SANGUE DEL CORDONE OMBELICALE: DALLA RACCOLTA ALLE APPLICAZIONI CLINICHE

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1 SANGUE DEL CORDONE OMBELICALE: DALLA RACCOLTA ALLE APPLICAZIONI CLINICHE Lorenza Lazzari, PhD Department of Regenerative Medicine Fondazione Ospedale Maggiore Policlinico Milan, Italy

2 Introduction Il sangue di cordone ombelicale viene usato come fonte alternativa di cellule staminali al trapianto di midollo osseo e più di 4000 trapianti sono stati effettuati in tutto il mondo durante gli ultimi dieci anni soprattutto in pazienti (80% pazienti pediatrici) affetti da patologie oncoematologiche

3 Goals of the Milano Cord Blood Bank Banking for unrelated transplantation Banking for related transplantation

4 Development of Milano Cord Blood Bank February 1993: first unrelated collection September 1993: first related collection September 1994: trained midwives start collection July 1997: ISO 9002 certification January 2004: FACT inspection March 31, 2007: unrelated related units cryopreserved and available for clinical use

5 CORD BLOOD COLLECTION

6 CB Collection Procedure Cord blood is collected by: Trained midwives or bank s personnel After vaginal or cesarean deliveries by a closed collection system 1 Vaginal delivery: Collection is performed with placenta in utero Cesarean delivery: Collection is performed after expulsion of the placenta, which is held at its edge and suspended with the fetal aspect and the cord down 3 2

7 Delivery suites collecting cord blood for the Milano Cord Blood Bank MCBB 13 2 MI-Vittore Buzzi MI-Mangiagalli CO-S.Anna CO-Valduce BS- Poliambulanza Cernusco Melegnano Melzo Sondrio Desenzano Manerbio Cavalese Riva del Garda Sondalo Varese

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10 PLACENTAL BLOOD UNIT

11 Scoperta delle cellule staminali nel sangue di cordone ombelicale (CB) Primo trapianto di CB Gluckman et al.: Hematopoietic reconstitution of a patient with Fanconi anemia by means of umbilical cord blood from an HLA-identical sibling. NEJM 1989;321:1174 Kurtzberg et al.: Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. NEJM 1996;335:157 Nakahata & Ogawa: Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors. J Clin Invest 1982;70:1324 Broxmeyer et al.: Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. PNAS USA 1989;86:3828

12 Le cellule staminali da CB sono più naïve e pertanto l attecchimento di globuli bianchi e di piastrine è più lento esponendo il paziente immunodepresso a maggiori rischi Caratteristiche biologiche delle cellule staminali del CB verso il midollo osseo (BM) CB contiene una frequenza maggiore di cellule staminali. Le cellule staminali da CB hanno un maggiore potenziale di proliferazione e di espansione. Nonostante una dose cellulare minore, le cellule staminali da CB attecchiscono e sostengono l ematopoiesi in vivo.

13 Donazione di CB: criteri di esclusione Storia familiare di malattie ereditarie Fattori di rischio nei genitori Sierologia positiva nei genitori Parto pre-termine (<34 settimane) Anormalità congenite Stress fetale

14 Final product standard (1) Assenza dei criteri di esclusione Consenso informato firmato Volume >60 ml e WBC >10 x 10 9 Congelamento entro le 36 ore dalla raccolta Negatività per HBsAg, TPHA, anti- HIV 1,2 anti-hcv, anti HTLV I-II (sul siero della madre)

15 Final product standard (2) Determinazione di: ABO e Rh HLA-A, B, DRB1 (unità e madre) Valutazione del potenziale clonogenico Anti-CMV e toxotest (sul siero della madre) Contaminazione batterica Disponibilità della documentazione relativa alla criopreservazione (-135 C or minore) Re-test del sangue materno a 6 mesi dal parto

16 DONOR EVALUATION

17 Exclusion criteria Family history of inherited diseases Risk factors in the parents Positive serology of the parents Pre-term delivery (<34 weeks) Congenital abnormalities Fetal distress

