1. CliniMACS® CD34 Reagent System
FDA Advisory Panel
Miltenyi Biotec
CliniMACS® CD34 Reagent System
September 23, 2011
With Sincere Thanks and Appreciation To:

2. Director, Regulatory Affairs Miltenyi Biotec Incorporated
The CliniMACS® CD34 Reagent System FDA Advisory Panel September 23, 2011
Nancy Johansen
Director, Regulatory Affairs
Miltenyi Biotec Incorporated

3. Agenda and Presenters
Company Overview, Product Introduction and Registration of the CliniMACS® CD34 Reagent System as a Humanitarian Use Device Overview of the Clinical Indication and Unmet Medical Need and Summary of the BMT CTN 0303 Clinical Trial Summary of the CliniMACS® CD34 Reagent System Performance Summary of the BMT CTN 0303 versus BMT CTN 0101 Data Analysis Protocol Findings Summary of Safety and Probable Benefit of the CliniMACS® CD34 Reagent System
Nancy Johansen
Director, Regulatory Affairs
Miltenyi Biotec Incorporated
Steven Devine, M.D.
Ohio State University
Arthur G. James Cancer Center
Carolyn Keever-Taylor, PhD
Medical College of Wisconsin
Marcelo Pasquini, M.D., M.S.
Center for International Blood and
Marrow Transplant Research (CIBMTR) Medical College of Wisconsin
Kai Pinkernell, M.D.
Head of Clinical Development
Miltenyi Biotec GmbH
Maybe summary instead of Review in both slides?

4. Miltenyi Biotec Corporate Overview
Founded in 1989 in Bergisch Gladbach, Germany
Roughly 1100 employees worldwide
Subsidiaries in 10 countries; N.A. Headquarters in Auburn, CA
Miltenyi Biotec sells products and services worldwide.
Employees:
D: >800
EU: 80
US: 100
Asia-Pacific: 60

5. CliniMACS® CD34 Reagent System
The overall function of the CliniMACS® CD34 Reagent System is to select CD34+ cells from heterogeneous hematologic cell populations
Thereby passively depleting T-cells

6. CliniMACS® CD34 Reagent System
CliniMACS® Tubing Sets
(Standard and Large Scale)
Standard:
0.6 x 109 CD34+ Cells
from 60 x 109 Cells
Large Scale: x 109 CD34+ Cells
from x 109 Cells
CliniMACS®plus Instrument
CliniMACS® PBS/EDTA Buffer

7. The Principle of the CliniMACS® CD34 Reagent System
Elution of the labeled cell fraction
Magnetic labeling
Magnetic Separation (elution of the non-labeled cell fraction)

8. Location of CliniMACS®plus Instruments in the USA
162 instruments within 97 institutions in the U.S.

9. CliniMACS® CD34 Reagent System Protocols (as of July 2011)
84 IDE protocols utilize the CliniMACS® CD34 Reagent System
Strict distribution procedures are in place to ensure that investigational products are provided only for FDA/IRB approved protocols

10. Installation, Training and Customer Support
CliniMACS® plus Instruments are installed by qualified Miltenyi personnel
Installation and Operational Qualification (IQ/OQ) is
performed at time of installation
All subsequent servicing and Preventative Maintenance are performed by qualified Miltenyi personnel
Customer Training
Performed by qualified Miltenyi personnel
Training validated by written test
Emergency Hotline Support
Monday through Friday (9 a.m. – 9 p.m. EST)
If pre-arranged, outside of normal business hours

11. CliniMACS® CD34 Reagent System Registration Pathway
BMT CTN Study Conducted
2011 HDE Submitted
CliniMACS® CD34 Reagent System Registration Pathway
1998 US MF Submitted
2010 DAP Finalized
2004 Pre-IDE Meeting
1997
CE marked
2005-HUD Designation
Dec 2009
FDA pre-HDE meeting

12. Humanitarian Use Device
The device is designed to treat or diagnose a disease or condition that affects fewer than 4,000 individuals per year in the U.S.
The device is not available otherwise, and there is no comparable device available to treat or diagnose the disease or condition; and
The device will not expose patients to unreasonable or significant risk, and the benefits to health from the use outweigh the risks
Clinical data must support “safety” and “probable benefit” argument
Exempt from “effectiveness requirement” consistent with the HDE requirements

13. Clinical Trial Supporting the HUD Registration of the CliniMACS® CD34 Reagent System*
Miltenyi supported a Phase II multi-center clinical trial sponsored by the BMT CTN (BB-IDE 11965) which enrolled 47 AML patients from October 2005-December 2008
Evaluated the use of the CliniMACS® CD34 Reagent System for selecting CD34+ cells from HLA-matched related donors for allogeneic stem cell transplantation after myeloablative therapy in patients with Acute Myeloid Leukemia (AML) in 1st or 2nd CR, without additional GVHD prophylaxis
* Miltenyi provided material goods only in the support of this study.
MBI has negotiated rights through the National Marrow Donor Program (NMDP) to cross reference the BMT CTN 0303 IDE submission for purposes of product registration

