CAR T-cell Therapy in Haematologic Malignancies

Transcription

1 CAR T-cell Therapy in Haematologic Malignancies Amit Khot Click to edit Master subtitle style Consultant Haematologist Peter MacCallum Cancer Centre, Melbourne

2 CAR Therapy Click to edit Master subtitle style

3 Chimeric Antigen Receptor T-Cells

4 History of CAR T-cell development Production of CAR T-cells Trials CLL, ALL, AML Future Development

5 Adoptive Immunotherapy using gene modified T-cells Ex vivo expanded tumour-infiltrating lymphocytes Gene modified T-cells Chimeric antigen receptors (CAR) Previously limited in vivo expansion of T-cells and disappointing clinical activity Melanoma (2/17), neuroblastoma (4/11), ovarian Ca (0/14)

6 Development of Clinical Scale T Cell Manufacturing Process 1987: Discovery that CD28 is gatekeeper for T cell proliferation 1993: CD3/CD28 beads first produced 1998: First CARs: CD4 zeta for HIV patients 2001: First cancer patients treated, non-gene modified 2010: First cancer CARs CART19 Bead addition Bead removal T cell infusion

7 Chimeric Antigen Receptors (CAR) 4-1BB (CD137)

8 CAR Therapy: Evolution of CARs with additional costimulatory domains this is hhh ext tt e x t th Sadelain, et al. Cancer Discov Classic CARs Sports CARs Muscle CARs Dotti et al. 2009

9 Adoptive Transfer of Gene-Modified T-cells expressing a chimeric T-cell receptor Chimeric receptor approach Tumour-reactive T-cells can be generated by retroviral transduction of peripheral blood-derived T-cells that can be expanded in vitro

10 Targeting Leukemia with Chimeric Antigen Receptor Modified T cells CARs combine an Ag recognition domain of antibody with intracellular signaling domain into single chimeric protein. Gene transfer (virus vector) to stably express CAR on T cells confers novel Ag specificity. Native TCR T cell CTL019 cell CD19 Dead tumor cell Tumor cell Anti-CD19 CAR construct

11 Advantages of Approach No requirement for MHC-restriction ScFv component can be modified to be directed toward a variety of TAA, thereby addressing antigenic heterogeneity within and between tumours Ability to simultaneously manipulate a range of T-cell functions concomitant with anti-tumour T-cell function (localization, expansion, survival) Limited potential for tumour escape by MHC/peptide loss.

12 CD19: An ideal tumor target CD19 expression is restricted to B cells and possibly follicular dendritic cells CD19 is not expressed on pluripotent bone marrow stem cells CD19 is expressed on the surface of most B cell malignancies Antibodies against CD19 inhibit growth of tumor cells preb-all B cell lymphomas and leukemias myelomas Stem Cell pro B pre B immature B mature B plasma cell CD19 CD22 CD20 Brentjiens / Sadelain

13 Dysfunctional T Cells in CLL Riches JC et al. Blood. 2013;121:

14 CLL Study Overview- ClinicalTrials.gov #NCT CD19+ heme malignancy eligibility tumor restaging CART19: αcd19-41bb-cd3ζ transgene CD8α linker CD19 V L GGGSx4 linker EF1α promoter CD19 V H CD8α hinge and transmembrane region TCR-ζ signaling domain 41-BB signaling domain Day 0 Day 0 1 Day 3 Day 5 Harvest Day (10±2) Leukocyte apheresis CD3/28+ selection of T cells with anti-cd3/anti-cd28 mab- coated magnetic beads Seed in gas permeable bags. Transduce with αcd19 4-1BBζ vector Vector wash out. Culture in gas permeable bags Culture in WAVE bioreactor Remove beads Harvest, Wash, concentrate Cryopreserve product in infusible cryomedia Production/cryopreservation of CART-19 cells FDA approved therapy Monitor for recurrence Relapse PBMC baseline assays Week -4 Apheresis #1 Week -1 Lymphocyte depleting chemotherapy a CART-19 infusion cells/kg Day 0, 1, 14 b (intrapatient dose escalation) PBMC endpoint analysis Week +4 (depending on last infusion) Monthly observation for 6 months Quarterly observation up to 2 years post infusion Follow-up for 15 years as mandated by FDA for gene transfer protocols Protocol Status: enrollment completed April 2013

