Melanoma Bridge Meeting
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1 Melanoma Bridge Meeting Improving Adoptive Immune Therapy with Genetically Engineered T cells David Stroncek, MD Chief, Cell Processing Section, DTM, CC, NIH 3 December 2015
2 Adoptive T Cell Therapy: Dose Matters 1x10 5 cells/kg 1x10 6 cells/kg CD19 CAR T Cells 1x10 7 cells/kg Anti-BCMA CAR T Cells? 1x10 8 cells/kg 1x10 9 cells/kg TIL
3 Need for T Cell Expansion Large doses required - TIL Vector is expensive start with low quantities of cells
4 Methods of Cell Expansion IL-2 + anti-cd3 IL-2 + anti-cd3 + irradiated pooled healthy donor lymphocytes IL-2 + artificial antigen presenting cells (K562 cells) IL-2 anti-cd3/anti-cd28 beads
5 Chimeric Antigen Receptors CAR CD19 CD28 CD3 Key Components: Antigen-binding (scfv) CD3-zeta Signal 1 Costimulatory Signal 2 (CD28, CD137 (41BB)) Advantages over T-Cell Receptors: Specific for a surface antigen Free of MHC restriction Signals for full activation are selfcontained Adapted from Lee DW et al, Clinical Cancer Research 2012
6 Manufacture of CAR-modified T cells Shannon L. Maude et al. Hematology 2014;2014: by American Society of Hematology
7 CAR T Cell Therapy at the NIH CD19 Children with acute lymphocytic leukemia (ALL) CD22 Children with CD19-negative ALL Anti-GD2 Children with osteosacrcoma and neuroblastoma Anti-BCMA (B cell maturation antigen) Adults with multiple myeloma
8 Results of Treating 20 Pediatric ALL Patients with Anti- CD19 CAR T Cells Lee DW et al, Lancet 2014
9 Other CAR T Cell Clinical Trials
10 CD19-CAR T Cell Manufacturing at the NIH Initial Impression Yields of CAR T cells were highly variable Some products failed to meet lot release criteria: 1 or 3x10 6 transduced cells/kg Potential Causes Poor quality T cells Prior chemotherapy Underlying disease Biological variability Quality of the apheresis product Inhibition by contaminating cells
11 Composition of Peripheral Blood Mononuclear Cell (PBMC) Concentrates PBMC Concentrates Lymphocytes Monocytes Granulocytes Natural Killer cells Red blood cells Platelets Composition of PBMC Concentrates from Healthy subjects (n = 41) Mean ± 1SD Range Lymphocyte (%) 68.4 ± to 83 Monocytes (%) 18.8 ± to 32 Granulocytes (%) 9.54 ± to 42
12 Myeloid Derived Suppressor Cells (MDSC) Found in the blood and tumor microenvironment of cancer patients Suppress T cell proliferation Monocyte (CD33+ CD14+ HLA-DR-) and granulocyte (CD33+ CD15+ HLA-DR-) phenotypes Presence in patients with hematologic malignancies is not certain
13 Reviewed CD19- and Anti-GD2-CAR T cell Processing Records Assessed product consistency and factors associated with poor outcomes How often will a patient enrolled in a clinical trial receive the therapy? Can the manufacturing process be improved?
14 CD19- and Anti-GD2-CAR T Cell Manufacturing Tumaini B, Lee DW, Lin T, Castiello L, Stroncek DF, Mackall C, Wayne A, Sabatino M. Cytotherapy Nov;15(11):
15 CD19-CAR T Cell Manufacturing Protocol: 12-C-0112 Phase I Study of T Cells Expressing an Anti-CD19 Chimeric Receptor in Children and Young Adults with B Cell Malignancies 28 Patients (July Dec 2014) First 22 patients: 11 day culture Patient 23 to 28: 7 day culture
16 First 28 CD19 CAR T Cell Products: Transduced T Cell Yield Cells in Final Product Mean ± 1SD Range T Cells (x10 6 ) 1,362 ± to 3,800 Transduced T Cells (x10 6 ) 1,084 ± to 2,990 Transduced T Cells (%) 68.3 ± to 96.8 Four Products Failed to Meet Dose Patient 2 (3.9 x10 6 transduced T cells) Patient 5 (19.4x10 6 transduced T cells) Patient 22 (0.0 transduced T cells) Patient 26 (2.4x10 6 transduced T cells) Transduced T Cells (106)
17 Composition of the Final CD19 CAR T Products (n=27); Proportion of CD3+ Cells and Transduced T Cells CD3+ (%) ± 8.9% Transduced T Cells (%) ±
18 Comparison of PBMC Concentrates that Resulted High and Low CD19 CAR T Cell Yields Met Dose Requirements (n=24) Did Not Meet Dose Requirements (n=4) Lymphocytes 75.3 ± 14.1% 42.3 ± 8.4% Monocytes 15.3 ± 10.8% 39.8 ± 12.9% Granulocytes 6.9 ± 8.6% 16.3 ± 12.2% p
19 Large Quantities of Monocytes and Granulocytes in PBMC Concentrates are Associated with Poor CD19- CAR T cell yields (n=22) Transduced T Cells (x106) r = Transduced T Cells (x106) r = Monocytes (%) Granulocytes (%)
20 Autologous PBMCs Depletion of Plastic Adherent Cells During the Production of Autologous Anti-CD19 CAR T Cells Plastic Adherence T Cell Isolation Anti-CD3/CD28 bead selection T Cell Stimulation Anti-CD3/CD28 beads + IL-2 Transduction Anti-CD19/CD28/CD3 zeta vector Expansion Continue culture with anti-cd3/cd28 beads + IL-2