14. CliniMACS® CD34 Reagent System Proposed Humanitarian Use Indication
“Humanitarian Use Device: Authorized by U.S. Federal law for processing allogeneic HLA-matched hematopoietic progenitor cells-apheresis (HPC-A) from a related donor to obtain a CD34+ cell enriched population intended for hematopoietic reconstitution following a myeloablative preparative regimen without the need for additional graft-vs-host disease (GVHD) prophylaxis in patients with acute myeloid leukemia (AML) in first or second morphologic complete remission”

15. Steven Devine, M.D. Professor of Internal Medicine Director, Blood and Marrow Transplant Program The Ohio State University Arthur G. James Cancer Center
BMT CTN 0303
Co-Study Chair

16. Acute Myeloid Leukemia (AML)
Most common leukemia diagnosis in adults1
U.S. incidence: ~ 12,330 new cases diagnosed annually
Mortality is roughly 8,950 cases per year
However, less than 2,500 patients progress to transplant
Allogeneic stem cell transplantation (SCT) is the single most effective therapy currently available for the prevention of relapse and shows significant survival benefit in AML patients with intermediate and poor risk cytogenetics in first complete remission (CR1)2
Acute myeloid leukemia (AML), also known as acute myelogenous leukemia or acute non-lymphocytic leukemia, is a clonal malignant disease characterized by an over-proliferation in the myeloid stem cell compartment. This results in an accumulation of myeloid precursor cells in the blood and the bone marrow, arrested in an early stage of maturation. These immature and dysfunctional cells in the marrow interfere with normal hematopoiesis by inhibition of normal differentiation, which leads to suppression of the erythroid, myeloid and megakaryocytic series, resulting in bone marrow failure. Leukemic cells may also spread outside the blood and bone marrow, and invade other parts of the body, including the central nervous system, skin, gums, and testes.
annual incidence rising from 3.8 cases per 100,000 to 17.9 cases per 100,000 adults aged 65 and older
High mortality (also for younger adults, when untreated) / poor survival (5 year relative survival is 21% overall and decreasing with higher age)
1 8/26/10
2 Koreth, J et al. JAMA 2009 16

17. Graft Versus Host Disease (GVHD) Complicates Allogeneic Transplantation From Matched Related Donors (MRD)1-6
Reduced incidence of leukemic relapse and overall survival often not realized due to complications caused by GVHD
Acute GVHD (aGVHD) risk is 35-45%
33-81% of these patients will develop chronic GVHD (cGVHD), resulting in post-transplant morbidity, mortality and reduced quality of life
Immunosuppressive agents used to prevent and treat aGVHD do not affect incidence of cGVHD
Previous studies demonstrate that T cell depletion reduces the risk of severe acute and chronic GVHD
46 Ferrara JLM, et al: Graft-versus-host disease. Lancet 373: , 2009.
47 Chao NJ, et al: Cyclosporine, methotrexate, and prednisone compared with cyclosporine and prednisone for prophylaxis of acute graft-versus-host disease. N Eng J Med 329: , 1993.
48 Devine SM, et al: Recent advances in allogeneic hematopoietic stem-cell transplantation. J Lab Clin Med 141:7-32, 2003.
49 Clift RA, et al: Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens. Blood 77: , 1991.
50 Nash RA, et al: Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors. Blood 96: , 2000.
51 Ratanatharathorn V, et al: Phase III study comparing methotrexate and tacrolimus (prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after HLA-identical sibling bone marrow transplantation. Blood 92: , 1998.
1 Ferrara J, et al. Lancet Chao N, et al. NEJM Devine S, et al. J Lab Clin Med 2003
4 Clift R, et al. Blood Nash R, et al. Blood, Ratanatharathorn V, et al. Blood 1998

18. Chronic GVHD of the Skin
Walker, I; 2011 BMT Tandem Meetings CBMTG Section; Clinical Trials Network Meeting.

19. Chronic GVHD of the Oral Mucosa
Walker, I; 2011 BMT Tandem Meetings CBMTG Section; Clinical Trials Network Meeting.

20. Chronic GVHD and Quality of Life
Quality of life (QoL) at 6 or 12 months is significantly worse in patients with acute or cGVHD after transplant1
QoL at 12 months improves unless cGVHD develops1
Significantly less patients return to work if they develop cGVHD after allogeneic transplantation2
Only 41% of patients with cGVHD return to work at 3 years, compared to 95% of patients without cGVHD2
1 Lee et al. BMT 2006; (38)
2 Wong et al. Blood. 2010; 115(12)

