CANCER GENE THERAPY WITH DESIGNER T CELLS: TOXICITY VERSUS ACTIVITY Richard P Junghans, PhD, MD Director, Biotherapeutics Development Lab Associate Professor of Surgery and Medicine Boston University School of Medicine Chief, Division of Surgical Research Roger Williams Medical Center Providence, RI, USA No commercial relationships to disclose.
Anti- CEA Normal T cells Designer T cells IgTCR = Immunoglobulin-T cell receptor CIR = Chimeric immune receptor
Carcinoembryonic antigen (CEA) o Expression High on tumor, low on normal Topological sequestration o High clinical relevance: On colorectal, breast, pancreas, lung, others More than 100,000 deaths/yr for CEA+ tumors
CLINICAL TRIAL Clinical Data: 1 st Generation Phase I Study of Anti-CEA Designer T Cells in Adenocarcinoma ( 1st generation ) FDA BB IND 7301
TCR Anti-Cancer T Cell Gene Therapy Gene-Modified TCR
Interventions o Phlebotomy/Apheresis o Isolate patient s peripheral blood mononuclear cells (PBMC) o Activate/transduce with IgTCR o Expand in IL2 o Harvest cells equal to dose; Infuse o Monitor for Toxicity/Response
Clinical Summary o Number of doses administered (24) o Patients treated (7): 5 colorectal, 2 breast o Doses sizes administered 1 x 10 9, 3 x 10 9, 1 x 10 10, 3 x 10 10, 1 x 10 11 cells
Pharmacokinetics Rapid Systemic Loss
CEA (NG/ML) 1600 1400 1200 Response: Proof of Principle T Cells CEA 1000 800 Increasing pain 600 400 Pain resolved 200 0-28 -21-14 -7 0 7 14 21 28 Day of Treatment BUT! Time-Limited in Duration
Pharmacodynamics o Immunogenicity: None. 0/7 with plasma reactivity against designer T cells o IL2 arm: Systemically active. Positive for NK cell expansion, T cells stable
Toxicity o Probably related or Definitely related No grade III toxicity, one grade IV toxicity (grade II fever >> grade IV SVT) No delayed grade III, IV toxicity Positive for low grade fevers, mild GI symptoms (<grade III) Transient hypoxemia (O 2 sat<90%) with high T cell dose
(-) IL 2 Toxicity Name Sex Age Diagnosis Dose Fever (24h) Adverse Events (Grade III-IV) Relatedness MS Dose 1 (6/10/98) Dose 2 (6/24/98) Dose 3 (7/8/98) F 75 Colon Ca 1x10 9 3x10 9 1x10 10-102.3 F Bilirubin GI Bleed * Death * Possibly (temporal only) # Possibly (temporal only) # RH Dose 1 (7/13/99) Dose 2 (7/27/98) Dose 3 (8/10/98) Dose 4 (8/26/98) M 55 Rectal Ca 1x10 9 3x10 9 1x10 10 3x10 10 102 F 101 F <101 F <101 F Anemia Possibly (temporal only) # DB Dose 1 (7/15/98) Dose 2 (7/29/98) Dose 3 (8/12/98) Dose 4 (8/26/98) M 54 Colon Ca 1x10 9 3x10 9 1x10 10 3x10 10 99.8 F Rigors 102.1 F Bilirubin (9/23/98;28 days) Possibly (temporal only) # HF Dose 1 (8/11/98) Dose 2 (8/25/98) Dose 3 (9/14/98) Dose 4 (10/14/98) Dose 5 (2/1/99) M 75 Colon Ca 1x10 10 3x10 10 1x10 11 1x10 11 1x10 11 - - - - - A Flutter* A Flutter Death * (2/3/99) Possibly (temporal only) Possibly (temporal only) # GT Dose 1 (6/8/99) M 69 Rectal Ca 1x10 11 103.8 F SVT/Hypotension* Rigors Probably (to fever) (+) IL 2 Name Sex Age Diagnosis Dose Fever (24H) Adverse Events (Grade III-IV) Relatedness JD Dose 1 (9/23/98) Dose 2 (10/5/98) Dose 3 (10/19/98) Dose 4 (11/2/98) F 39 Breast CA 1x10 9 3x10 9 1x10 10 3x10 10 102 F 102 F EM Dose 1 (11/4/98) Dose 2 (11/16/98) Dose 3 (11/30/98) F 47 Breast CA 1x10 9 3x10 9 1x10 10 101.2 F Bilirubin Death* Possibly (temporal only ) # Possibly (temporal only) # * Event generating SAE report # Current progression
Patient GT o 69M, rectal ca, baseline RBBB Febrile 103.8F (39.9C) within 6 hrs SVT, responsive to IV beta blockers blood pressure, 20 mm drop asymptomatic o blood cultures neg, cell cultures neg o expect it: >>immune response indicators recommend as pharmacodynamic measure o avoid predisposing cardiac conditions
