Rationale for cord blood banking from hematopoietic stem cell transplant to regenerative medicine. Milan November 2008

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1 Rationale for cord blood banking from hematopoietic stem cell transplant to regenerative medicine Milan November 2008

2 Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLAidentical sibling Gluckman E, Broxmeyer HE, Auerbach AD, Freidman HS, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, Cooper S, English D, Kurtzberg J, Bard J, Boyse EA. N Engl J Med 1989;321:

3 Twenty years later

4 History of cord blood transplantation 1988 First Cord blood transplant Clinical observation that GVHD was reduced even in HLA incompatible CBT Establishment of Cord blood banks Feasibility of HLA incompatible unrelated cord blood transplants 1997 Role of cell dose and HLA 2000 Retrospective comparisons between UBMT and UCBT in children and adults 2003 Criteria of cord blood choice and indications 2004 Double cord blood transplants 2005 Reduced intensity conditioning 2005 Presence of non hematopoietic stem cells source for regenerative medicine

5 Unrelated Cord Blood Transplantation by region ( ) n=~ (estimated number based on CIBMTR, Eurocord and Japanese CB registries) North America South America Europe Australia Asia M E/ Africa

6 Eurocord missions EUROCORD is an international registry on behalf of the European Blood and Marrow Transplant group (EBMT), including European and non European centres. EUROCORD collaborates with CBBs by validation of data and sending statistical analysis of the cord blood transplants performed with their respective units to every associated CBBs EUROCORD registry based analysis has developed crucial reports supporting the concept of establishing cryopreserved cord blood banks for clinical use.

7 Number of CBT / year reported to Eurocord Related n=473 Unrelated n=3835 *Still collecting data March

8 Single Unrelated CBT according to the recipient age/year reported to Eurocord Children n=2036 Adults n=1690 *Still collecting data March

9 EUROCORD Double Unrelated CBT / year reported to Eurocord Reduced intensity Conditioning CBT / year reported to Eurocord * * March *Still collecting data

10 Major breakthrough in the use of cord blood 1. Comparison with HLA matched unrelated transplants same results than with mismatched cord blood in adults and children 2. Role of cell dose and HLA 3. Double cord blood 4. Reduced intensity conditioning 5. Non hematopoietic stem cells in cord blood

11 Searching and identifying an alternative stem cell donor Information on A + B + DRB1 typing (%) Median search time (months) Donors identified but not available (%) Rare haplotypes represented (%) Main limiting factor to graft Ease acquisition of rearranging date of cell infusion Potential for immunotherapy Potential for viral transmission to Potential recipient for congenital disease transmission Risk for the donor Main problems to be overcome Main criteria to be considered UBMT HLA identity Difficult Yes Yes No Low GvHD Grewal et al, Blood 2003 Rocha et Locateli, BMT 2008 UCBT ~ 80 < 1 ~ 1 20 Cell dose Easy No No Yes No Graft failure, delayed immune recovery Haplo HSCT 100 immediate None Not applicable Poor mobilization Easy Yes (limited) Yes No Low Delayed immune recovery, lack of T cell mediated GVL effect

12 Single UCBT in children with malignancies n=903 Event free survival according to disease status at CBT Early phase: 45%+/ 4 Intermediate : 38%+/ 3 Advanced : 32%+/ 3 p<0.001

13 Cord Blood vs Bone Marrow Pediatric Study from CIBMTR and NYBC M. Eapen et al. The Lancet 2007 ALL and AML Transplanted in in the U.S. BM (n=116) CB (n=497) Median follow up of survivors BM=5 and CB=4 years

14 Leukemia free Survival Pediatric Study 100 Adjusted P robability, % CB matched (n=35) 60% CB 1 Ag MM high (n=157) 45% BM matched (n=116) 38% CB 2 Ag MM (n=267) 33% CB 1 Ag MM low (n=44) 35% Months

15 Summary Pediatric Study Relative to matched BM Matched and 1 AG MM CB Lower GVHD Similar rates of TRM, relapse and LFS 2 AG MM CB Lower GVHD Higher rates of early TRM Lower rates of relapse Similar rates of LFS

16 Children with high risk acute leukaemia with an indication of allogeneic HSCT and without an HLA identical sibling donor Search for an alternative donor HSCT simultaneously in cord blood banks and donor registries < 3 months between remission and transplant Unrelated Cord Blood Transplant HLA matched (6 out of 6) * cord blood unit and not less than 1.0 x10 7 nucleated cells/kg at infusion 1 or 2 out of 6 HLA differences* and 3.0 x10 7 nucleated cells/kg at infusion Unrelated Bone Marrow Transplant Allele matching of HLA A, B, C, DRB1, if donor available < 3 months between remission and transplant Allele mismatching of one or two alleles out of 8 to be considered in absence of cord blood graft Rocha Lancet 2007