15 Clinical trial overview z1 CAR Native TCR T cell CTL019 cell Native TCR CD19 2 Gene transfer Lymphodepleting chemotherapy days 4 Tumor cell 3

16 Cells (x10-3 /mm 3 ) UPN R/B R/B B Clinical Responses CARs WBC ALC UPN 01 Day Day Corticosteroids started UPN 03 Days from Infusion Pre-Therapy 3 Months Porter NEJM 2011

17 CAR Persistence and Expansion in PBMC in CLL: Delayed onset tumor lysis syndrome CART-19 persistence and trafficking blood (subject #3): Hospitalized for tumor lysis syndrome copies / g gdna Day (post infusions) Porter et al. NEJM, 2011

18 A copies/mg gdna Pharmacology and Pharmacokinetics of CTL CART 19: Blood Day (post infusion) UPN 01 UPN 02 UPN 03 CART19 cells proliferate 2 to 4 log10 in all patients in vivo CD8 Day CAR moiety expressed for at least 6 months 2. Sustained antibody delivery with a single infusion of engineered T cells 3. CARs expressed for at least 2 years

19 In Vivo CTL019 Expansion Correlates with Response copies/ug genomic DNA CRs Day from infusion CHP copies/ug genomic DNA PRs Day from infusion copies/ug genomic DNA NRs Day from infusion Patients who achieve CR have >1000 CART19 by day 20 CRs have been sustained in 10 of 11 patients

20 Sustained B Cell Aplasia 24 Months Post-Infusion UPN01 base 12 months 18 months UPN03 base 6 months 15 months Michael Kalos

21 Tocilizumab Anti-Cytokine Therapy for CRS 104 Temp (deg F) a 7, 6a 7. 12p 7. 6p 8. 12a 8, 6a 8. 12p 8. 6p 9. 12a 9, 6a 9. 12p 9. 6p a 10, 6a Tocilizumab, d p 10, 6p a 11, 6a p 11, 6p a 12, 6a p 12, 6p a 13 6a p 13, 6p a 14, 6a p 14, 6p Page 21 CLL Pt Dr. P. Wood NORD CTL019 Confidential David Porter, MD

22 Pilot Trial of CTL019 for Relapsed, Refractory CLL (ASH Abst ) Major responses 8/14 (57%) Page 22 CR 4/14 (29%) PR 4/14 (29%) No dose:response relationship Median CTL019 dose 1.45 ( ) v 1.3 x 10^8 ( ) in responders vs non-responders No dose:toxicity relationship

23 Outcome Of CD19 CAR T-cells In CLL Overall response rates 40% Patients with CR had no minimal residual disease (MRD) Responding patients had persistence of CAR T cells more than several months post-rx Patients with persistence of CAR T cells had Persistent absence of normal B cells Profound hypogammaglobuinemia IgM and IgA levels of 0 Required maintenance therapy with IVIg Page 23 Porter DL, et al. Blood 122:873 ASH Proceedings 2013, 2014, 2012

24 Emma Whitehead refractory relapse of B-ALL April 16, 2012 NEJM 368:1509

25 Rapid Induction of Remission in pre-b ALL: pt #1 Marrow T Cells day +6 day +23 Deep remission induced in 23 days No chemotherapy was given Status: CR (12+) MRD <0.01% cells Blasts (ALL) Stephan Grupp, NEJM 2013

26 Serum Cytokine (fold change from baseline) Serum Cytokine (fold change from baseline) Serum Cytokine (fold change from baseline) Serum Cytokine (fold change from baseline) Serum Cytokine (fold change from baseline) Cytokine Release Syndrome (CRS) CHOP-101 CHOP-100 CHOP IL-1b IL-6 IFN-g 100 TNF-a IL-2R 10 IL-2 IL-10 1 IL-1RA Steroids Daysafter Daysafter Infusion Infusion Etanercept Tocilizumab Grupp et al, NEJM Dr. P. Wood NORD CTL019 Confidential 26 ASH 2012 Pipeline Review CONFIDENTIAL December 7, 2012

