNT Cell alone WT-1 CD8 T WTiNT Cell WTiNT Cell + TMZ (lymphodepletion) htert CD8 T TERTiNT Cell - TERTiNT Cell + CTX + IL-2 NY-ESO-1 CD8 T ESOiNT Cells - ESOiNT Cell + CTX + IL-2 MAGE-A3 CD8 T MAGE3iNT Cells - HPV E6/E7 CD8 T HPViNT Cells - MAGE3iNT Cell + CTX + peptide vaccine HPViNT Cell + CTX + peptide vaccine Lymphomas Nasopharyngeal cancers Gastric cancers Glioblastoma, Leukemias Lung, gastric, & pancreatic cancers Sarcomas Ovarian cancers Lung cancers Sarcomas Cervical cancers

40 Milestones for the NCC T Cell Therapeutics Establishment of CTL production protocol Installation of cgmp facility, NCC cgmp translation of production protocol IND filing of EBViNT cells Pilot production Phase I of EBViNT Cells, Lymphomas KFDA inspection of cgmp facility, NCC IND filing of WTiNT cells Phase I of WTiNT Cells, Glioblastomas IND filing of TERTiNT cells Thank Everyone Involved!!

41 Limitations and Supplement Approaches to NCC CTLs Limitations 1. HLA restriction 2. Peripheral repertoire restriction 3. Manufacturing period Approaches to overcome the limitations 1. High avidity TCR gene-modified T cells 2. CAR (chimeric antigen receptor)-modified T cells

42 IV-1. 제 1 세부과제 High avidity TCR-expressing CTL (etcl) and Chimeric Antigen Receptor (CAR)-modified CTL 42

43 IV-1. 제 1 세부과제 자가유래 T 세포치료제의제한점 1. 비정상적혈액상태 T 세포치료제제조불가능 2.Mixture of low & high avidity TCR T 세포치료제효력감소 3. 긴제조공정 피험자사망시치료불가능 High avidity TCR 유전자확보 1.T 세포치료제적용환자비율증가 적용대상증가 2. 강력한 1차항암면역반응유도가능 완치효과증가 3. 제조공정단축 적용대상증가 43

44 High Avidity TCR Gene-modified T Cells

45 IV-1. 제 1 세부과제 추진전략및방법 I Cloning epitope-specific CD8 T cells 45

46 IV-1. 제 1 세부과제 추진전략및방법 II Development of high avidity TCR mini-library Single cell cloning Epitope-specific CD8 T (10 50 clonotypes) htert, WT1, NY-ESO1 Mutated tumor Ags ~4 weeks Single clone selection TCR mini-library Non-mutated tumor Ag htert WT1 NY-ESO1 Mutated tumor Ag p53, p53, k-ras, b-raf, b- Selection In vitro tumor killing assay etcr T cell production TCRa/b cloning Recombinant virus expressing the TCRa/b gene catenin, PIK3CA, JAK2 Discard 46

47 IV-1. 제 1 세부과제 추진전략및방법 III Personalized etcr T cells #1 #2 #3 #4 #5 A*02 A*24 A*02 A*24 A*02 A*24 TCRa/b Mini-library hter T WT1 NY-EOS1 TCRa/b 유전자도입 T 세포분리 TCR 유전자도입세포분리 대량증식 투여 암조직또는혈액 - 적용대상증가 - 강력한 1 차항암반응 - 짧은제조기간 47

48 IV-2. 제 2 세부과제 Chimeric Ag Receptor T cells Perforin Granzymes Ab ScFv Signaling 4-1BB CD3z Antigen Cancer Stem Cells CD8 + T cells IFN-g, TNF-a, IL-2 48

49 MAbs to produce CAR-modified T cells 1. Anti-MVR (malignancy variant receptor) 2. Anti-CSCR (cancer stem cell receptors)

50 CD19 Anti-MVR-BBz Chimeric Antigen Receptor Construct A ALL patient s PBMCs Surface 951/control Isotype Anti-MVR 951/MVR Anti-MVR-BBz CD8-leader (GLY 4 SER) 3 linker CD8-hinge/TM 4-1BB-Cyto CD3z-Cyto VH VL Anti-MVRscFv Signaling domain Anti-MVRscFv Lentiviral vector pelps-mvr-bbz 4-1BB CD3z MVR-BBz

51 Count Specific tumor cell lysis by anti-mvr CAR-T cells. BC-1 B-lymphoma CFSE Low LCL B-lymphoma CFSE High Anti- MVR No T cells Con T cells Anti-MVR-T cells 4 hrs 18 hrs CFSE

52 Summary I BB-based CTL therapeutics has been developed. 2. EBV-specific CTLs showed a therapeutic effect against NK/T lymphoma. 3. Effective cancer therapy requires both active tumor killing and nullification of tumor-promoting factors. 4. Improvement of 4-1BB CTL therapy Conversion of Treg to Teff Enrichment of central memory CD8 + T cells Shortening manufacturing period 5. etcr-ctl and CAR-modified CTL can overcome some limitations associated with peripheral repertoire-dependent CTL therapy