Adoptive cell therapy using genetically modified antigen-presenting cells Naoto Hirano Ontario Cancer Institute Princess Margaret Cancer Centre University of Toronto UofT-USP Oncology Conference November 15, 213
Adoptive transfer of antitumor T cells Establishes antitumor immunologic memory enabling life-long rejection of cancer cells.
Can we generate antitumor T cells in vitro which possesses an intrinsic capacity to persist as memory T cells in humans?
T cell grafts T cell clones Advantages Known Ag specificity and avidity (monoclonal). Disadvantages Higher chance of antigen escape. T cell lines Known Ag specificity with unknown avidity (polyclonal). Possibly not high avidity. Tumor-infiltrating lymphocytes Polyclonal Ag specificities; lower chance of antigen escape. Not available from all patients; specificities not completely defined. TCR-transduced T cells Off-the-shelf agent. Higher chance of Ag escape (monoclonal); possible TCR mispairing. CAR-transduced T cells Known Ag specificity and high avidity. Unexpected on/off target toxicites.
K562-based artificial antigen presenting cells (aapc) to generate antigen-specific CD8 + T cells IL-2 CD83 CD83L CD8 CD28/CTLA4 aapc-a2 aapc HLA-A2 TCR CD8 + T cell 1 ICAM-1 LFA-1 LFA-3 CD2 HLA-A2 CD8 CD83 ICAM-1 LFA-3 1 1 1 1 IL-15 8 8 8 8 8 6 6 6 6 6 4 4 4 4 4 2 2 2 2 2 K562: HLA class I/II -, CD8 ±, CD83 -, CD86 -, ICAM-1 +, LFA-3 +, PDL1/2 - (Hirano et al. Blood, 26)
Long-lived MART1 CTL generated with aapc MART1 specific CTL line Ficoll MART1 tetramer (Butler et al. Clin Cancer Res, 27)
Central memory ~ effector memory phenotype of antigen-specific T cells generated with aapc CD25 + CD27 + CD45 CD45 RA - CD28 +- CD62L +- CD69 + CCR7 RO + +-
Phase I study of the adoptive transfer of MART1/ Melan-A CTL for malignant melanoma (ClinicalTrials.gov, NCT512889) Eligibility HLA-A*2:1 Stage IV or unresectable stage III melanoma Absolute lymphocyte count >1,/µl Accessible tumor for excision/biopsy Exclusion Immunosuppressive therapy <3 days Active autoimmunity requiring immunosuppression
Generation of clinical grade CTL with aapc 33 aapc 33 Pulsed with MART1 peptide at 1 μg/ml Cytotoxicity A2 + PBMC from single pheresis Positively selected CD8 + T cells (CliniMacs) 1 1 1 1 1 1 Multimer staining Interferon-γ ELISPOT Large scale stimulations every 7 days (3 weeks)
Generation of MART1 CTL using clinical grade aapc 33 MART1 Tetramer Stain MART1 Tetramer Positive Cells
Study timeline CTL Generation 1 CTL Generation 2 Leukapheresis 1 Infusion 1 Infusion 2 Leukapheresis 2 Leukapheresis 3 Day -21 Day Day 14 Day 35 Day 7 CTL generation Leukopheresis, select for CD8 + T cells, and 21 day culture with irradiated, MART1 peptide pulsed aapc 33 in IL-2 and IL-15 Two 35 days cycle 21 days of CTL culture 2nd infusion of CTL on day 35 Two dose levels Cohort one: 2 x 1 8 cells/m 2 Cohort two: 2 x 1 9 cells/m 2 No in vivo modulation No cytokine or vaccine therapy No lymphodepletion (Butler et al. Sci Transl Med, 211)
