Flow Cytometric Applications for Immunotherapy of Cancer

Flow Cytometric Applications for Immunotherapy of Cancer Bone Marrow Transplantation and Cellular Therapy Program and Divisions of Neuro-oncology and Neurosurgery University of Alabama at Birmingham

Basic concepts General and iatrogenic suppression of the cancer patient s immune environment and tumor derived immunosuppresion - the empire strikes back Is there life for the immune system after chemo/radiotherapy? Immunotherapy strategies - cytokines, cells, genes, viruses and vaccines T cell therapy and innate immune strategies - a new hope?

Used with permission from The Wall Street Journal, WSJ.com. Copyright 200 Dow Jones & Company, Inc. All rights reserved. Flow Cytometry is evolving from the standard fare of lymphocytes and CD34

Flow Cytometric Immune Monitoring Pre-transplantation immune status - immunophenotyping, immune function, cytotoxicity Graft qualification - composition/potency Post-transplantation/postimmunotherapy monitoring - immunophenotyping, activation and cytotoxicity Minimal residual disease assessment in hematopoietic malignancies

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Tumor-derived Immunosuppresion and Immune Escape Down-regulation of MHC Class I Secretion of immunosuppressive proteins Induction of regulatory T cells (CD3+CD4+CD25+FoxP3+). MSC population of tumor stroma Interference with costimulatory factors such as down regulation of CD40L NK inhibitory receptors on MHC Class I - cells

T Cell Absolute Count in Patients with GBM pre- and post- Therapy 1.6 Absolute lymphocyte count x 10 6 1.4 1.2 1.0 0.8 0.6 0.4 Control PRE Early PostOP Late PostOP 0.2 0.0 CD3 CD3+CD8+ CD3+CD4+ Test

Proliferative capacity of gd T cells in healthy volunteers vs GBM patients.

T cells do not normally invade the parenchyma of GBM.

Circulating Regulatory T cells in Patients with Gliomas

Immune Suppression TGF-b Interference in multiple steps in cell cycle - inhibition of c-myc, cdk2 and cdk4 synthesis, induction of cdk inhibitors, inhibition of cyclin A synthesis Inhibition of antigen presentation by dendritic cells Inhibition T cell proliferative response to antigens, mitogens, and activating monoclonal antibodies. Abrogation of cytotoxic activity. T cell apoptotic death

Immune System Recovery

UPN 97 T CELL RECOVERY % OF TOTAL LYMPHOCYTES 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 0 200 400 600 800 1000 1200 1400 1600 DAYS POST BMT CD3 CD3+CD4+ CD3+CD8+

6 6 6 6 6 6 1600 1400 1400 1200 1200 EP vs CD3TCD EP vs CD3CON 1000 EP vs ABTCD EP vs ABCON 1000 800 800 600 600 400 400 Absolute Lymphocyte Count x 10 200 0 1 2 3 4 5 6 Post-BMT Interval Absolute Lymphocyte Count x 10 200 0 1 2 3 4 5 6 Post-BMT Interval 700 1000 600 EP vs CD4TCD EP vs CD4CON 800 EP vs CD8TCD EP vs CD8CON 500 400 600 300 400 200 Absolute Lymphocyte 100 0 1 2 3 4 5 6 Post-BMT Interval Absolute Lymphocyte 200 0 1 2 3 4 5 6 Post-BMT Interval 120 30 100 25 EP vs GDTCD 80 EP vs CDCON 20 EP vs CD4RATCD EP vs CD4RACON 60 15 40 10 Absolute Lymphocyte Count x 10 20 0 1 2 3 4 5 6 Post-BMT Interval Absolute Lymphocyte Count x 10 5 0 1 2 3 4 5 6 Post-BMT Interval

500 500 EP vs CD56TCD EP vs CD56CON EP vs CD19TCD EP vs CD19CON 400 400 Absolute Lymphocyte Count x 10 6 300 200 100 Absolute Lymphocyte Count x 10 6 300 200 100 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Post-BMT Interval Post-BMT Interval

CD4+ T Cell Regeneration Following Intensive Chemotherapy Young children have a more rapid recovery of CD4+ T cells (initially CD4+CD45RA+). Thymic dependent CD4+ T cell regeneration occurs primarily in young children. Older patients have deficiencies in the thymic pathway of CD4+ T cell recovery that could impact on host immunocompetence.