21. Chronic GVHD Treatment
The treatment of cGVHD with long-term corticosteroids increases the risk of cataract formation, avascular necrosis, and osteoporosis1
The 10-year survival is less than 5% for patients affected by severe cGVHD
There are no effective options for the prevention or treatment of chronic GVHD
1 Horowitz, ME., and Sullivan, KM. Blood Reviews (2006); 20: 15–27

22. Unmet Medical Need Served by The CliniMACS® CD34 Reagent System
The incidence and severity of GVHD are most effectively reduced by ex vivo T cell depletion (TCD) of the allograft
There is currently no approved method for ex vivo TCD for allogeneic SCT in the United States
If approved, the CliniMACS® CD34 Reagent System will be the only FDA approved method of CD34+ enrichment and passive TCD available

23. Clinical Study Rationale
Use of ex vivo T cell depletion (TCD) has been limited by
logistical difficulties and variability in TCD methods
lack of an FDA-approved method
concerns regarding potential risk of graft rejection and leukemic relapse
A multi-center trial of TCD in AML patients in complete remission (CR1/CR2) using standard eligibility criteria and a uniform method of TCD was warranted

24. HLA-Identical Sibling-Matched, CD34+ Selected, T cell Depleted Peripheral Blood Stem Cells Following Myeloablative Conditioning For First or Second Remission Acute Myeloid Leukemia (AML): Results of Blood and Marrow Transplant Clinical Trials Network*
(BMT CTN) Protocol 0303
S Devine, S Carter, R Soiffer, M Pasquini, P Hari, A Stein, H Lazarus, C Linker, E Stadtmauer, E Alyea, C Keever-Taylor, and R O'Reilly
* Devine, S et al; BBMT, 2011

25. Blood and Marrow Transplant Clinical Trials Network (BMT CTN)
Established: Sept. 2001; renewed July 2011
Funded by NHLBI/NCI
20 Core Center cooperative agreements
1 DCC cooperative agreement
>80 affiliate centers access trials through DCC
Goal of the Program:
Provide the infrastructure needed to allow promising HCT therapies to be developed/ evaluated in high quality multicenter studies. 25

26. BMT CTN Centers, 2011 >100 centers have enrolled >3900 patients since 2003
= PBMTC Centers
= Core Centers
= Affiliate Centers 26
Mmh06_16.ppt

27. BMT CTN 0303 Historical References
Single center studies show reduced GVHD without increased relapse rates in AML patients receiving T cell depleted (TCD) allografts in CR1-3
One randomized, prospective, multi-center TCD trial showed no increase in relapse in AML patients receiving TCD allografts4
1 Aversa et al. Blood Cells Mol and Disease 2008
2 Pappadopoulos et al, Blood, 1998
3 Soiffer et al; Blood 1997
4 Wagner, J. et al. Lancet 2005 27

28. Randomized, Prospective, Multi-center T Cell Depletion (TCD) Vs
Randomized, Prospective, Multi-center T Cell Depletion (TCD) Vs. Methotrexate and Cyoclosporine (M/C) Trial
Unrelated Bone Marrow
Wagner et al., Lancet 366:733, 2005

29. BMT CTN 0303 Statistical Sampling and Time Points
The sample size was 45 patients, wherein 47 patients were enrolled and 44 completed treatment
There were no blinding or randomization aspects to this trial
The median follow up of the patients was 34 months
(Range: months)

30. BMT CTN 0303 Study Eligibility
AML in CR1 or CR2
Age 18-65
HLA-identical sibling available
No more than 2 induction cycles of chemotherapy required to induce remission
No more than six months from CR to transplant (three months for CR2)
Other standard organ function criteria
No uncontrolled bacterial/fungal/viral infections
Karnofsky performance status > 70% 30

31. BMT CTN 0303 Study Endpoints
Primary Endpoint:
Disease-Free Survival (DFS) at 6 months >75%
Secondary Endpoints:
Acute and chronic GVHD
Overall Survival (OS)
Disease-Free Survival at 2 years
Transplant-Related Mortality (TRM)
Relapse
Engraftment/graft failure
Infusional Toxicities
Incidence of EBV reactivation and PTLD
Proportion of grafts containing > 5 x 106 CD34+ cells/kg and < 1 x 105 CD3+ cells /kg

32. Eight Centers Enrolled Patients onto BMT CTN 0303
Site
Patients Accrued
Dana Farber/Partners Cancer Center 18
Ohio State University 8
Memorial Sloan Kettering Cancer Center 7
Medical College of Wisconsin
City of Hope National Medical Center 3
University Hospitals of Cleveland 2
University of CA, San Francisco 1
University of Pennsylvania
TOTAL 47
44 patients were evaluable on study 32