18 DONOR CONSENT

19 Mother s informed consent is requested in regard to: Collecting cord blood Banking the cord blood unit Using the cord blood for unrelated transplantation Performing serology tests Being available for a 6 months check Storing data in the registry Storing samples

20 MICB FACILITY

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22 LABORATORIO RICERCA DI UNITA COMPATIBILI E CONTROLLO DEI DATI

23 LABORATORIO PROCESSAMENTO DEL SANGUE PLACENTARE

24 LABORATORIO DI CRIOCONSERVAZIONE

25 SOFTWARE DI CONTROLLO DELLA SALA DI CRIOCONSERVAZIONE

26 QUADRO DI GESTIONE E DI CONTROLLO

27 THE BANKING PROCESS

28 ACCETTAZIONE DELLA SACCA DI SANGUE PLACENTARE

29 SOFTWARE DI GESTIONE DELLE UNITA DI SANGUE PLACENTARE

30 DETERMINAZIONE DEL VOLUME DI SANGUE RACCOLTO

31 CONSERVAZIONE DELLA SACCA DURANTE LE FASI DEL PROCESSAMENTO

32 UNIT CHARACTERIZATION

33 Characterization (1) Informed consent Clinical history of the mother, the father and their families Volume > 60 ml NC count before volume reduction >1000x10 6 Volume reduction: bottom-and-top procedure NC count after volume reduction >800x10 6 Cryopreservation: volume to volume final DMSO 10% Check 6 months after delivery

34 Characterization (2) ABO and Rh (unit) Serological (class I) and genomic HLA (class I-II) on unit and maternal sample Contamination with maternal DNA evaluation (upon request) Clonogenic potential: CFU-GM, CFU-GEMM, BFU-E, CFC colonies CD34+ cell count Nucleated red cell count Hemoglobin screening for thalassemia and hemoglobinopathies Sterility test: aerobic and anaerobic bacteria and antibiogram

35 Characterization (3) Serological screening on maternal serum collected at delivery: HBsAg, anti-hiv 1-2, anti-hcv, HCV-RNA, HIV- RNA, TPHA, ALT, anti-htlv I-II, anti-cmv, anti- Toxoplasma, anti-hbc Serological screening on maternal serum collected 6 months after delivery: HBsAg, anti-hiv 1-2, anti-hcv, TPHA, ALT, anti- HTLV I-II, anti-cmv, anti-toxoplasma Serological screening on the unit: HBsAg, anti-hiv 1-2, anti-hcv, anti-htlv I-II CMV-DNA on the unit (if anti-cmv IgM positive)

36 Characterization (4) Quality control on a thawed sample from a unit s segment cryopreserved and stored : Viability Nucleated cell count Clonogenic potential: CFU-GM, CFU- GEMM, BFU-E, CFC colonies Sterility test: aerobic and anaerobic bacteria and antibiogram HLA-A,B,DRB1 confirmatory typing

