Cell Therapy Liaison Meeting. Carl June, M.D.

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1 Cell Therapy Liaison Meeting Personalized Medicine Carl June, M.D. Professor of Pathology and Lab Medicine Abramson Cancer Center University of Pennsylvania October 17, 2008 AABB Headquarters Bethesda, MD

2 Overview: Adoptive T Cell Therapy Enable vaccination in immunosuppressed patients Reconstitute fludarabine induced immunodeficiency Therapy of progressive multifocal leukoencephalopathy (PML)

3 Hematologic Malignancies: Treatment Related Immunodeficiency Cytotoxic therapies cause severe and persistent immuodeficiencies and lymphopenia Immune reconstitution after hematopoietic stem cell transplant (HSCT) characterized by: Rapid (1-2 months) recovery of B cells Impaired T lymphocyte recovery (CD4 > CD8) Sustained antibody levels post-hsct require recovery of competent T cells Review of qualitative and quantitative aspects of immune recovery after auto PBSCT: Gauillame et al, Blood 92, 1471, 1998 Older patients have poor response to chemotherpy: more toxicity with FCR. Immune reconstitution important?

4 Treatment Related Immunodeficiency: Consequences Opportunistic infections Varicella, etc. Vaccine responses poor or absent ASBMT/CDC guideleines (Dykewicz, MMWR, 2000) Even when immunization is delayed until 12 months, antibody responses to capsular polysaccharide vaccines are poor and are unlikely to prevent disease -Only 19% of all enrolled patients developed protective antibody concentrations after the 24-month immunization (Guinan et al, 1994) Implications for tumor vaccines Prolonged lymphopenia may have implications for increased risk of relapse The worst: Progressive Multifocal Leukoencephalopathy

5 Prognostic Implications of Early Lymphocyte Recovery Post PBSCT for NHL Overall Survival NHL after PBSCT (104 patients) ALC recovery by day 15 Months Porrata et al. Blood 2001; 98: 579

6 General Approaches for Adoptive T Cell Therapy J Clin Invest :

7 Cell Culture Approaches for Adoptive T Cell Therapy

8 Clinical Scale T Cell Culture Process Total manufacturing costs: $15,650 Levine et al. Science. 1996; 272:1939 Levine et al. J Hematotherapy 1998: 7:437 Wave Bioreactors

9 Cell Manufacturing Flow Chart -Fresh Final Product Lymphocyte Apheresis $1,425 Day 0 Cryopreservation Thaw + CytoMate Wash $204 CD3/28 Positive Selection (SOP 0226) $1,161 Day 3 Day 5 Stimulation with CD3/CD28 Beads and Seed Culture in Baxter Lifecell Flasks D3 Pre-H: Maintain Culture in Baxter Lifecell Flasks D5 Load and Culture in Wave Bioreactor $922 $4,986 Remove Beads MaxSep Day 11 or Harvest Day Harvest Fenwal QC Testing and QA release Fresh Infusible Product $625 $3,400 Other costs: Labor, Equipment maintenance contracts, environmental monitoring, facility fees ~$4,600

10 Multiple Myeloma Plasma cell neoplasm characterized by serum monoclonal Ab, osteolytic lesions, pathological fractures, anemia, hypercalcemia 15% of hematologic malignancies Autologous transplants are highly effective for tumor reduction (first line therapy), but cures are infrequent. GVM/GVT: Allogeneic transplants can induce cures, but treatment-related risks are high.

11 Myeloma Phase I/II Adoptive Transfer Trials T Cell In Vitro Activation and Expansion to Infuse Cells Randomize Pneumococcal Vaccine (PCV) T Cell Collection Mobilization Stem Cell Collection High-dose Melphalan Stem Cell Transplant T Cell Infusion (Day +12 or +2) PCV vaccinations (Days +14, +42, +90) Study Day Rapoport et al. Nat. Med. 2005; 11: 1230 Immune Assessment Studies 100

12 CD3/28 Stimulated T Cell Infusion Leads to Rapid Lymphocyte Recovery Lymphocyte Count (per mm3) 5,000 4,000 3,000 2,000 1,000 T cell infusion CD3/28 T cells n = patients Controls (n=23-27) First successful randomized adoptive transfer trial Day of Transplant Gp 1+3 N=17 P = Gp 2+4 N=16

13 CD3/28 Stimulated T Cell Infusion Boosts Protective Antibody Levels Prevnar vaccines Pneumococcal IgG (mcg/ml) Gp 1,2 only T Cells Gp 1,3 T Cells Gp 1,3 T Cells Gp 2,4 T Cells Gp 2,4 Group 1 Group 2 Group 3 Group Days Group 1 vs other groups days 42 to 180: P<0.01

14 Adoptive transfer of vaccine primed T cells augments immunity in lymphodepleted hosts o First successful randomized multicenter adoptive immunotherapy trial: feasibility! o Accelerated recovery of CD4 and CD8 counts to normal levels by day 42 (P=0.004) o Protective antibody levels established by day 30 o Adoptive transfer of vaccine primed T cells appears to facilitate establishment of CD4 T central memory cells o Ongoing trial demonstrates striking schedule dependent effects of T cell transfer: day 2 vs day 12 post busulfan. Rapoport et al. Nat. Med. 2005; 11: 1230

15 UPCC 05402: Fludarabine-Cyclophosphamide With Immune System Support Using Costimulated Autologous T-cells in Patients With Previously Treated Relapsed/Refractory Follicular Lymphoma Rationale for phase I trial: (1) Prolonged lymphopenia and risk of opportunistic infections after fludarabine (2) Could Treg depletion tip the balance to increased effector function (i.e. less anergy) CD10 DR bcl-2 CD19 sig t(14;18) CD20 CD22 CD80 bcl-2 PI: Steven Schuster, M.D.