27 copies/mg gdna mg gdna copies/mg gdna gdna Daysafter Infusion Efficient Trafficking of CTL019 T Cells to CNS in ALL Marrow Blood CHOP-100 CHOP-101 1% marking Morphology Blood (Day of 10) CARs In Vivo Blood Day Marrow Daysafter Infusion Daysafter Infusion CSF Blood (Day 10) CSF (Day 23) Daysafter 80 Infusion Daysafter Infusion Stephan Grupp, NEJM CSF Day 23

28 SSC CD34 B Cell Aplasia and Emergence of CD19 Escape in ALL Pt #2 CHOP 101 Baseline CHOP 101 Day < CHOP 101 Day CD CD45 Patient #2: Ablation of CD19 expressing cells and in vivo expansion of CD19 negative ALL post CTL019

29 Dec 9, 2012

30 Pediatric CTL019 ALL Study High Response Rate (n=13) Duration of Response (as of 18 May 2013) Months CHP100 CR CHP101 CHP103 CR, transient PR CHP104 CR CHP105 CR adult ALL #1 CHP108 CHP110 CHP111 CHP112 CHP113 NR CR CR CR CR CR, MRD 0.1%, transient ORR 11/12 (92%) CR 10/12 (84%) PR 1/12 (8%) NR 1/12 (8%) CHP114 CHP CR 30 ASH 2012 Pipeline Review CONFIDENTIAL December 7, 2012 Stephan Grupp, NEJM 2013

31 CART19 Associated Cytokine Release Syndrome 6/7responding patients developed a CRS High fevers, myalgias, nausea, hypotension, hypoxia, etc.. Very high levels of IL6 IFN-g, modest TNF-a Mild increases in IL2 5/11 patients with NR had CRS (p=0.15) Rapidly reversed with tocilizumab (3) when needed. Treatment for CRS day 2-11 Will early treatment for CRS abrogate response? CRS associated with HLH/MAS (Hemophagocytosis, hemolysis, DIC, ferritin >500,000, altered MS) 31 ASH 2012 Pipeline Review CONFIDENTIAL December 7, 2012

32 CART19 Associated Cytokine Release Syndrome 6/7responding patients developed a CRS High fevers, myalgias, nausea, hypotension, hypoxia, etc.. Very high levels of IL6 IFN-g, modest TNF-a Mild increases in IL2 5/11 patients with NR had CRS (p=0.15) Rapidly reversed with tocilizumab (3) when needed. Treatment for CRS day 2-11 Will early treatment for CRS abrogate response? CRS associated with HLH/MAS (Hemophagocytosis, hemolysis, DIC, ferritin >500,000, altered MS) 32 ASH 2012 Pipeline Review CONFIDENTIAL December 7, 2012