17 Single UCBT in adults with malignancies n=697 Event free survival according to disease status at CBT Early phase: 42%+/ 4 Intermediate : 35%+/ 4 Advanced : 24%+/ 3 p<0.001

18 Comparison of Matched unrelated bone marrow and mismatched unrelated cord blood transplant in adults with acute leukemia Eurocord study New England Journal of medicine V. Rocha et al, 2004

19 MULTIVARIATE ANALYSIS Hematopoietic Recovery & GVHD UBMT UCBT RELATIVE RISK P=0.01 * * * * P> P< P= ANC >0.5 x 10 9 /L Platelets >50 x 10 9 /L Acute GVHD Chronic GVHD * Reference Group

20 UBMT UCBT MULTIVARIATE ANALYSIS TRM, RELAPSE AND LFS * P=0.29 * P=0.46 * 0.70 RELATIVE RISK TRM Relapse Leukemia Free Survival * Reference Group

21 Leukemia free Survival ADJUSTED PROBABILITY, % Laughlin et al, NYBC / IBMTR: NEJM BM, matched (n = 367) UCB, 1 Ag MM (n = 150) BM, 1 Ag MM (n = 83) YEARS

22 Strategy of alternative stem cell donor in adults with malignant disorders High resolution HLA typing of the adults To be considered Haplo T depleted in AML Simultaneous search Cord Blood Banks Bone Marrow donor registries NC dose collected to be increased with number of mismatches (single or double) >2.5x10 7 /kg NC > 3.5x10 7 /kg NC >1.5x10 5 /kg CD34 >2x10 5 /kg CD34 HLA: 0 1/6 HLA: 2/6 UCBT <8/10 or >3 mths delay for AL) HLA 9 or 10/10 UBMT

23 Minnesota algorithm of cord blood selection for transplantation For patients with hematological disorders who need a haematopoietic stem cell transplant, but lack a HLA matched related or unrelated donor CB unit 5 6 out of 6 HLA MM* NC cryopreserved dose >2.5 3 x10 7 /kg Yes No Single UCBT Double UCBT Each unit *HLA A,B antigen typing, DRB1 allele typing **Partial matching between the 2 CB units 5 6 out of 6 HLA MM** NC dose >1.5 x107/kg Double umbilical cord blood transplantation. Majhail NS, Brunstein CG, Wagner JE.Curr Opin Immunol Oct;18(5):571 5

24 What did we learn from the clinical studies Engraftment is delayed and depends on number of CD34+ cells in the graft optimal dose >2x10 5 /kg GVH is reduced GVL is functional Immune reconstitution is delayed Long term survival is improved because of the better quality of hematologic and immunologic reconstitution

25 HLA and cell dose in UCBT in patients with malignant disorders (n=925) Graft characteristics 1 antigen or allelic mismatch (for DRB1) (n=389) CLASS I (A,B) 67% CLASS II (DRB1 HR) 33% 2 antigen or allelic mismatch (for DRB1) (n=377) CLASS I (n=142) 38% CLASS II (n=28) 7% CLASS I+II (n=207) 55% Median nb of nucleated cells before freezing: 4.4 x 10 7 /kg ( ) (n=820) Median nb of nucleated cells infused: 3.1 x 10 7 /kg ( ) (n=925) Median nb of CD34 infused: 1.4 x 10 5 /kg ( ) (n=667) Median nucleated cell loss after thawing: 26% (1 70%)

26 Multivariate variate analysis analysis in malignant diseases Neutrophils engraftment Favourable prognostic factors Platelets engraftment Favourable prognostic factors HLA 6/6 or 5/6 Early and intermediate phase of the disease Cells infused > 2x 10 7 /Kg HLA 6/6 or 5/6 Early and intermediate phase of disease Cells infused > 2x 10 7 /Kg Patient s neg. CMV serology TRM Favourable prognostic factors Relapse Favourable prognostic factors HLA 6/6 or 5/6 Early and intermediate phase of the disease Cells infused > 2x 10 7 /Kg HLA 4/6 or 3/6 Early and intermediate phase of the disease

27 Neutrophils recovery according to the number of CD34 infused (n=925)* CIF neutrophils recovery >=1.4x10e5/kg <1.4x10e5/kg p= *CD34 data available in 667 cases Days