Temperature Heart Rate GT Vital Signs Temperature Heart Rate 105 104 103 102 101 100 99 98 97 400 350 300 250 200 150 100 50 96 0 12:18 13:00 13:40 14:34 14:50 15:20 17:34 22:34 0:10 5:00 11:00 18:00 21:00 22:00 15:00 22:00 24:00:00 2:00 Time (Hour)
CEA (NG/ML) 1600 1400 1200 T Cells CEA 1000 800 Increasing pain 600 400 Pain resolved 200 0-28 -21-14 -7 0 7 14 21 28 Day of Treatment
GVHD in bowel Apoptotic bodies at base of crypts
GT biopsy: no apoptotic bodies Conclude: cytokine syndrome
CEA targeting by TCR Parkhurst et al, ASGCT 2010 o Cloned murine TCR for HLA-A2 presented CEA peptide o TCR gene transfer (not CAR ~ 1 st gen), 3 subjects Lymphodepletion 2-4 x 10^8 cells 1-20% engraftment o Toxicity against bowel, all had grade 2/3 diarrhea T cell infiltration epithelial destruction o Some tumor partial response noted o However, concluded not safe to target CEA What is the difference???
MHC class I on enterocytes Topology MHC I: Primarily basal expression
CEA on enterocytes Antigen sequestration: CEA confined to microvilli
Lesson on Ag selection o What are normal tissues expressing? o What is topology of expression? Epithelial cancers Antigens Expression CEA Lumenal Class I Basal Her2 Basal CA IX Basal
Liver, etc Infuse Causes of Activity Loss? Tissues T T * Blood T #1 #2 T * Cancer
New Directions: Hypotheses o Tumor eradication would follow if EITHER #1. T cells persisted systemically [QUANTITY] ( bypass co-stimulation ) OR #2. T cells expanded intratumorally [QUALITY] ( provide co-stimulation )
Hypothesis 2: 2 nd Generation Provide co-stimulation Incorporate Signal 2 into designer T cells
Immunology 101
T Cell Activation Antigen Presenting Cell MHC B7 TCR CD28 1 2 T Cell o o o Gene expression - Cytokines (IL-2, 4, IFN-, etc) - Surface molecules (CD25, CD40L, etc) Cytotoxicity Proliferation
Designer T Cells First Generation o IgTCR chimeric immunoglobulin T cell receptor Tumor Cell Antigen Ig TCR 1 Modified T Cell CD28 MIPCEA Advantage: IgTCR provides Signal 1: adequate T cell cytotoxicity. Disadvantage: Lacking Signal 2, undergoes Activation-Induced Cell Death (AICD) after killing target cells. [HYPOTHESIS]
Signals o Signal 1: Activated: T cells kill tumor >> and die by AICD Resting: anergy o Signal 1+2: Activated: T cells kill tumor >> and proliferate Resting: activation, see above
1 st and 2 nd Gen Constructs IgTCR Ig28TCR ( Tandem ) VL VH VH VL VL VH VH VL 28 28 Signal 1 Signal 1+2
2nd Gen T Cell Tumor-Induced Proliferation Expansion on MIP101 Expansion on MIPCEA 1.0E+07 CEA(-) 1.0E+07 CEA(+) 8.0E+06 8.0E+06 6.0E+06 4.0E+06 UnTd IgTCR Tandem 6.0E+06 4.0E+06 UnTd IgTCR Tandem 2.0E+06 2.0E+06 0.0E+00 Day 0 Day 3 Day 7 0.0E+00 Day 0 Day 3 Day 7 Day Day Emtage et al. Clin Cancer Res 2008
Adjuvant model: Superior in vivo Tumor Suppression by 2nd Gen T Cells Tumor size (mm ) 2 Tumor size (mm ) 300 MIP101 300 MIPCEA 250 CEA(-) 250 CEA(+) 200 150 Untransduced IgTCR Tandem 200 2Untransduced IgTCR 150 Tandem 100 100 50 50 0 1 3 5 7 9 11 13 0 1 3 5 7 9 11 13 Day post tumor cell injection Day post tumor cell injection
Phase Ia/Ib Trial of 2 nd Generation Anti-CEA Designer T Cells in Adenocarcinoma FDA BB IND 10791
Hypotheses o 2nd gen Signal 1+2 designer T cells will escape AICD and proliferate intratumorally o Expanding designer T cells in tumor will maintain anti-tumor activity until tumor elimination
-IL2 Pt # Cohort 1x10 9 1x10 10 1x10 11 #1 #2 #3 #4 #5 #6 I II X X X T Cell Dose, Number of Cells X X X Phase Ia Safety Study Design #7 #8 #9 III X X X Any CEA+ cancer Funded by FDA Orphan Products Division