33 Published clinical trials employing CAR T cells targeting CD19+ B cell malignancies Effi cacy and safety of CAR based therapy Table I. Comparison of treatment strategies and outcome of ve centers that launched CD19 CAR T-cell adoptive therapy. Institution NCI [20,21] MSKCC [22] Upenn [6,23] COH [3] BCM [5] Registered no. NCT NCT , NCT NCT NCT NCT CD19 disease CLL and NHL CLL and B-ALL CLL NHL NHL Patient population Disease status Advanced, progressive CLL: chemotherapyresistant; ALL: Advanced, chemotherapyresistant Relapsed or refractory Relapsed or refractory relapsed Preconditioning Cyclophosphamide None vs. Variable * Fludarabine No and udarabine cyclophosphamide IL-2 infusion Yes No No Yes No Gene transfer approach Retrovirus Retrovirus Lentivirus Electroporation Retrovirus CAR signaling endodomain CD28 CD3 z CD28 CD3 z CD137 CD3 z CD3 z CD3 z vs. CD28 CD3 z CAR generation 2nd generation 2nd generation 2nd generation 1st generation 1st and 2nd generations Loss of normal B cells Yes Yes Yes No Yes Infused total cell number / kg / m / m 2 Infused CAR T-cell number / kg / m 2 Persistence of CAR T-cells 20 days 6months 0 day 8 weeks 6 months 1 day 1st: 4 weeks 3 months; 2nd: 6 weeks 6 months Outcome CR (1), PR (6), SD (1), NE (1) PD (4), SD (3), NE (1), B-cell aplasia (1) CR (2), PR (1) SD (1), PD (1) PD (4), SD (2) CAR, chimeric antigen receptor; NCI, National Cancer Institute; MSKCC, Memorial Sloan-Kettering Cancer Center; UPenn, University of Pennsylvania; COH, City of Hope; BCM, Baylor College of M edicine; IL-2, in terleukin-2; CLL, chronic lymphocytic leukemia; NHL, non-hodgkin lymphom a; ALL, acute lymphoblastic leukemia; PR, partial remission; CR, complete remission; SD, stable disease; PD, progressive disease; NE, not evaluated. * One with bendamustine, one with bendamustine and rituximab, one with cyclophosphamide and pentostatin. Numbers in parentheses are patient numbers. One patient at NCI treated with CAR-T cell infusion twice, one patient at MSKCC cancelled CAR T-cell infusion because of intervening complications; thus total number in outcome row does not add up to patient population in these two centers. outcome of CAR T-cell treatment and prognostic factors are listed in Table II. e results showed that patients bene ted from lymphodepletion before CAR T-cell infusion no association with the persistence of CAR T-cells ( 4 weeks, 7/ 9 vs.13/ 18, p 1.000) and peak percentage ( 0.1%, 4/ 18 vs. 4/ 9, p 0.375). Comparison of e cacies between the Xu, Leuk Lymph 2013:54:255

34 Identification of the LeY Antigen as a target antigen and Production of the vector encoding the Humanized Anti- LeY-CD28-zeta Chimeric Receptor Tumour Ag LewisY, a carbohydrate antigen over-expressed on >60% of epithelial, endometrial and gastric carcinomas (Hakomori 1984; Sakamoto, Furukawa 1986; Miyake M 1992) LeY expressed at low levels in normal tissue (gut mucosa, exocrine pancreas, ciliated epithelium, type II pneumocytes, activated neutrophils, hair follicles and CD34+ hematopoietic progenitor cells (Hellstrom 1990; Kitamura, Stockert et al. 1994; Zhang, Zhang et al. 1997) Fully humanized anti-ley antibody (hu3s193) characterized in vitro (Scott, 2000) and in a phase I study: 12 patients with epithelial cancers (Scott 2004). Extensive pre-clinical toxicology studies: pre-clinical and phase I study, there was no bone marrow toxicity, hemolysis, no human-anti-humanantibody (HAHA) or human-anti-mouse-antibodies (HAMA) response Tracking studies with 111Indium(In)-labelled Ab: Uptake at tumour sites but not in normal tissues (Scott 2004).

35 Identification of the LeY Antigen as a Potential Target The LeY antigen expressed on early myeloid progenitor cells and on corresponding CD34-positive leukaemia cell lines (Cao 2001) AML-patients : 15/33 were positive for LeY expression (46%) The MFI of the positive samples was 54.4 compared to an MFI of 2.2 for the lymphocytes. Flow cytometry showing moderate Le Y expression on AML cell line K562

36 Generation of Modified T-cells DNA coding for the humanized scfv derived from hu3s193 Chimeric receptor consisting of the extracellular humanized scfv, recognizing the LeY Ag, linked to an extracellular CD8 hinge region, a transmembrane and cytoplasmic CD28 signaling domain, and zeta signaling chain

37 Manufacturing facility CBCT (Centre for Blood Cell Therapies) Includes harvesting Specifically designed for compliant cell therapies Process development & tech transfer Conventional and negative clean pressure Complies with AS /350 (ISO ISO5/7)