37 MANIPOLAZIONE DELLA SACCA SOTTO CAPPA A FLUSSO LAMINARE

38 CENTRIFUGAZIONE DELLE SACCHE

39 SEPARAZIONE DEL BUFFY-COAT

40 CONTROLLO DELL IDENTITA DELLA SACCA TRAMITE BARCODE

41 ALLESTIMENTO DELLE PROVETTE E DELLE SACCHE PER L ESECUZIONE DEI TEST

42 PREPARAZIONE DEI CAMPIONI PER LA CARATTERIZZAZIONE DELL UNITA

43 PREPARAZIONE DELLA SOLUZIONE DI CONGELAMENTO

44 AGGIUNTA DELLA SOLUZIONE DI CONGELAMENTO

45 TRASFERIMENTO NELLE SACCHE DI CONGELAMENTO

46 SALDATURA DEI COLLARINI PER CONTROLLO DI QUALITA

47 UNITA CONGELATA IN 3 SACCHE CORREDATE DI CAMPIONI

48 CONGELAMENTO IN CONGELATORE A DISCESA PROGRAMMATA

49 DOCUMENTAZIONE DELLA CURVA DI CONGELAMENTO

50 SALA CRIOBIOLOGICA

51 CONSERVAZIONE IN AZOTO LIQUIDO

52 VALUTAZIONE DEL POTENZIALE CLONOGENICO INCUBAZIONE DELLE PIASTRE

53 VALUTAZIONE DEL POTENZIALE CLONOGENICO LETTURA DELLE COLONIE AL MICROSCOPIO

54 DATA MANAGEMENT

55 SELEZIONE DI UNITA COMPATIBILI E PRODUZIONE DEL REPORT

56 VALIDAZIONE DELLE UNITA

57 UNIT TRANSPORTATION

58 PREPARAZIONE DELL UNITA DA SPEDIRE

59 LE SACCHE VENGONO POSTE NEL DRY SHIPPER

60 APPLICAZIONI CLINICHE

61 Milano Cord Blood Bank 309 Unrelated Units released till

62 CORD BLOOD TRANSPLANTATIONS

63 WHY EXPANDING? Cell number too small for larger adults expand early progenitors long-term hematopoiesis Prolonged tempo to platelet engraftment expand more mature progenitors short term engraftment

64 LABORATORY STUDIES: EX VIVO EXPANSION (1) Extensive expansion and maintenance of hematopoietic progenitor cells in the presence of TPO, FLT-3, IL-6, SCF and their engraftment potential in NOD/SCID mice (BJH 2001) Cryopreservation and thawing do not modify ex-vivo expansion of cord blood stem cells (BMT 2001)

65 LABORATORY STUDIES: EX VIVO EXPANSION (2) Comparison of serum-free media (Transfusion, 2001) Repopulation in secondary and tertiary recipients indicates stability of transgene expression and long-term self-renewal potential of transduced HSCs, suggesting that retroviral gene transfer into HSCs, followed by ex-vivo expansion, could facilitate longterm engraftment of genetically modified HSCs (Stem Cells, 2006)

66 CLINICAL GRADE CELL MANIPULATION Process description from idea to transplant 1. Laboratory studies Purification Culture Characterization 2. Animal studies Ex vivo expansion 3. Regulatory issues Facilities Staff training Quality control Approval by regulatory body Translation and validation Transplant Year

67 EX-VIVO EXPANSION OF HEMOPOIETIC PROGENITORS FROM CORD BLOOD FOR ALLOGENEIC HEMOPOIETIC TRANSPLANTATION IN PEDIATRIC RECIPIENTS Participants Cell Factory, MTC, Fondazione Osp. Maggiore, Milano Clinica Pediatrica, IRCCS Policlinico S. Matteo, Pavia Clinica Pediatrica, Università degli Studi, Torino Divisione Universitaria di Oncologia ed Ematologia, Ordine Mauriziano, Istituto per la Ricerca e la Cura del Cancro, Candiolo (Torino)

68 Primary aims 1.To evaluate the safety of ex-vivo expanded CB cells 2.To evaluate the influence of ex-vivo expanded CB cells on the engraftment kinetics AIMS Secondary aims 1.To evaluate the influence of ex-vivo expanded cells on the immunologic reconstitution, on the probabilty to develop GvHD and GvHL 2.To evaluate long term hemopoietic engraftment after ex-vivo expansion

69 EX VIVO EXPANSION PROTOCOL (1) For the selection of CD34+ cells from small volume thawed CB samples, we adapted and validated the use of a closed, clinicalgrade automated device (CliniMACS, Miltenyi Biotech).