33. BMT CTN 0303 Patient Characteristics
Patient Age
Mean (range)
46.3 (21-59 )
Donor age
46.2 (16-63)
Gender
Male
16 (36%)
Female
28 (64%)
Leukemia stage
CR1
37 (84%)
CR2
7 (16%)
Cytogenetic Risk (CR1/CR2)
Favorable
0 / 2
Intermediate
25 / 3
Unfavorable
10 / 1
Unknown*
2 / 1
* Unknown cytogenetic risk due to lack of metaphase during testing 33

34. Patient Conditioning Regimen
BMT CTN 0303
Patient Conditioning Regimen
Day of Tx
TBI 1375 cGy*
11 total doses; administered on days -9 through -6
5mg/kg
2.5 mg/kg
60mg/kg
CliniMACS® CD34-enriched cells

35. BMT CTN 0303 Donor Mobilization & Leukapheresis
Received daily G-CSF for mobilization following screening and enrollment
Leukapheresis performed according to institutional standards
Daily leukapheresis with subsequent CD34+ cell selection using the CliniMACS® CD34 Reagent System continued until a post-selection target dose of > 5.0 x 106 CD34+ cells/kg and < 1 x 105 CD3+ cells/kg was met

36. BMT CTN 0303 Steering Committee Laboratory Representative
Carolyn Keever-Taylor, PhD Professor of Medicine Director BMT Laboratories Division of Hematology and Oncology Medical College of Wisconsin
BMT CTN 0303
Steering Committee Laboratory Representative

37. The Manuscript Entitled: “Characteristics of CliniMACS® System CD34-Enriched T Cell-Depleted Grafts in a Multi-Center Trial for Acute Myeloid Leukemia-Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Protocol 0303” has been submitted and accepted for publication by the peer reviewed journal, “Biology of Blood and Marrow Transplantation”    

38. Secondary Endpoint: CliniMACS® CD34 Reagent System Performance
Leukapheresis collections from a Matched Related Donor were performed in order to obtain a minimum of ≥ 2.0 x 106 CD34+ cells/kg
Target of > 5.0 x 106 CD34+ cells/kg and < 1 x 105 CD3+ cells/kg
Up to three collections were allowed to achieve the minimum CD34+ cell dose
The CliniMACS® Tubing Sets (Standard or Large Scale) were used based on starting nucleated cell counts

39. Cellular Testing Requirements
Cell Viability (7-AAD), TNC and CD34+ cell content
At product receipt
After platelet and antibody wash
On CliniMACS® CD34-enriched fraction
CD3+ T cell content
Other cellular testing on CliniMACS® CD34-enriched fraction only
CD14+ monocytes
CD19+ or CD20+ B cells
CD56+ NK Cells
Similar point is to identify testing requirements. Not on slide but will indicate that Flow was expected to have been validated and labs were to have participated in proficiency programs and be CLIA certified.

40. Donors and Products 47 patients enrolled, 44 proceeded to transplant
86 products collected
Total lots (cells from one tubing set) assessed=84
Collections pooled for 2 patients
4 sites processed from 9 to 34 lots
4 sites processed ≤ 4 lots
May not be needed, but sets stage for data

41. CliniMACS® CD34 Reagent System Post-Processing Performance N = 84
% CD34+ Recovery
% CD34+ Purity
Log10 TCD
% Viability
Mean
66.06
93.0
4.78
96.57 SD
±20.25
±8.3
±0.55
±3.84
Min
29.9
61.5
3.2
74.0
Max
125.6
99.8
5.9
100.0
Overall outcome for all sites. Could be dropped.

42. Center to Center Statistical Analysis
Sites processing ≥ 9 lots compared individually (N=4)
Sites processing ≤ 4 lots pooled (N=4)
Multivariate analysis used a linear mixed effect model to account for repeated measures (≥ 2 lots for most patients)
Pairwise center comparisons were performed with Tukey-Kramer adjustment for multiple comparisons
Not sure what Adam will present. This as much as I would be willing to say about the stats

43. Pre-Processing Cell Counts
We first looked to see if the starting products were similar for each of the groups and found that there were some differences in each of the parameters. The median TNC, CD34+ cells, and CD3+ cells were highest for center two at which the first two donors were mobilized using 16 mg/kg G-CSF initially required by the protocol. Subsequent donors were mobilized at 10 mg/kg. The figure shows the products that split. Viability by 7-AAD also differed between center 4 and most of the other centers by having more products with a lower viability. However, this center performed it’s analysis using a flow method that does not include wash steps, which is known to results in somewhat lower viability using 7-AAD, since dead cells are not washed away.