28 Months UCBT malignant disorders (n=925) Relapse according to number of HLA CI of Relapse p= or 1 HLA mimsmatch 2 or 3 HLA mismatches

29 Months UCBT malignant disorders (n=925) Disease free survival according to number of HLA Event Free Survival P= differences 2 differences 0 1 difference

30 Overall survival UCBT in non malignant disorders (n=268) Overall survival according to HLA and cell dose 2 and 3 HLA diff and cell dose < 3.5 (n=30) Months 0 and 1 HLA diff and cell dose >= 3.5 (n=11 2 and 3 HLA diff and cell dose >= 3.5 (n=62) 0 and 1 HLA diff and cell dose < 3.5 (n=28) P<

31 Cord blood units 2 HLA disparities > 3,0 x 10 7 TNC/kg UCB Transplantation Malignant disease Cell dose 3,0 x 10 7 TNC/kg at collection 2,0 x 10 7 TNC/kg at infusion Non malignant disorders : more graft failure Avoid HLA mismatch Avoid units < 3,5 x 10 7 TNC/kg + 2 or more HLA incompatibility No single unit 3,5 x 10 7 TNC/kg double in protocols Wagner et al, Blood 2002 Gluckman et al, Exp Hematol 2004

32 Questions Is cell dose more important than HLA? What is the level of resolution of HLA required? Is matching for HLA class I more important than class II Is it important to perform HLA C typing? Is a one HLA mismatched unrelated donor better than a one HLA mismatched cord blood? How to measure stem cell content and viability?

33 Inhibitory KIR ligand incompatibility and unrelated cord blood stem cell transplantation (UCBT) for patients with acute leukemia Roel Willemze, Celso Arrais Rodrigues, Myriam Labopin, Guillermo Sanz, Gérard Michel, Gérard Socié, Bernard Rio, Anne Sirvent, Marc Renaud, Luiz Madero, Mohamad Mothy, Christelle Ferra, Federico Garnier, Joan Garcia, Lucilla Lecchi, Gesine Kögler, Yves Beguin, Cristina Navarette, Marc Boogaerts, Irina Ionescu, Karim Boudjedir, Andrée Laure Herr Bellon, Eliane Gluckman and Vanderson Rocha on behalf of Eurocord Netcord and the ALWP of the EBMT

34 Cumulative incidence of relapse KIR ligand compatible KIR ligand incompatible

35 Leukemia free survival p=0.005 KIR ligand incompatible n=69 55%±7 at 2 yrs KIR ligand compatible n=149 31%±4 at 2 yrs

36 Multivariate analysis P VALUE HR 95.0% CI Overall Survival ALL vs AML 0,43 0, >1 HLA difference 0,004 0, KIR ligand MM 0,004 2, Leukemia free survival ALL vs AML 0,08 0, >1 HLA difference 0,08 0, KIR ligand MM 0,0016 2, Relapse incidence ALL vs AML 0,160 1, >1 HLA difference 0,050 0, RIC vrs MAC 0,007 2, KIR ligand MM 0,050 0,

37 Unrelated, single unit, cord blood stem cell transplantations from KIR ligand incompatible donors in patients with acute leukemia in CR resulted in a lower relapse incidence and in a higher leukemia free and overall survival These results were more evident for patients with AML

38 Future progresses in cord blood transplant

39 New protocols for cord blood transplant Graft facilitation Unmanipulated Double cord transplant Co infusion of : Ex vivo expanded cells Intra bone unit Third party Mesenchymal cells Conditioning non myeloablative vs myeloablative

40 Double cord blood transplant Wagner JE for the Minnesota group

41 Double Unit Selection 0 2 mismatch 0 2 mismatch Minimum allowed UCB cell dose #1 1.0 x 10 7 NC/kg 0 2 mismatch UCB #2 Minimum allowed cell dose 0.5 x 10 7 NC/kg Goal : maximize graft cell dose 1 o Endpoint: Donor Engraftment

42 Sustained Neutrophil Engraftment All patients p =.84 Double Single Incidence Days

43 Overall Survival CR1 & CR2 1.0 Probability I II I I II I I I II I I I I I I I I II I Years p = I I I I Double 72% (56 88%) Single I I 47% (51 75%)

44 Double cord blood transplant Conclusions from JE Wagner Double UCBT promotes engraftment, achieving rates comparable to single UCBT with adequate cell doses: mechanism unknown additive effect? Risk of grade 2 4 agvhd is higher after double UCBT (although no difference in risk of grade 3 4 agvhd) Risk of cgvhd is similar Reduced risk of relapse is associated with Double UCBT Early disease status (CR1 & 2) No Benefit from agvhd