70 EX VIVO EXPANSION PROTOCOL (2) CD34+ cells were cultured at a concentration of 5x10 3 cells/ml in the presence of a serum-free medium (CellGro SCGM, CellGenix), IL-6 (10ng/ml), SCF (50ng/ml), Flt3 (50ng/ml) and TPO (10ng/ml) and 10% AB allogeneic plasma, for 2 weeks. The expansion was carried out in our GMP class B facility

71 PATIENTS Patient #1 A 6-year old boy with beta-thalassemia was given 2 cord blood units derived from 2 HLA-identical fraternal (i.e. non syngeneic) twins (1 unmanipulated CB and 1 CB after ex vivo expansion) Patient #2 A 7-year old girl with sickle cell disease was given 2/3 of the cord blood unit derived from an HLA-identical sibling unmanipulated and 1/3 after ex vivo expansion Patient #3 A 8-year old boy with myelodisplastic syndrome was given 2/3 of the one-mismatch HLA unrelated cord blood unit unmanipulated and 1/3 after ex vivo expansion

72 Ex vivo expansion: patient #1 Pre-thawing: NC 168.8x10e6, CD34+ cells 0.96x10e6 Post selection: CD34+ cells 0.36x10e6, purity 88% Post expansion: Vitality 97% NC 124.6x10e6. Fold expansion: 623 CD34+ cells 9.97x10e6 Fold expansion: 56.6 CD133+ cells 9.06x10e6 CD19+ cells 0.007x10e6 NK+ cells 0.007x10e6 CD x10e6 CD61+ cells 1.4x10e6

73 Clinical evaluations: patient #1 Neutrophil and platelet (>20,000/mL) recovery occurred on day +16 and +32, (median time needed for both neutrophil and platelet reconstitution after CB transplant is usually days and days), respectively He did not have GvHD (acute or chronic)

74 Clinical evaluations: patient #1 Through the evaluation of STRs, both the ex vivo expanded and the unmanipulated units contributed to hematopoietic reconstitution. DNA derived from the ex vivo expanded unit was found also in peripheral blood, in CD4-enriched lymphocytes and in CD19- enriched B cells. The patient is alive and well at +27 months

75 Ex vivo expansion: patient #2 Pre-thawing: NC 376.7x10e6, CD34+ cells 2.06x10e6 Post selection: CD34+ cells 0.59x10e6, purity 67% Post expansion: Vitality 96.5% NC 125x10e6. Fold expansion: CD34+ cells 13.5x10e6 Fold expansion: 30.5 CD133+ cells 10.1x10e6 CD19+ cells 0.008x10e6 NK+ cells 0.075x10e6 CD x10e6 CD61+ cells 1.35x10e6

76 Comparison of CD34+ cell content with and without expansion 20 Patients #2 15 without expansion with expansion x10e CD34+ cells

77 Clinical evaluations: patient #2 Neutrophil and platelet (>20,000/mL) recovery occurred on day +18 and +24, respectively She did not have acute GvHD and she did not experience fever Through the evaluation of STRs, more than 90% of the circulating leukocytes are of donor origin The patient is alive and well at +20 months

78 Ex vivo expansion: patient #3 Pre-thawing: NC 358.1x10e6, CD34+ cells 0.82x10e6 Post selection: CD34+ cells 0.23x10e6, purity 22% Post expansion: Vitality 91% NC 58.3x10e6 CD34+ cells 11.6x10e6 Fold expansion: 56.6 CD133+ cells 10.4x10e6 CD19+ cells 0.005x10e6 NK+ cells 0.029x10e6 CD x10e6 CD61+ cells 2.67x10e6

79 Comparison of CD34+ cell content with and without expansion 20 Patient #3 without expansion 15 with expansion x10e CD34+ cells

80 Clinical evaluations: patient #3 Neutrophil and platelet (>20,000/mL) recovery occurred on day +29 and +45, respectively He had grade II agvhd and limited cgvhd of the oral cavity The patient is alive and well, with full donor chimerism, at +17 months

81 CONCLUSIONS Clinical grade GMP expansion of CB progenitor cells is feasible and safe. The expanded cells are able to contribute to long-term hematopoietic reconstitution. The method herein described is now open to adult patients in the context of a phase I/II clinical trial.