44. Post Processing Outcomes
Here the post processing outcomes are shown by center, and although some differences are seen these are mostly limited to a one or at most two centers. CD34 recovery was significantly lower for center 4 as compared to center 3 and 1, and center 1 had significantly higher recovery than center two, though this was clearly affected by the four lots that exceeded 100% recovery. Purity and log TCD were less variable, with very high purities achieved for most products with a few outliers at center 2, 4, and the combined Log TCD significantly differed only between center 3 and 4, and again viability was generally high, with the only significant difference seen for center 3 with a uniformly high viability near 100%.
All centers were able to process grafts that met the study criteria

45. CD34+ Cells x 106/kg of Patient Weight Infused
CD34+/kg
CD34+ Target Dose
CD34+ Minimum Dose
CD34 infused. Nicely shows that not only were the targets met, but they were mostly exceeded.
I have not presented anything regarding the number of collections for each patient to achieve the doses, Figure that would be part of the clinical presentation, but let me know.
All patients received the minimum CD34+ dose (> 2.0x106 cells/kg)
84.1% of patients received > 5 x 106 CD34+ cells/kg 45

46. Patient Weight Infused
CD3+ Cells x 105/kg of
Patient Weight Infused
CD3+/kg
Upper limit of CD3+ dose
This represent the cells infused. All final graft products were under the upper limit.
No patients received more than 1.0x105 CD3+ cells/kg 46

47. Final Cellular Product Summary
Parameter
Result
Median CD34+ dose
7.92 x 106/kg
Range
x 106/kg
Median CD3+ dose
0.7 x 104/kg
0.1 – 8.3 x 104/kg
Median Log10 TCD
4.9 logs
3.2 – 5.9 logs
The grafts contained a median of nearly 8e6 CD34+ cells/kg, with only 0.7e4 T cells. The products were sterile, as tested by gram stain and culture, with endotoxin below the detectable limits in all cases. No significant infusional related toxicities were observed.
All gram stains/14 day cultures were negative
All endotoxin < 5.0 EU/kg
No significant infusion related toxicities observed

48. Conclusions - Secondary Endpoint CliniMACS® CD34 Reagent System Performance
All sites, and all products met and most exceeded study goals for:
CD34+ cell infusion dose > 2 x 106/kg
84% met the goal of > 5 x 106/kg
CD3+ cell infusion dose < 1 x 105/kg
The performance of the CliniMACS® CD34 Reagent System was stable and reproducible, resulting in a consistently high degree of CD34+ cell enrichment, T cell depletion and sterility in a multi-center setting

49. Steven Devine, M.D. Professor of Internal Medicine Director, Blood and Marrow Transplant Program The Ohio State University Arthur G. James Cancer Center
BMT CTN 0303
Co-Study Chair

50. Primary Endpoint 6 Month Disease-Free Survival of >75%
6 months (95% CI )

51. Secondary Endpoints Neutrophil/Platelet Engraftment
Analysis N
Median Days to Engraftment
Day 30
Estimate
Day 100 Estimate
Platelet Engraftment >20K/µL 44
16 days
93.2%
(95% CI: )
97.7 %
92-100)
Cumulative Incidence of Neutrophil Engraftment
>500/µL
12 days
100% )
No primary graft failures
One secondary graft failure at Day +54 after initially engrafting on Day +12 51

52. Stopping guideline of <30% TRM at 1 year was not exceeded
Secondary Endpoint Cumulative Incidence of Transplant-Related Mortality (1yr)
All Patients By CR1/CR2
1yr
(95% CI: )
Stopping guideline of <30% TRM at 1 year was not exceeded
TRM at 2 years was 19.9% (95% CI: )

53. Secondary Endpoint Cumulative Incidence of EBV Reactivation
All Patients By CR1/CR2
2 yrs
13.6%
(95% CI: )
2 yrs
2 yrs
8 patients treated for EBV DNA levels >1000 copies/mL
One case of PTLD with subsequent death
EBV monitoring performed weekly

54. Secondary Endpoint Cumulative Rate of Relapse
All Patients By CR1/CR2
2 yrs
2 yrs
1 yr
2 yrs
* N=7 patients treated in CR2; 4 patients relapsed
(95% CI: %)

55. Secondary Endpoint - Cumulative Incidence of Acute GVHD Grades II-IV
All Patients By CR1/CR2
100 days
(95% CI: )
No Grade IV acute GVHD observed
Published acute GVHD risk 35-45% 55

56. Secondary Endpoint - Cumulative Incidence of Acute GVHD Grades III-IV
All Patients By CR1/CR2
100 days
(95% CI: 0 – 10.8%)
No Grade IV acute GVHD observed

57. Secondary Endpoint Cumulative Incidence of Chronic GVHD (2 yrs)
Includes Limited and Extensive
All Patients By CR1/CR2
2 years
(95% CI: )
Published chronic GVHD risk 33-81%

58. Secondary Endpoint Cumulative Incidence of Extensive Chronic GVHD (2 Years)
All Patients By CR1/CR2
2 years
(95% CI: ) 58