38 Generation of modified T-cells Retroviral expression systems are used to stably express transgenes in primary T-cells The c-dna construct has been fully sequenced for verification The humanized anti- LeY scfv-cd28-ζ receptor has been cloned into the psamen retroviral vector and transfected into the packaging line PG13 In psamen, expression of the construct is driven by the long terminal repeat (LTR) promoter region of the Moloney murine leukemia virus (MMLV). The utility and safety of the psamen vector in clinical trials has been established (Parker, Do et al. 2000) This vector and packaging system has been used to isolate high titre producer cells for a number of different scfv receptors

39 Safety considerations Replication deficient retrovirus: removal of all structural genes and replacing them with the genes of interest: the gene for the chimeric T-cell receptor Generation of replication competent retrovirus by homologous recombination between the retroviral vector and the helper plasmid sequences in the packaging line which could eventually lead to development of a lymphoproliferative disorder Auto-immunity - Graft versus host disease (GVHD) - against any LeY positive cell population: GIT, pancreas, type II pneumocytes, neutrophils, CD34+ haematopoietic progenitors

40 Preclinical data Successful introduction of the chimeric receptor into primary human T-cells control anti-ley The psamen vector transduced human PBL at high frequencies. A representative FACS of transduced PBL shows transduction efficiency of 55% upon incubation with the anti- Le Y - scfv-cd28-ζ vector (anti- Le Y ) as opposed to empty vector (control). Staining was done with the anti-anti-le Y idiotype antibody followed by a PE-conjugated anti-mouse IgG antibody. Staining control consisted of anti-igg alone. The histogram lines are superimposed in the control sample. Both, CD4 and CD8 cells were transduced

41 Preclinical data Transduced anti-ley T-cells proliferate in response to chimeric receptor ligation Le Y -specific transduced human lymphocytes are able to proliferate. A proliferation assay showed T-cell proliferation upon chimeric receptor stimulation with immobilised anti-idiotype antibody, but not with an irrelevant (anti-tag) antibody. The Y-axis shows counts per minute (cpm) of tritiated thymidine.

42 Preclinical data Transduced anti-ley T-cells secrete IFN-γ in response to tumour cells control anti-ley 7000 IFN-g (pg/ml) MCF-7 RPMI 8226 LP-1 U266 OPM-2 NCI- H929 media Co-culture with the moderately LeY expressing MM cell line RPMI resulted in strong IFN-γ secretion

43 Preclinical data LeY-expressing tumour cells are lysed by anti-ley transduced T-cells % specific lysis % specific lysis (c) 40:1 20:1 10:1 5:1 2.5:1 (d) RPM I-8226 LP-1 NCI H-929 OPM -2 U266 40:1 20:1 10:1 5:1 2.5:1 Effector:Target ratio Le Y -specific transduced human T lymphocytes are cytotoxic upon stimulation with cells expressing the Le Y antigen. T-cell transductants were cytotoxic to the Le Y -expressing MCF- 7, Panc89, A431, and HT29 epithelial tumour cell lines and the MM cell line RPMI-8226, but not to cell lines not expressing Le Y (Colo205 and the other MM cell lines) (a + c). The control (empty vector transduced T-cells) are shown for comparison (b + d). This is a representative experiment out of three. The assays were run as triplicates.

44 Preclinical data Systemically delivered anti-ley T- cells inhibit tumour growth in mice Adoptively transferred anti-le Y T- cells can eradicate Le Y+ tumour in vivo. OVCAR-3 tumour cells were injected subcutaneously into NOD-scid mice followed by treatment beginning on either day 0 (a) or day 7 (b) after tumour inoculation. Tumours were rejected over a period of days in response to four intravenous injections of anti-le Y T-cells (circles). Tumours grew progressively in nontreated mice (triangles) and in mice receiving control T-cells transduced with empty vector (squares). Tumour inhibition experiments were performed three times with similar results. Anti-Le Y T-cells inhibited tumour growth to a significantly greater extent when compared to control T-cells (P2=0.002, Mann- Whitney test).