82 THANKS Cell Factory - Rosaria Giordano - Tiziana Montemurro - Elisa Montelatici - Gabriella Andriolo - Viviana Lo Cicero - Valentina Parazzi - Francesca Chelli Milano Cord Blood Bank - Lucilla Lecchi - Ilaria Ratti - Silvia Giovanelli - Mina Garcea - Manuela Brasca - Paolo Rebulla

83 THANKS Department of Regenerative Medicine

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85 Banking process at the Milano Cord Blood Bank (1)

86 Banking process at the Milano Cord Blood Bank (2) 8 9 Quality control on the thawed segment

87 Transportation (non-frozen) Methods shall be designed to protect the integrity of the unit and the safety of facility personnel Sealed secondary plastic bag Shipping container shall be made of material adequate; and shall be carry specific labels Transport records shall permit the tracing of CB unit from the collection facility to its final destination Transport records shall document: the identify of responsible for shipping, date and time of shipping and receipt of the unit Shipping list identifying each unit enclosed Transport records shall be maintained indefinitely 1 2 Milano Cord Blood Bank 2004

88 Acceptance Criteria Absence of exclusion criteria Written informed consent Collection < 36 hours Volume > 60 ml NC > 1000x10 6

89 Volume reduction Bottom-and-Top procedure Buffy-coats collected from cord blood units fractionated in Bottom-and-Top Compoflex bags processed with Compomat G4 (NPBI)

90 Freezing solution addition 3 freezing bags with segments 1 2 Freezing solution: Dextran 80% DMSO 20% (final DMSO 10%)

91 Cryopreservation Controlled rate freezer Quarantine 2 1

92 Donor evaluation 6 months after delivery

93 6 months check program (1) A bank volunteer calls the mother who donated cord blood 2 months before, to remind her of the retesting scheduled at 6 months who donated cord blood 6 months before, to schedule her visit at the Cord Blood Bank or at her delivery suite

94 6 months check program (2) Serology is repeated on a fresh mother s blood sample Mother s serum is tested for HBsAg, anti-hiv 1-2, anti-hcv, syphilis, ALT, HTLV I-II, anti-cmv, anti-toxoplasma The blood samples are frozen and stored A bank physician interviews the mothers to check their medical history form collected at donation and to collect the baby s medical history

95 6 months check program (3) The newborn s medical history and health status at 6 months are collected through mother s interview according to a protocol to identify: inherited diseases blood-borne diseases untoward effects of cord blood collection Routine pediatric assistance is accessible to mothers through the National Health Service

96 6 months check program (4) The bank sends the results of serology to the mother If checks are uneventful, the unit exits quarantine and can be released If the 6 months check cannot be carried out, the unit is flagged with this information If the unit is selected, the requesting physician is informed

97 Results of check at 6 months Of the 2315 mothers with negative serology at delivery, who attended the 6 months check, 1 showed indeterminate anti-hcv seroconversion (c22). HCV-RNA determination negative by RT-PCR

98 Local Data Management A software was developed in house with Microsoft Visual Basic and Oracle to perform: Data storage and management Search of compatible units Patient s follow up

99 Before Release

100 Before release Quality Control on the thawed segment: Viability (trypan blue) Nucleated cells count CFU-GM Aerobic and anaerobic bacterial contamination Confirmatory typing Review of the records Mother s HLA typing

101 Unit Transport

102 Transport of Cryopreserved Units Transport in a dry shipper Temperature below 135 C Temperature monitoring device Documentation to avoid X- ray control 2 1

103 Unit Thawing

104 Process Management How to ensure that the frozen product we release is the same product as the one we have collected and characterized? HLA type cord blood unit newborn s mother

105 Cord Blood Bank Facility Adequate space Adequate equipment Adequate staff Job description Training Competence evaluation

106 Cord Blood Bank Cryobiology Area Security Monitoring and alarm system Frozen storage at below 135 C Quarantine storage Written disaster procedure