59. Secondary Endpoint Disease-Free Survival at 2 Years
All Patients By CR1/CR2
By Stage
1 yr
2 yrs
2 yrs
2 yrs
Check graph #2 by CR1/CR2 the line is partially missing
Historical data estimates 2 year DFS < 60% in CR11-3
2º endpoints were > 70% for CR1 and > 60% for CR2
1 Suciu Blood Brunet et al, Hematologica 2004
3 Cornelissen et al, Blood 2007

60. Secondary Endpoint Overall Survival at 2 Years
1 yr
2 yrs 60

61. Adverse Events (AEs) Toxicities were reported as AEs
No Unexpected Grade 3-5 AEs were reported
AEs were due to regimen related events and toxicities common to allo HCT and could not be attributed to the CliniMACS® CD34 Reagent System
The most common toxicities within 1 year post-transplant:
Regimen related Grade 3 or 4 GI toxicity (40.9%)
Abnormal liver function (36.4%)
Graded using the NCI’s CTCAE version 3.0

62. Causes of Death by 2 Years Post-Transplant
Recurrence 6
Infection 4
Idiopathic Pneumonia
Organ failure
Other* 2 1
PTLD
Total 16
* Possibly cardiac related 62

63. BMT CTN 0303 Conclusions
HCT following myeloablative preparative regimen for patients with AML in CR1 or CR2 can be performed in a multicenter setting using the CliniMACS® CD34 Reagent System without additional post-transplant pharmacologic GVHD prophylaxis
All 1º and most 2º endpoints were met, demonstrating:
81.8% Disease-Free Survival 6 months post TX
No primary graft failure; consistent neutrophil and platelet engraftment
Acute GVHD grades II-III <23%. No Grade IV aGVHD
Chronic GVHD at 2 years 19%; extensive 6.8%
TRM <20% at 2 years
Overall risk of relapse was low at 23.7% at 2 years
The CliniMACS® CD34 Reagent System consistently produced a graft with > 2 x 106 CD34+ cells/kg and < 1 x 105 CD3+ cells/kg with no reported device related toxicities

64. Principal Investigator Data Analysis Protocol
Marcelo Pasquini, M.D., M.S. Assistant Professor of Medicine Medical College of Wisconsin Assistant Scientific Director Center for International Blood and Marrow Transplant Research (CIBMTR)
Principal Investigator
BMT CTN 0101 versus BMT CTN 0303
Data Analysis Protocol

65. A Single Arm, Multi-center Phase II Trial of Transplants of HLA-Matched, CD34+ Enriched T cell Depleted Peripheral Blood Stem Cells Isolated by the CliniMACS® System in the Treatment of Patients with AML in First or Second Morphologic Complete Remission
(BMT CTN) Protocol 0303
A Randomized Double-blind Trial of Fluconazole versus Voriconazole for the Prevention of Invasive Fungal Infections in Allogeneic Blood and Marrow Transplant Patients
(BMT CTN) Protocol 0101

66. The Data Analysis Protocol (DAP)
Performed according to FDA approved Data Analysis Protocol # which was a prospective statistical comparison of retrospective data
This comparison of the 0101 and 0303 data has been submitted for publication. The manuscript is currently under review.

67. Enrollment Period BMT CTN 0101 versus BMT CTN 0303
Enrolling Centers by Protocol, and Their
Contribution to Each Study 15 3 5
23% of 0101
47.5% of 0303

68. Data Analysis Protocol (DAP) Objective
Test the hypothesis that T cell depletion using the CliniMACS® CD34 Reagent System as the sole form of immune suppression (BMT CTN 0303) demonstrates a comparable safety profile as compared to conventional GVHD prophylaxis post-transplant (BMT CTN 0101) in AML patients in CR1/CR2 receiving a Peripheral Blood Stem Cell (PBSC) allograft from a matched related donor

69. The Following One Year* Endpoints Were Statistically Compared
Disease-Free Survival (DFS)
Overall Survival (OS)
Engraftment
Graft Failure
Transplant-Related Mortality (TRM)
Relapse
Incidence and Severity of Acute GVHD (aGVHD)
Incidence and Severity of Chronic GVHD (cGVHD)
* BMT CTN 0101 follow-up was limited to 1 year

70. Eligibility Criteria for 0303 was Matched in 0101 Patient Subsets
Patient Age 18-65
Unmodified PBSC Allograft
HLA identical sibling donor
AML in CR1 or CR2
All cytogenetic risk patients included in 0101
Only CR2 patients in 0303 enrolled favorable cytogenetic risk patients