A Subject 7 Pre-infusion Post-infusion Multimer Adoptive transfer of MART1 CTL results in long-term increases in circulating MART1 T cells day day 35 day 7 day 126.2% 6.5% 6.4%.3%.19% CD8 B MART1 multimer + CTL (% CD8).4.3.2.1 Subject 2 6 4 2 1 Subject 7 C MART1 multimer + CTL (% CD8).6.5.8.6.4.2 P =.1 P =.1 Day 35 65 95 35 65 95 125 Infusions. Preinfusion day 14 day 49
Persistent MART1 T cells are phenotypically memory T cells Multimer CD45RA Subject 2 Pre-infusion.4% CD8 1.11% 1 5 8 26 CD62L 84 12 day 1 Post-infusion 54 day 12.8% 8 14 33 45 Multimer CD45RA.2% Subject 7 5 Pre-infusion CD8 49 9 37 CD62L 5.58% day 1 2 8 91 Post-infusion.3% 4 day 7 19 4 24 53 day 258.11% 32 11 53 Subject 8 Pre-infusion Post-infusion B Multimer CD45RA.3%.46% CD8 17 38 2 4 21 24 25 69 CD62L day 1 day 66.6% 16 1 5 3 45 34 day 156.8% 4 5 55 36 CD45RA - CD62L + expression on MART1 multimer + CTL (%) 75 5 25 P =.2 Pre-infusion day 7
Persistent MART1 T cells are functionally memory T cells Subject 2 IFN- SFU/1 6 CD8 + T Cells 15 1 P =.1 5 Control MART1Control MART1 Pre-infusion day 56 IFN- SFU/1 6 CD8 + T Cells 75 5 25 P =.2 A375 Malme-3M day 56 Subject 7 IFN- SFU/1 6 CD8 + T Cells 2 15 1 P =.2 5 Control MART1Control MART1 Pre-infusion day 7 IFN- SFU/1 6 CD8 + T Cells 2 15 1 5 P =.4 A375 Malme-3M day 7 Subject 8 IFN- SFU/1 6 CD8 + T Cells 5 25 P =.3 Control MART1Control MART1 IFN- SFU/1 6 CD8 + T Cells 5 25 P =.4 A375 Malme-3M Pre-infusion day 66 day 66
Infused MART1 CTL traffic to tumor sites Post-infusion day 5 Pre-infusion A CD8 1 1 11 12 13 CD8 1 5Vb14A 5Vb2B 5Vb2A 13.2% No V primer 1 1 2 Foxp3 5Vb14A 5Vb2B 1 Expanded MART1 CTL 5Vb2A Multimer 12 Fresh TIL.54% No RNA 1 3 Multimer B No V primer CD4 Clonotypic CDR3 UbcH5B CD8 Graft 1 Graft 2
Complete resolution of metastatic melanoma following adoptive transfer A Pre-infusion Post-infusion day 74 day 14
No. Patient response to adoptive therapy Age/ sex Advanced cancer Day 7 status Time to next therapy 1 74M liver, adrenal, spleen, lung, skin Died on day 51 2 69M lung, skin 3 49F lung, adrenal Progressive disease Mixed response Day 13 Day 146 4 68M muscle, lung, mediastinum, cardiac Stable Day 14 5 66M multiple lymph nodes Partial response 54+ months (complete response without further therapy) 6 55M lung Stable Day 287 7 7F lung, skin Progressive disease Day 335 8 8M lung, mediastinal nodes Stable Day 372 9 64M lung, skin Progressive disease Day 146 As of November, 213
Basic Mechanisms of T Cell Stimulation and Inhibition Stimulation Inhibition
Anti-CTLA4 mab (Ipilimumab) Removes the brakes on the immune response. Induced 1-15% objective clinical response in advanced melanoma patients (Hodi et al. N Eng J Med, 211). Approved by FDA in 211 and Health Canada in 212.