Summary 2 nd Generation CEA o 5 patients treated 1 st cohort completed 2 nd cohort in process o Safety, no SAEs (no new risks with 2 nd gen) o Responses one minor (brain and lung) one SD 12+ months o Continue in escalation o Need to assess value of IL2 supplementation
Hypothesis #1 T Cell Engraftment Auto-Transplant: Engraft designer T cells via lympho-expansive capacities of the body after lympho-depletion treatments (Bypass co-stimulation)
NMA Melanoma TILs Tumor Harvest Melanoma CD8+ TIL X Non-myeloablative (NMA) Conditioning Hematologic Recovery 6/13 major responses Tumor Response Dudley et al Science 2002;298:850
Prostate Cancer T Cell Harvest CIR+ Ex vivo gene therapy Anti-PSMA designer T cells Non-myeloablative (NMA) Conditioning +IL2 low dose (outpatient) Hematologic Recovery CIR+ CD3+ Tumor Response
Prostate Specific Membrane Antigen (PSMA) o Surface membrane glycoprotein 100,000 Daltons o Unrelated to PSA o Normal prostate epithelium and vasculature (reportedly on type II astrocytes?) o Elevated expression in metastatic lesions and hormone refractory disease o High clinical relevance: 28,000 deaths per year from PSMA+ prostate tumors o Antibody (3D8) from G. Murphy and A. Boynton
PSMA on prostate cancer
Normal tissue PSMA Tasch et al. Crit Rev Immunol 2001 Prostatic epithelium and vasculature Dispensable white gray And brain??!! Kinoshita et al. W J Surg 2006 Sacha et al. Neuroscience 2007
Thought: If DTC toxic against brain, then best to have a format that allows T cell suppression and escape T cell quantity: Can be controlled with infusion Engraftment with vast numbers hard to control But: No 1 st gen infused will be therapeutically successful T cell quality (potency): After remove steroid suppression: 1 st gen DTC won t reactivate 2 nd gen will Conclude: 1 st gen safe with engraftment (2 nd gen safe with infusion)
Phase I Study of Autologous Transplantation of Anti-PSMA Designer T Cells after NMA Conditioning in Prostate Cancer BB IND 12084
Treatment Schema Designer T cells -------- modify T cells ------ microbiologic testing --------- NMA T cell infusion T cell collection ------ G-CSF ----- PSC collection ------- chemotherapy ----------- start IL2 ------ end IL2-21* -20* -16* -8-2 0 +28 Study Day Biopsy* CTX 60 mg/kg d-7, d-6 Fludarabine 25 mg/m2 d-5 to d-1
Phase I Study Enrollment Plan o T Cell Dose, Number of Cells o Pt # Cohort 10.9 10.10 10.11. o #1 X o #2 I X o #3 X o #4 X o #5 X o #6 X o #7 X o #8 II X o #9 X o #10 X o #11 X o #12 X o (Bx) o #13 X o #14 III X o #15 X o #16 X o #17 X o #18 X omonitoring Safety Designer T cell persistence/expansion in blood In tumor Tumor response Funded in part by US Army
Counts per ul Counts per ul Peripheral Blood Recovery o Chemo d-8 to d-2 o T cells d0 o IL2 start d0-d28 o ANC=0 x5-8 d Recovery ANC>500 6000 5000 4000 3000 2000 1000 0 Pt #1 ANC and ALC over Time T cells -8 0 8 16 24 days ANC ALC 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 T cells -8 0 8 1 days AC ALC
IL2 (pg/ml) IL2 via Continuous Infusion IL2 of JN 2000 1800 1600 1400 1200 1000 20 IU/ml 800 600 400 200 0 prescreen preinfusion post- 0 hr post 1 hr post 3 hr post 8 hr post 24 hr post 48 hr post-day 5 post-day 7 post-day 14 post-day 21 post-day 28 75,000 IU/kg/d ~ 3 MIU/m2/d
Engraftment 1.1% 2.5% Blood sample Day +14 #1 CIR+ CD3 61% #2 CIR+ 7.3% Dose CD3+
PSA PSA PSA Response 3.50 T cells 3.00 2.50 2.00 1.50 Conditioning d-8 to d-2 T cells infused d0 Low dose IL2 d0 to d28+ 1.00 0.50 0.00 16 14 chemo -20-10 0 10 20 30 40 50 Days T cells 12 10 8 6 4 2 0 chemo -20-10 0 10 20 30 40 50 60 70 80 Days