71. Patients meeting eligibility Patients meeting eligibility
DAP Patient Selection
BMT CTN 0101
BMT CTN 0303
Patients Randomized
N=600
Patients Enrolled
N=47
No Transplant
N=1
Received Transplant
N=599
Received Transplant
N=44
No Transplant
N=3
Eligibility Criteria
Age 18-65
HLA Match-Sibling
PBSC
AML in CR1 or CR2
Patients meeting eligibility
N=84
Patients meeting eligibility
N=44
AML CR1
N=65
AML CR2
N=19
AML CR1
N=37
AML CR2
N=7

72. Baseline Characteristics
% of patients
0303 (N=44)
p-Value
Significantly More Females in 0303
Female
44%
63.6%
0.04
Male
56%
36.4%
Patient Ages Similar in Both Protocols
Median Age
45.1
48.5
0.14
% > 50 yrs of Age
32.1%
43.2%
0.22
Karnofsky Performance Status Similar in Both Protocols
100%
22 (26.2%)
17 (38.6%)
0.36
> 90%
46 (54.8%)
> 80%
13 (15.5%)
8 (18.2%)
> 70%
3 (3.6%)
2 (4.5%)

73. Cytogenetic Risk Profile By Protocol
Control
(0101)
(N=84)
N (%)
CD34+ Enriched (0303)
(N=44)
p Value
Favorable 9
(10.7%) 2
(4.5%)
0.34
Intermediate 55
(65.5%) 28
(63.6%)
Unfavorable 12
(14.3%) 11
(25.0%)
Unknown 8
(9.5%) 3
(6.8%)
Slightly more unfavorable cytogenetics in 0303 than in 0101;
overall, statistically similar
* Slovak M et al. Blood SWOG/ECOG classification used for profile determination.

74. Conditioning Regimen BMT CTN 0101 versus BMT CTN 0303
# (%)
0303
(N=44) 
Conditioning Regimen
TBI based
43 (51)
44 (100)
Busulfan based
41 (49)
ATG
7 (8)
GVHD Prophylaxis
T cell depletion
Tacrolimus based
48 (57)
Cyclosporine A based
37 (43)

75. Day +30 Neutrophil Engraftment (> 500/µL)*
100% in CD34+ (0303)
96.4% in Control (0101)
p=0.002
3 primary graft failures in the Control (0101)
0 primary graft failures in the CD34+ Enriched (0303)
1 secondary graft failure in each trial
*Cumulative Incidence

76. Day +100 Platelet Engraftment (≥ 20,000/µL)*
97.7% in CD34+ (0303)
88.1% in Control (0101)
p=0.228
*Cumulative Incidence

77. Transplant-Related Mortality*
All Patients by Protocol CR1/CR2 and Protocol
p=0.62
16.7% in Control (0101)
13.6% in CD34+ (0303)
*Cumulative Incidence

78. Incidence of 12 Month Relapse*
20.3% in Control (0101)
20.6% in CD34+ (0303)
*Cumulative Incidence

79. 12 Month Relapse* CR1/CR2 By Protocol
57.1%
CR2
p=N/A**
31.6%
p=0.02
17%
CR1
p=0.57
13.7%
**Statistical calculations not performed due to low patient numbers
4/7 in 0303 and 6/19 in 0101 relapsed
*Cumulative Incidence

80. 12 Month Disease-Free Survival
65.7% in CD34+ (0303)
63% in Control (0101)
p=0.59

81. 12 Month DFS in CR1/CR2 By Protocol
72.8%
66.1%
p=0.39
CR1
52.6%
p=0.7
p=N/A*
CR2
28.6%
* Statistical comparisons were not performed on CR2 patients
due to low patient numbers

82. DFS Stratification by Age > 50 CR1 and CR2
73.7% 0303 > 50
68.3% 0101 < 50
59.7% 0303 < 50
p=0.88
51.9% 0101 > 50

83. 12 Month Overall Survival
All Patients by Protocol CR1/CR2 and Protocol
77.3% in
CD34+ (0303)
73.8% in
Control (0101)
p=0.61
p=0.87

84. Acute GVHD Grades II-IV*
p=0.068
39.3% in Control (0101)
22.7% in CD34+ (0303)
*Cumulative Incidence

85. Acute GVHD Grades II-IV in Patients < 50 Years of Age*
47.4% in Control < 50 (0101)
p=0.028
24% in CD34+ < 50 (0303)
*Cumulative Incidence

86. Acute GVHD Grades III-IV *
p=0.31
9.5% in Control (0101)
4.5% in CD34+ (0303)
*Cumulative Incidence

87. Limited/Extensive Chronic GVHD*
p=0.0004
49.9% in Control (0101)
15.9% in CD34+ (0303)
1 year post-hoc power for the comparison between 0101 and 0303 was 98%
*Cumulative Incidence