45 Direct intra bone marrow injection C of Cord Blood Cells to overcome delayed/failure to engraft Francesco Frassoni Centro Cellule Staminali e Terapia Cellulare Transplant Center and Cord Blood Unit Ospedale San Martino Genova

46 Day +30 bone marrow aspirate Injected site CONTROLATERAL non injected site 100% donor 100% donor

47 Graft facilitation new approaches Ex vivo Expansion Cytokines Cocktail FLT3+TPO+SCF Cytokines Cocktail + copper chelator (Gamida) + co incubation with MSC ( Viacell inc)) + Valproic acid (Arcese) Homing molecules SDF 1 agonist (Z. Lapidot Israel) CXCR 4 agonist (Dr Wang, Chemokine therapeutics Corp) CD26 inhibitor (Diprotin A) HE. Broxmeyer USA Increase expression of Wnt with by manipulation of cytokines (Dkk). Regulation of generation of microenvironment niches Parathyroid hormones D. Scadden Harvard et M. Andreef MD Anderson USA

48 Non hematopoietic stem cells in cord blood Possible use in regenerative medicine

49 Source of cord non hematopoietic stem cells Blood Cord endothelium Wharton jelly Placenta

50 ES like cells in cord blood A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. Kögler G, et al. J Exp Med Jul 19;200(2): Morphological and molecular characterization of novel population of CXCR4+ SSEA 4+ Oct 4+ very small embryoniclike cells purified from human cord blood: preliminary report. Kucia M,et al. Leukemia (2): Production of stem cells with embryonic characteristics from human umbilical cord blood. McGuckin CPet al, Cell Prolif. 2005;38(4):245 55

51 Non hematopoietic Stem cells in cord blood ES like MSC EPC Liver Pancreatic islets Retina Neurones

52 Perspective in regenerative medicine Replacement Hematopoietic stem cells Gene modified cells Drug like effect MSC MSCs anti inflammatory IL 1 receptor antagonist Wnt signaling Driving traffic in the right direction

53 Stem cells in cord blood Questions Are cord blood stem cells different from ES cells or adult cells What is their immunogenicity What is their differentiation potential What is their tumour potential How do they compare with IPs?

54 Future of cord blood banking

55 Indication of Cord blood banking Unrelated for allogeneic cord blood transplant for hematological diseases Directed for siblings of a patient with leukemia or another disease curable by allogeneic hematopoietic stem cell transplant Autologous for potential use later in life

56 CORD BLOOD BANKING Arguments for autologous banking Individual right to store own cells Unique opportunity for collection Biological insurance CB cells can be stored long term Knowledge on CB biology evolves rapidly CB has unique properties and contains unique cell populations with therapeutic potential Possibility for therapeutic application in the mid long term : regenerative medicine

57 CORD BLOOD BANKING Public/allo vs private/auto Proven benefit Quality : accreditation Public money, non profit Continuity guaranteed Solidarity Equal access Balanced information Global inventory Unproven benefit Quality variable Private money, for profit Continuity not guaranteed Compete with allo Unequal access Biased information No inventory

58 CORD BLOOD BANKING Regenerative medicine No (not yet?) proven benefit of regenerative cell therapy, and even less so with CB Degenerative diseases occur at relatively old age : what will be cell viability and potential after a storage time of years? Will standards in 50 years be compatible with current collection and storage practice? What new knowledge/treatment will be there in 50 years that will render cell therapy obsolete? What type of cell/property does CB offer that is not available in adult BM or other tissues?

59 CORD BLOOD BANKING Hybrid auto/allo CB banking Coexistence : mother chooses between auto or allo banking; auto business funds allo banking (Stemcyte) Auto allo : unit stored as auto, but must be released for allo if requested (Spain) Split banking : 20% of volume for auto and 80% for allo (Virgin)

60 CORD BLOOD BANKING WMDA statement Supports establishment of public CBB based on altruistic and voluntary cord blood donation. Very low likelihood that an autologous CB unit will be used for transplantation. Currently no clear proof that these cells will be able to be used for regenerative medicine or to treat other diseases. Must ensure that family receives impartial and accurate information about risks and benefits of private storage and sign informed consent. All CBB subject to the same standards. Promotion or general funding of autologous or related CB storage without medical indication for directed donation should not be supported by governments.

61 Eurocord, Hôpital Saint Louis, Paris, France

62

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