21 Monocyte Depletion Improves CD19 CAR T Cell Yield: Manufacturing Cells a Second Time for 2 Patients Patient 22 s Fresh PBMCs CD3+ Cell Antibody-Bead Selection CD19-CAR T Cell Product TNC Percent Transduced T Total Transduced Cells Cells 38.1 x10 6 NT 0 Patient 26 s Fresh PBMCs CD3+ Cell Antibody-Bead Selection CD19-CAR T Cell Product TNC Percent Transduced T Total Transduced Cells Cells 19.8 x % 2.4x10 6 Patient 22 s Thawed PBMCs CD3+ Cell Antibody-Bead Selection + Plastic Adherence Monocyte Depletion CD19-CAR T Cell Product TNC Percent Transduced T Total Transduced Cells Cells 186 x % 147x10 6 Patient 26 s Thawed PBMCs CD3+ Cell Antibody-Bead Selection + Plastic Adherence Monocyte Depletion CD19-CAR T Cell Product TNC Percent Transduced T Total Transduced Cells Cells 171 x % 160x10 6
22 More Rigorous Monocyte Depletion Improves CD19-CAR T Cell Yields: Anti-CD3/CD28 Beads plus Plastic Adherence (n=13) Results in Greater Yields than Depletion of Monocytes by Anti-CD3/Anti-CD28 Beads (n=6) 253±227x10 6 vs 394±187x10 6 ; p= Transduced T cells (x106) Antibody-Bead Antibody-Bead + Plastic Adherence
23 Anti-GD2 CAR T Cell Manufacturing Protocol: 14-C-0059 A Phase I Trial of T Cells Expressing an anti-gd2 Chimeric Antigen Receptor in Children and Young Adults with sarcoma and neuroblastoma expressing GD2 PI: Crystal Mackall 11 patient have been treated First 6 patient: standard processing CD3/CD28 bead selection Most recent 5 patients: modified processing Plastic adherence CD3/CD28 bead selection
24 Monocyte-like MDSCs in Pediatric Sarcoma Patients Zhang H et al. Blood. 2013;122(7):
25 Expansion of Anti-GD2-CAR T Cells (n=6) is Less Than That of CD19-CAR T Cells (n=22) A B T Cells (x106) Transduced T Cells (x106) Anti-CD19 Anti-GD2 0 Anti-CD19 Anti-GD ,945 ±1,213x10 6 vs 159±106x10 6 ; p= ,369± 889x10 6 vs 127±102x10 6 ; p=
26 Comparison of Anti-GD2- and CD19-CAR T PBMC Concentrates: Anti-GD2 CAR T PBMCs Contain More Monocytes CD19 CAR (n = 22) Mean ± 1SD Anti-GD2 CAR (n = 6) Mean ± 1SD Lymphocytes (%) 70.8 ± ± Monocytes (%) 18.0 ± ± Granulocytes (%) 8.4 ± ± p
27 Anti-GD2 CAR T Cell Yields: Effect of Monocyte Depletion by Plastic Adherence 3000 T Cell Expansion 183±106x10 6 (n=6) vs 1,404±1136x10 6 (n=5) p= Transduced T Cell Expansion 147±102x10 6 (n=6) vs 576±437x10 6 (n=5) p= T Cells (x106) Transduced T Cells (x106) Antibody-Bead Antibody-Bead + Plastic Adherence Antibody-Bead Antibody-Bead + Plastic Adherence
28 Monocyte Depletion Improves Anti-GD2 CAR T Cell Yield: Manufacturing a Patients Cells a Second Time Patient 9 s Fresh PBMCs CD3+ Cell Antibody-Bead Selection TNC CD19-CAR T Cell Product Percent Transduced T Cells Total Transduced Cells 45.3 x % 4.8x10 6 Patient 9 s Thawed PBMCs CD3+ Cell Selection + Ficoll + Plastic Adherence Monocyte Depletion >400-fold change TNC CD19-CAR T Cell Product Percent Transduced T Cells Total Transduced Cells 5,200x % 1,986x10 6
29 Enriching PBMC Concentrates for Lymphocytes Selection of CD4+ and CD8+ cells using monoclonal antibodies conjugated to magnetic beads Miltenyi CliniMACS Plus and Prodigy
30 Elutriation: Principles Centrifugation Separation is based on cell density Counter-Flow Elutriation Separation based on size Expected order of elutriation Platelets RBCs Lymphocytes Granulocytes Monocytes
31 Elutra Terumo BCT Modified blood cell separator (Spectra) Closed system disposables Operation time is approximately 45 minutes; total time about 1 hour and 15 minutes
32 Anti-GD2 CAR T Cell Yields: Effect of Monocyte Depletion by Plastic Adherence (n=5) and Elutriation (n=2) 1600 Transduced T Cells (x106) Plastic Adherence Elutriation
33 Conclusions Some autologous PBMC concentrates collected from patients with ALL, sarcoma or neuroblastoma contain large quantities of monocytes and granulocytes Monocytes and possibly granulocytes can inhibit T cell expansion Rigorous depletion of PBMCs of monocytes and granulocytes improves T cell expansion
34 Cell Processing at the NIH We are implementing more rigorously depletion of granulocytes and monocytes from PBMC concentrates for CAR T cell manufacturing Evaluating monocytes removed from the PBMC concentrates Other manufacturers appear to moving toward the use of highly purified lymphocyte preparations
35 Acknowledgements NIH, Cell Process Section Minh Tran Sue Ellen Frodigh Jiaqiang Ren Vicki Fellowes Jianjian Jin Virginia DavidOcampo Marianna Sabatino NCI Pediatric Oncology Branch Trey Lee Terry Fry Crystal Mackall
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