88. Patients with Infections
Infectious Complications
Control
(0101)
N=84
CD34+ Enriched (0303)
N=44
# of Infectious Reports
# of Patients
1-3 44 20
4-5 7
6-10 6 3
>10 2
Total # of Patients with Infections
(% of Patients) 57
(67.9%) 32
(72.7%)

89. Types of Infections Seen By Protocol
Control
(0101)
N=84
CD34+ Enriched (0303)
N=44
# of Infections and
(# of Patients)
Bacterial
(48)
(25)
Viral
46 (30)
(24)**
Fungal
9 (9)*
(5)
Protozoal
0 (0)
(1)
Other
4 (3)
(0)
Total Infection Events
162
112
* Represents confirmed infections only; 10 possible and 3 presumptive infections not included
** 8 of 24 viral infections in CD34+ enriched cohort (0303) were due to EBV reactivation  

90. Percentage of all deaths due to infection were
Infection Events
Control
(0101)
N=84
CD34+ Enriched
(0303)
N=44
Maximum Severity by Patient
# of Patients
(% of Patients)
None
(33.3%)
(27.3%)
Moderate
(35.7%)
(34.1%)
Severe
(27.4%)
(29.5%)
Life Threatening/Fatal
(3.6%)
(9.1%)
Percentage of all deaths due to infection were
18.2% in 0101 and 20% in 0303

91. Causes of Death at One Year Post-Transplant
Control
(0101)
N and % of Deaths
CD34+ Enriched (0303)
Recurrence
9 (40.9%)
3 (30%)
Acute GVHD
(4.5%)
Chronic GVHD
Infection
4 (18.2%)
2 (20%)
Interstitial Pneumonia
Veno-occlusive Disease
1 (4.5%)
ARDS
Organ Failure
5 (22.7%)
PTLD
1 (10%)
Total
22 (23.1%)
10 (18.9%)

92. DAP Statistical Comparison Summary
The incidence of chronic GVHD at one year post-transplant was significantly lower for recipients of CD34+ enriched allografts (p= )
Between patients receiving CD34+ enriched versus unmanipulated grafts, there was no significant difference in:
Platelet and Neutrophil Engraftment, Acute GVHD, DFS, OS, TRM, and Relapse
Low numbers of CR2 patients in both cohorts make statistical comparisons of relapse inconclusive

93. The DAP results support a comparable safety profile for CD34+ enriched transplants as compared to patients receiving unmanipulated grafts and conventional GVHD prophylaxis but with a significant reduction in cGVHD
DAP Conclusion

94. Kai Pinkernell, M.D. Head of Clinical Development Miltenyi Biotec GmbH
Overall Conclusions of Safety and Probable Benefit of the
CliniMACS® CD34 Reagent System

95. Performance and Safety Conclusions CliniMACS® CD34 Reagent System
The CliniMACS® CD34 Reagent System consistently produced a graft with > 2 x 106 CD34+ cells/kg and < 1 x 105 CD3+ cells/kg
84% of the grafts contained > 5 x 106 CD34+ cells/kg
There was no significant difference between CD34+ enriched and unmanipulated allografts for:
Platelet and Neutrophil Engraftment, Acute GVHD, DFS, OS, TRM, and Relapse
While 0303 patients experienced more infectious episodes/patient (112/44 patients in 0303 versus 162/84 patients in 0101), these did not translate to higher infectious death rates (18.2% of deaths in 0101 vs. 20% in 0303)
Increased incidence of EBV reactivation reports most likely due to stringent protocol specified monitoring of EBV in 0303

96. Probable Benefit Conclusions CliniMACS® CD34 Reagent System
The CliniMACS® CD34 Reagent System was shown to consistently yield CD34+ enriched, T cell depleted, sterile cellular grafts in a multi-center setting
Low rates of both acute and chronic GVHD without the risks associated with traditional pharmacologic GVHD prophylaxis while maintaining consistent engraftment, excellent DFS and OS, low TRM, and a low incidence of relapse
Significantly reduced incidence of chronic GVHD without compromising survival

97. Safety and Probable Benefit Objectives Met
The CliniMACS® CD34 Reagent System is safe, and has a probable benefit in significantly reducing the incidence of chronic GVHD while eliminating the need for pharmacologic GVHD prophylaxis for AML patients in complete remission, undergoing PBSC transplantation from a matched related donor

98. Acknowledgements BMT CTN NHLBI/NCI CIBMTR/EMMES/NMDP
Investigators and Contributors
Steven Devine
Marcelo Pasquini
Carolyn Keever-Taylor
Richard O’Reilly
Robert Soiffer
John Wingard
Adam Mendizabal, EMMES
Shelly Carter, EMMES
Nancy DiFronzo, NHLBI
Mary Horowitz, CIBMTR
Nancy Poland, NMDP
Many, Many others
The patients and their families who have made these trials possible

99. Thank You