16 Effect of Fludarabine on T Cells Keating MJ, et al. Long-term follow-up of patients with chronic lymphocytic leukemia (CLL) receiving fludarabine regimens as initial therapy. Blood 92: , 1998

17 Follicular Lymphoma Study Design Relapsed / refractory FL (grade 1 or 2) Staging / PBL studies / apheresis Cryopreserve CF x 4-6 cycles Thaw / Monodeplete / CD25 Deplete Response Assessment / PBL Studies CR / CRu / PR / SD Co-stimulate and Expand In Vitro T-Cell Infusion (Day 0) Harvest PBL Studies / DTH Testing (Day 60) Response Assessment (Day 120) Anti-CD25 coated Epoxy beads Follow - up

18 Protocol Treatment CHEMOTHERAPY cyclophosphamide (250 mg/m2) days 1 3 fludarabine (25 mg/m2) days 1 3 T - CELL INFUSION DOSE LEVELS 1 - <5 x 10 9 CD3 + cells > 5-10 x 10 9 CD3 + cells

19 Subject Characteristics n = 13 Median age (years) 49 (range: 32 68) Sex (male : female) 5 : 8 Median number of prior therapies 2 (range: 1 3) Rituximab refractory 12 (92%) Stage II, II X, III, IV 1 (7.5%), 1 (7.5%), 7 (54%), 4 (31%) Bone marrow involved 1 (7.5%) Elevated LDH 2 (15%)

20 Enrollment and Results 13 subjects enrolled between May 2003 and March subjects removed after 2 CF cycles due hematologic toxicity; 1 subject removed after 4 CF cycles because of disease progression. 10 subjects received 4 CF cycles: 10 CD25-depleted, CD3/CD28-costimulated T-cell products generated: fold CD3 + cell expansion: median 26 (range 6 79) 10 T-cell products infused after 4 CF cycles: x 10 9 CD3+ cells x 10 9 CD3+ cells

21 Increase in T-Cell T Counts by Dose Level CD8 cells CD4 cells <5x10e9 >5x10e9 Dose Level median Day 30 T cell counts <5x10e9 >5x10e9 Dose Level p = (Wilcoxon signed-rank test)

22 Total CD4 and CD8 Counts over Time 700 n = T cell infusion cells / ul median CD4 median CD Pre-Rx month Long term correction of lymphopenia

23 Progressive Multifocal Leukoencephalopathy (PML) First described in 1958 in 2 CLL Pts, 1 HD Pt multiple CNS demyelinating lesions, rapid, fatal outcome Caused by ubiquitous polyoma virus, JC 35 cases reported post rituximab for lymphoma at Lugano. 32 died within 1 year. Occurs in up to 5% of AIDS Pts Before HAART, 1yr survival in HIV+ was 10% Natalizumab- humanized mab vs α4 integrin

24 T cells Transfer for Progressive Multifocal Leukocephalopathy (PML) 61 yo M w/cll since 1996 Previous therapy fludarabine, rituxan, cytoxan Relapsed /2007- Brain lesions may be Progressive Multifocal Leukoencephalopathy. Progressed on MRI in November Blood received 12/17/07 for pre-clinical evaluation of compassionate use T cell infusion Compassionate use IND

25 Case Report: Therapy of iatrogenic PML in CLL 10/23/07 11/3/07 Courtesy Igor BID

26 Gated FS/SS, then Viaprobe neg, CD19 neg, CD8 pos PBMC 12/17/ D0-PBMC D0-PBMC D0-PBMC % <0.02% <0.02% CEF JCV p36 JCV p100 Final Product: Expanded T-cells G1-PRE-H G1-PRE-H G1-PRE-H % 0.06% 0.05% CEF JCV p36 JCV p100 JCV p36, p100 courtesy Igor BID

27 PBMC 12/17/ D0-PBMC D0-PBMC D0-PBMC % <0.02% <0.02% PBMC , 7 days post-infusion PBMC PBMC PBMC % 0.09% 0.26% PBMC , 13 days post-infusion PBMC PBMC PBMC % 0.26% 0.20% PBMC , 27* days post-infusion PBMC PBMC PBMC % 0.08% 0.04% CEF JCV p36 JCV p100

28 Pt Current Status and next steps CD4/CD8 ratio increased from 0.5 to 1 after infusion Candida DTH, originally negative, now marginally positive (3mm) MRI: stable to improved 10 months post infusion. CLL stable disease Ongoing: longitudinal eval of JCV Large T Ag response, Treg levels

29 Points for Discussion Adoptive transfer of autologous costimulation leads to long term immune reconstitution in 1) myeloma; 2) follicular lymphoma/cll and 3) PML Clinical trial costs can not be supported with grants PML is orphan disease, nearly uniformly fatal in non- HIV setting Lack of biotechnology industry for T cell based therapies to facilitate technology transfer and enable wide availability. Personalized medicine requires randomized efficacy data. How to get there?

30 Collaborators and Acknowledgements U. Penn Bruce Levine Ed Stadtmauer Steve Schuster David Porter Richard Carroll Nicole Aqui Robert Vonderheide Elena Perez Carmine Carpenito Michael Milone Jim Riley U. Maryland Aaron Rapoport Alan Cross Dean Mann Robert Edelman Support NIH NCI Leukemia & Lymphoma Society