Specific Features Of Thymic - Independent T Cell Regeneration T cell diversity is limited by the initial T cell inoculum in the graft. Host antigenic exposure provides a critical means of peripheral T cell expansion. This raises the issue of potential limitation of the T cell repertoire versus that produced by thymicdependent T cell regeneration.

CD4+ T Cell Function in NSCLC Patients Following Induction Chemotherapy 2D Graph 1 700 Stimulated CD4+ ATP Release 600 500 400 300 200 100 0 9622 10249 UPN Pre Chemo Post-Chemo

T Cell Function 100 Days Following Allog TCD BMT

CD4/CD69 CD8/CD69 R2 R5 REST R3 R6 SEB R4 R7 PHA

Minimal Residual Disease Monitoring

MRD Monitoring - Issues Requires highly trained technical staff familiar with multiparameter Boolean gating in flow cytometry analytical software. Best done on the same day as immune recovery testing to establish immune status and to plan for cellular therapy if warranted. Element of faith evidence of things not seen

Specialized topics

A multi pronged approach to cancer immunotherapy Type Frequency Enhance survival T cell immunity NK/LAK DC vaccination Costimulation: OX40, 4-1BB In situ maturationcd27, CD40 (mrna/dc) Antigen Defined tumor-associated antigens Mixtures (ampl. RNA) Induction of immunity + Persistence of immunity Prevent attenuat CTLA-4 Immune suppression Immune escape Tregs B7H-1, TGFβ, IL-10 ClassI/II down-regulation KIR Fas/FasL

Flow Cytometry Quality Control CD34 April G. Durett Flow Cytometry Facility Center for Cell & Gene Therapy

Role of Flow Cytometry and Cell Sorting Expansion in the areas of evaluation of graft preparation assessment of cellular therapies monitor immunological recovery Expanded role requires increased technical proficiency increased Quality Control and Assurance

Flow Cytometry Facility Certified High Complexity CAP Accreditation College of American Pathologists Unannounced inspections every 2 years Increasing focus on QC / QA Guidelines / Checklists Flow Cytometry Hematology General Laboratory CLIA Certification Clinical Laboratories Improvement Act Texas Department of Health

Quality Control Documentation The Mantra Documentation Documentation

Quality Control for Flow Cytometry Involves all steps and aspects of procedure Sample collection, labeling, transport Process sample integrity & staining Reagents Equipment Data Management Dot-plot analysis Final reports Personnel

Quality Control - Reagents Monoclonal Antibodies should be titrated with cells of interest prior to use Insufficient MAb Antigenic sites not saturated Excess MAb Increase non-specific binding and quenching Non-titrated antibodies may result in both decreased staining intensity and percent positive populations

Quality Control Monoclonal Antibodies Fluorescence (geometric mean channel) µl CD34(8G12) per 5x10 5 Cells 0 10 20 30 40 1600 1200 800 400 Manufacturer recommended dosage per 10 6 Cells 0 0 200 400 600 800 1000 ng CD34(8G12) per 5x10 5 Cells

CAGT Staining Protocol Cell Concentration 10 5 10 6 in 100µL MAb saturated dose in 100µL Reaction Time 15 minutes in dark Reaction Temp 18ºC C to 22ºC Optimize each variable to establish lab standard

Staining Protocol 3-part Diff T & B Cells T & NK Cells Stem Cells Graft Evaluation Panel CD45 FITC CD45 FITC CD45 FITC Lineage1 FITC CD14 PE CD19 PE CD16 PE CD56 PE CD34(8G12) PE CD3 PerCP CD3 PerCP CD45 PerCP Analysis Trigger gate = CD45 + cells Logical Sequential Gating [Boolean]

Quality Control Staining Protocol Lymphs from 3-part 3 Diff = Light Scatter Lymphs from T, B, & NK tests T + B + NK = Lymphs from 3-part 3 Diff Commercial Reference Controls Assay value within manufacturer s s range Accuracy, precision, & variability tracked with Levy-Jennings plot

Quality Control - Instruments Maintenance Logs for EACH Instrument Cleaning Calibration Reference controls Instrument specific parameters Scheduled specific maintenance monthly, quarterly, semi-annual, annual Problem Solution log Levy-Jennings plots