Summary Prostate o Five patients treated Safely administered; toxicity due to conditioning Neutropenic fevers, hospitalization for antibiotics Malaise, fatigue (IL2) 50 and 70% reduction in PSA in two subjects o More doses to follow in escalation 10^10 10^11 o Limitations??? 1st generation: signal 1 only, After resting, do not reactivate 2 nd generation in preclinical o Conclude: PSMA safe for DTC targeting
Considerations for Antigen Targeting o The target Normal tissues that express Essential/Dispensible? Topology of expression (epithelial) Unknowability is central to safety testing design Safety with Ab targeting no assurance for T cells o The method of targeting Potency: Co-stimulation, can reactivate after suppression Quantity: Infuse vs engraft Infusion allows control of patient exposures (DLI)
T cells homing in on target THE END
Table 1. Features of designer T cell versions On Contact with Antigen. CIR Activated T cells Resting T cells 1st gen Signal 1 killing, AICD anergy 2nd gen Signal 1+2 superactivation, killing, activation, killing, cytokines, proliferation cytokines, proliferation
Table 2. Features of T cell administration methods Conditioning Risk? In blood In tumor Cost* Infuse None No Transient Transient $5-10K Engraft Chemo, XRT Toxic Stable Sustained $60-100K *Clinical, non-manufacturing costs of patient treatment and monitoring. (Manufacturing ~$15K for 10^11 dose; less for lower doses.)
[ QUALITY ] Table 3. Options matrix for designer T cell Strategies (2x2) [ QUANTITY ] Administration method Infuse Engraft Designer 1st gen 1 2 T cell version 2nd gen 3 4 0 is untested state Strategy 1 = 1 st gen infused Strategy 2 = 1 st gen engrafted Strategy 3 = 2 nd gen infused Strategy 4 = 2 nd gen engrafted
Table 4. Likely toxicity response of auto-reactive DTC to immune suppression on different Strategies On steroids Off steroids Comment 1 st gen only 1 st gen; 0 1 Suppress Suppressed don t reactivate 0 2 Suppress Suppressed don t reactivate 2 nd gen 2 nd gen reactivate; 0 3 Suppress Suppressed small numbers* 0 4 Suppress Autoimmune very large numbers *Known from BMT that dosage of allo-reactive T cells in donor lymphocyte infusions (DLI) critical to safety. Low dose controlled endogenous mechanisms (Treg, etc). High dose uncontrolled except with max suppress, lethal.
Safe paths of Strategy Escalation for exposures to designer T cells 1. Avoid maximum exposure for first-inhuman DTC tests for Ag (Strategy 4) 2. Once a Strategy safely tested for Ag, other Strategies safe for that Ag 3. But NOT all paths advisable (---) 0 is untested state Strategy 1 = 1 st gen infused Strategy 2 = 1 st gen engrafted Strategy 3 = 2 nd gen infused Strategy 4 = 2 nd gen engrafted
Optimal paths of Strategy Escalation for exposures to designer T cells All paths to 2 nd gen engrafted pass through 2 nd gen infused 1. Lower hazard 2. Lower cost 3. Better science: Test hypothesis of more signals to create effective immune response (without engraftment) NB: No 2 nd gen DTC has yet been tested by infusion to full doses with appropriate cytokine support; hypothesis of sufficiency OR deficiency still untested. 0 is untested state Strategy 1 = 1 st gen infused Strategy 2 = 1 st gen engrafted Strategy 3 = 2 nd gen infused Strategy 4 = 2 nd gen engrafted
Considerations for Antigen Targeting o The target Normal tissues that express Essential/Dispensible? Topology of expression (epithelial) Unknowability central to safety testing design Safety with Ab targeting no assurance for T cell o The method of targeting Co-stimulation, can reactivate after suppression Infuse vs engraft Infusion allows control of patient exposures (DLI)
T cells homing in on target THE END