A Anti-CTLA4 mab therapy promotes the expansion of infused CTL as memory T cells Pre-infusion Post-infusion day 74 day 167 day 224 day 537 CTLA-4 (+) Multimer CD45RA CD8 1.4% 1 5 CD62L 84.9%.11%.17% 7.8% 8 12 23 11 9 13 2 6 47 21 44 56 23 59 26 1 8 6 4 2 V 14 69% B 25 P =.1 25 P <.1 IFN- SFU/1 5 PBMC 2 15 1 5 Control MART1 Control MART1 IFN- SFU/1 5 PBMC 2 15 1 5 A375 Malme-3M Pre-infusion day 537 day 537
Durable responses following CTLA4 blockade αctla-4 Subject 2: response 16 months αctla-4 Subject 3: response 61+ months
Patient Response to Anti-CTLA4 mab Therapy No. Age/ sex Time to next therapy Next therapy Outcome after next therapy Duration of response 1 74M -- Died without next therapy -- -- 2 69M Day 13 Ipilimumab (1 mg/kg) Partial response 16 months 3 49F Day 146 Ipilimumab (1 mg/kg) Partial response 61+ months 4 68M Day 14 BRAF inhibitor Stable 3 months 5 66M No further therapy No further therapy No further therapy 54+ months (complete response) 6 55M Day 287 High dose IL-2 Death due to sepsis -- 7 7F Day 335 Ipilimumab (3 mg/kg) Stable 6 months 8 8M Day 372 Ipilimumab (3 mg/kg) Stable 5 months 9 64M Day 146 Ipilimumab (1 mg/kg) + bevacizumab Partial response 43+ months As of November, 213
Other immunotherapeutics that can be combined with aapc-based adoptive T cell therapy TCR gene transfer A2/NY-ESO-1,A24/WT1 Checkpoint blockade CTLA-4, PD-1, PDL1 mab Cytokine administration IL-7, IL-15, IL-21 Vaccines DC, peptide, protein, DNA, RNA, adjuvant Non-myeloablative lymphodepletion Radiotherapy Small molecule inhibitors
Conclusions Artificial APC-based adoptive T cell therapy can establish antitumor immunologically memory in patients with advanced cancer without lymphodepletion or cytokine therapy. Checkpoint blockade can enhance pre-existing antitumor immunologically memory in patients with advanced cancer.
Effective combination immunotherapy with minimal toxicities in vivo T cell in vivo T cell immunity in vivo T cell immunity in vivo T cell immunity immunity Anti tumor Depletion Anti tumor Establishment Anti tumor Enhancement Anti tumor Lymphodepletion Adoptive transfer Vaccine Checkpoint blockade Cytokine
Acknowledgments Dana-Farber Cancer Institute Marcus Butler Philip Friedlander Makito Tanaka Stephen Hodi Osamu Imataki Linda Drury Alla Berezovskaya Lisa Brennan Mary Mooney Marisa Flavin Andrew Murray Donna Neuberg Genita Metzler Kristen Stevenson Matthew Milstein Lee Nadler Massachusetts General Hospital Donald Lawrence Martin Mihm Brigham and Women s Hospital Sara Russell Michael Jaklitsch Elsa Velasquez Ontario Cancer Institute/Princess Margaret Cancer Centre Munehide Nakatsugawa Shlomit Boguslavsky Yuki Yamashita Diana Gray Toshiki Ochi Valentin Sotov Kenji Chamoto Shinya Tanaka Marcus Butler Tingxi Guo
Acknowledgments Grant Support Immunotherapy Fund 1 S. Craig Lindner Fund for Cancer Research Rudolf E. Rupert Foundation for Cancer Research Cancer Research Institute/Ludwig Institute for Cancer Research Cancer Vaccine Collaborative Friends of the Dana-Farber Cancer Institute Dunkin Donuts Rising Stars Program American Society of Hematology National Institute of Health/National Cancer Institute Ontario Regional Biotherapeutics Program (ORBiT) Ontario Institute for Cancer Research Campbell Family Institute for Breast Cancer Research Princess Margaret Cancer Foundation