45 Preclinical data Anti LeY T-cells were not pathogenic in mouse model % survival Group 1 Group Days since irradiation and injection of T cells Adoptively transferred anti-le Y T-cells do not affect survival of sub lethally irradiated mice 45 BALB/c mice with the common leukocyte antigen CD45.2 were irradiated with 2.5 Gy. Control mice in Group 1 (n=15) were monitored only. Mice in Group 2 (n=30) received an intravenous transplantation with 1x10 7 T- cells transduced with an anti LeY chimeric receptor gene. The follow-up of >2 years showed identical survival of mice in both groups.

46 Investigational Product The investigational product used in the study was the vector: Anti-LeY- scfv-cd28-ζ vector, a non-pathogenic, replication-incompetent retroviral vector specifically designed for this study under GMP-conditions.

47 Study design A Phase 1, open-label, single centre study Sample Size: 6 patients Primary Endpoints Adverse events according to NCI CTCAE v3 Acute GVHD Chronic GVHD

48 Secondary Endpoints Immunologic activities Percentage of infused labelled cells localizing in bone marrow ( SPECT scan) Presence of anti-ley positive T-cells in both PB and BM Percentage of anti LeY positive T-cells in BM or PB Serum IFN-γ and IL-2 levels Presence or absence of autoimmune disease

49 Secondary Endpoints Anti-tumour activities Overall response Time to progression Time to treatment failure Duration of response Overall survival

50 Secondary Endpoints Pattern of tracking of labelled re-infused T-cells Location of labelled T-cells (from SPECT scan) Persistence of anti-ley T-cells Persistence of anti-ley T-cells as detection of 1% of mononuclear cells in PB, BM or extramedullary disease to be anti-ley-positive on day 7 or later after transfusion of the T-cell product by FC or PCR LewisY expression on disease LeY expression assessed with FC in PB and BM samples

51 Exploratory End-points Concentration of the cytokines IFN-γ, IL-2, IL-4, IL-6, IL-10, TNF in PB and BM The total number of transduced T-cells in culture on day 0 Duration of detectability of the transduced T-cells in PB and BM Proportion of central memory phenotype T-cells in the T-cell culture on day 2 Indication of any immunologic activities Overall clinical response status

52 Subjects Patients with acute myeloid leukaemia or highrisk MDS who had persistent disease following chemotherapy with a fludarabine containing regimen (FCR). To receive T-cell product within days following the establishment of persistent disease status.

53 Subjects Newly diagnosed AML/ high-risk MDS with poor prognosis Age > 65 years Age years with any of the following single cytogenetic abnormalities: -7, -5, trisomy 8, abnormal 3q, t(6;9), t(9;22) or t(9;11), normal karyotype with FLT3-ITD Age years with a complex aberrant karyotype defined as >4 cytogenetic abnormalities Any age with relapsed or refractory disease

54 AML/MDS 1 st Eligibility Screening 8 weeks Steady-state apheresis PBMC 24 months FCR-chemotherapy 3-6 weeks Reassessment of Disease To be eligible, patient must have detectable disease 9 days 2 nd Eligibility Screening 7 days Start T-cell culture (day -12) 10 days Fludarabine-Conditioning: 2 days (day -2, day -1) Next day 3 rd Eligibility Screening & Infusion of T-cell product (day 0) Safety & Response

55 Results 6 patients enrolled. 5/6 planned infusions safely completed No major safety issues after TSMC review for 3 patients

56 Summary of Cryopreserved Apheresis Products Total Volume (ml) Total cells MNC Cells per bag LeY x x 10 9 LeY x x 10 8 /3.9 x 10 8 LeY x x 10 9 /4.1 x 10 9 LeY x x 10 8 / 1.1 x 10 9 LeY x x 10 9

57 Markers expressed on anti Lewis Y gene modified T-cells at Day -2 Marker LeY.001 (%) LeY.002 (%) LeY.004 (%) LeY.005 (%) LeY.007 (%) CD CD CD4 + CD Anti-LewisY TCR