Quality Control Flow Cytometer Optical Alignment use of manufacturer recommended particles Laser Current / Power PMT voltages Compensation use of manufacturer recommended particles Establish base-line cellular compensation All data evaluated for acceptable ranges and variability using Levy-Jennings

Quality Control Flow Analyst Competency Comprehensive testing following initial training Observation by supervisor Wet lab testing Dry lab testing Written quiz Communication Knowledge Annual follow-up testing Service Astuteness Technique

Quality Control Flow Analyst Standardized Proficiency Testing College of American Pathologists [CAP] Required for accreditation Pass >80% Blind samples Flow Cytometry CD34 2 samples / 2 times per year Flow Cytometry Lymphocytes 3 samples / 3 times per year Hematology WBC, RBC, PLT 5 samples / 3 times per year

CD34 The Beginning Beckman-Coulter Technical Bulletin Hester & Durett ~1991 MDACC

Relationship of CFU and Cell Surface Markers CD34 CD90 CD109 Quiescent Stem Cell CFU-GEMM CD34 CD109 CD117 CD33 CD38 CD34 CD90 CD109 CD117 CD71 CD38 Cycling Stem Cell T-Lymphoid Progenitor BFU-E CD34 CD109 CD117 CD71 CD45RO Erythroid Lineage BFU-MK CD34 CD109 CD117 CD61 CD38 Megakaryocytic Lineage CFU-GM CD34 CD109 CD117 CD13 CD38 CD38 B-Lymphoid Progenitor CD34 CD90 CD19 CD38 HLA-DR TdT B-cell Lineage Granulocyte Lineage CD34 CD90 CD2 CD5 CD7 CD38 TdT NK Lineage T-cell Lineage

Flow Cytometry Facility Dedicated to CAGT and BMT CD34 Monitoring Graft Evaluation HPC BM UCB Quality Assessment of Graft Processing Procedures Cell Specific Selection - Depletion Infusion dose Quality Assessment of Cell Therapy Products & Vaccines B-cell depletion efficacy Release criteria for specific cellular subsets Immune Reconstitution following Transplant or Cellular Therapy

Development of Prediction Algorithms Circulating Peripheral Blood CD34 Determine optimal time for apheresis Reduce number of apheresis procedures Reduce number of products cryopreserved Achieve protocol directed CD34 target values Provide overall cost reduction

Relationship between Circulating Peripheral Blood CD34 concentration and Stem Cells collected per Apheresis Apheresis Collection Total Lin NEG CD34 + Cells 10 10 10 9 10 8 10 7 10 6 Dependent upon mobilization Dependent upon apheresis parameters Software TBP Interface stability 10 5 1 10 100 1000 Lin NEG CD34 + Cells per µl L Peripheral Blood

Assessment of Cellular Damage R1 R2 R3 R1 = 81% 7AAD NEGATIVE healthy intact cells Side Scatter R2 = 7% 7AAD MEDIUM cells with ruffled membranes & vacuolated cytoplasm R3 = 12% 7AAD BRIGHT nuclear material 7AAD [ViaProbe] R1 + R2 = Trypan Blue

Assessment of Cellular Damage 92% Good 92% 7AAD 7AAD NEGATIVE 81% Bad 81% 7AAD 7AAD NEGATIVE 38% Ugly 38% 7AAD 7AAD NEGATIVE Side Scatter 7AAD

Efficacy of Cell Specific Selection and Purging Procedures CD34 Recovery 120% 100% 80% 60% 40% 20% 0% 10 7 10 8 10 9 10 10 CD34 Load Mass CD34 Purity 120% 100% 80% 60% 40% 20% 0% CD34 Recovery is inversely related to CD34 load mass [r -0.546] CD34 Purity is directly related to CD34 load mass [r +0.709]

Efficacy of Cell Specific Selection and Purging Procedures Determine composition of graft post-processing processing 100% Positive Fraction - Cell Purity 80% 60% 40% 20% 0% AUTO ALLO NMDP Grans CD19 CD34

Summary Flow Cytometry has an important role in graft evaluation and transplant follow-up Current regulatory environment requires ongoing Quality Control / Quality Assurance Of products for transplantation Of the evaluation methods and instruments Of analysts involved in evaluation