58 LewisY Unlabelled Finished Product Participant Viability (%) Required Cell Dose Total Transduced Cells (Identity) LeY x x 10 8 LeY x x 10 8 LeY x x 10 8 LeY x x10 8 LeY x x10 8 Acceptance Criteria 70% 5.0 x 10 8 For Information Only Meet Criteria Yes Yes NA

59 LewisY Indium-111 Labelled Finished Product Participant Cell Number Dose Activity at Release Time (MBq) Sterility (BaCT) LeY x No growth LeY x No growth LeY x No growth LeY x No growth LeY x No growth Acceptance Criteria 5.0 x No growth at 14 days Meet Criteria Yes Yes Yes

60 Testing, Acceptance Criteria, and Results Parameter Acceptance Criteria Result Meet Acceptance Criteria Sterility No growth No growth Yes T-cell Receptor Gamma Gene rearrangement assay Negative Negative No new monoclonal population was identified Yes Volume 100 ml 100 ml Yes Storage temperature 0 C Yes Gram Stain Negative Negative Yes Endotoxin <2.5 EU/mL <2.5 EU/mL Yes Mycoplasma Negative Negative Yes

61 Results No grade 3/4 toxicity related to infusion All patients with progressive disease D21 to 19 months 2 patients: morphologic remission 5 months and 19 months post infusion, with evidence of stable cytogenetic MRD in bone marrow

62 Blood Counts LeY 04

63 Patient LeY04 Day 6 Day 22 H&E CD33 CD3

64 Figure 2A Day 6 Day 22 CD4 CD8

65 LeY transgene copies/1000 cells Patient LeY-04: LeY transgene positive cells in the PB to day 28 and in a skin infiltrate. Genomic DNA prepared from patient GMP grade product (product), PB and BM mononuclear cells and PCR performed for the LeY transgene (arrowed). bp bp C

66 LeY 01 LeY02

67

68 111 In-labelled T cells traffic to bone marrow

69 Conclusions Adequate mononuclear cell collection is possible in this high risk patient group prior to definitive therapy Target T-cell numbers achieved in all patients The products manufactured under GMP all met quality release criteria 5 patients were treated with no major adverse effects

70 Conclusions After adoptive transfer, LeY-T cells migrate to the BM and persist Adoptively transferred LeY-T cells express the LeY CAR Molecular Therapy 2013

71 Other CAR Targets in Development Sadelain Cancer Discovery, 2013; 3:388

72 Questions for Ongoing Studies Why are CAR T cells not rejected? What is the mechanism of long term persistence? What is relationship of tumor burden to CAR T cell proliferation? What is the long term safety of CAR T cells? Can CAR T cells work in solid tumours

73 Questions Scale manufacturing Off the shelf products and standardisation Multiple antigen targets: sequentially/ concurrently Smart CARs that can discriminate target on normal tissue from same target on cancer Viral vector CRS

74 Off Target Toxicity Cytokine Release Syndrome No acute infusion toxicities Patients often experience delayed, grade IV cytokine release syndrome (CRS) High fevers Nausea Myalgias Capillary leak syndrome Hypoxemia Hypotension Some patients with grade IV CRS causing life-threatening hemodynamic/respiratory instability can respond to tocilizumab (anti-il- 6R) Porter DL, et al. Blood 122:873 ASH Proceedings 2013, 2012

75 Not All CAR-T Cells Are Alike Naïve (T N ) Memory Stem-Cell (T MSC ) Central Memory (T CM ) Effector Memory (T EM ) Effector (T E ) CD45RA+ CD62L+ CD28+ CD95- CD45RA+ CD62L+ CD28+ CD95+ CD45RO+ CD62L+ CD28+ CD95+ CD45RO+ CD62L- CD28+/- CD95+ CD45RO+ CD62L- CD28+/- CD95+ Persistence of CMV-specific T CM w/o Lymphodepleting Rx Berger, C et al J Clin Invest 118:294, 2008

76 Thank You