Michael Y. Shapira

1. An investigator initiated, prospective, randomized, controlled study in order to evaluate the effect of ATG (Fresenius) on engraftment, GVHD and GVL effect in haplo-identical allogeneic SCT Michael Y. Shapira

2. Introduction • This is a prospective phase III randomized, control trial assessing the effect and need for polyclonal rabbit anti-T lymphocyte antibodies (Fresenius ATG) administered during the conditioning of allogeneic haploidentical stem cell transplantation by examining its effect on engraftment, GVHD and GVL effect.

3. Rationale • The rationale for this protocol is based on the need to optimize current induction treatment of haploidentical allogeneic stem cell transplantation (haplo-SCT) in order to improve the outcome. • Data from myeloablative protocols suggest that ATG before SCT significantly reduces the risk for grade III-IV aGVHD. This does not translate to a reduction in TRM due to increased infections risk and thus survival is unchanged.

4. Rationale • The incidence of extensive chronic GVHD was also shown to be significantly reduced in patients receiving ATG in the myeloablative setting. • Since ATG has a mean elimination half-life of 29.8 days, effective levels may stay in patients’ blood for up to 8 weeks post SCT.

5. Rationale • As ATG is non-specific, it kill graft’s T-cells as well as NK cells. This may impair engraftment and GVL as in haplo-SCT NK recovery is crucial for GVL induction. Enhanced immunosuppression may also increase the incidence of post-transplant lymphoproliferative disease or other malignancies. On the other hand, ATG may suppress graft’s residual T-cells and prevent haplo GVHD. Thus the role ATG in haplo-SCT is questionable. Most centers do use ATG, while few don’t.

6. Rationale • The only way to determine if the inclusion of ATG to haplo-SCT induction protocol results in the best outcome is to conduct a randomized prospectively controlled study. • The study is aimed at to evaluate the effect of Fresenius ATG on engraftment, GVHD and GVL effect in haplo-SCT.

7. Primary objectives To assess the effect of Fresenius ATG on the engraftment following haplo-SCT: • Day of day of neutrophil engraftment (ANC>0.5x109/L) • Day of platelet engraftment >20x109/L.

8. Secondary objectives • Acute GVHD occurrence. • Time to acute GVHD. • GVHD grade. • Disease free survival at 100 days. • Day of platelet engraftment >50x109/L. • Overall survival at 100 days. • Infections incidence. • Transplant-related mortality (TRM). • Transplant-related toxicity (TRT).

9. Methods • 50 patients with mismatched (3/6-4/6 HLA matching) donors will be included. Patients will be randomized for two groups: Patients who will get rabbit anti-human T lymphocyte globulin (Fresenius ATG) as a part of pre-transplant conditioning (during the last 4 days of conditioning (day-4 to day-1)). Patients who will not get ATG as a part of pre-transplant conditioning.

10. Inclusion criteria • Patient age 3-70 years old with a disease necessitating allogeneic SCT. • Patients must have a mismatched donor willing and capable of donating peripheral blood stem cells and/or bone marrow progenitor cells using conventional techniques, and lymphocytes if indicated (mismatched defined as 3/6-4/6 HLA matching). • Each patient / patient's guardian must sign written informed consent.

11. Inclusion criteria • Patients must have an ECOG PS ≤ 2; Creatinine <2.0 mg/dl; Ejection fraction >40%; DLCO >50% of predicted; Serum bilirubin <3 gm/dl; elevated GPT or GOT >3 x normal values.

12. Exclusion criteria • Not fulfilling any of the inclusion criteria. • Active life-threatening infection. • Overt untreated infection. • Known hypersensitivity to ATG. • HIV seropositivity, Hepatitis B or C antigen positivity with evidence of active hepatitis. • Pregnant or lactating women.

13. Exclusion criteria • Donor contraindication (HIV seropositive confirmed by Western Blot Hepatitis B antigenemia; positive HCV antibodies with positive HCV PCR; evidence of bone marrow disease; unable to donate bone marrow or peripheral blood due to concurrent medical condition). • Inability to comply with study requirements.

14. Conditioning protocol • All patients will be prepared by the haplo-SCT protocol, based upon the following elements: • I.V. fludarabine 30mg/m2/day (on days -9 to -4); I.V. cyclophosphamide 60mg/kg/d (on days –8 to –7), with or without I.V. ATG according to randomization group (on days –4 to –1). ATG dose: will be a cumulative dose of 40 mg/kg ATG (10 mg/kg/day x 4 days). Each day’s infusion time will be 8 hours.

17. T-cell depletion • Separation of CD34+ stem cells is done using CliniMACS (CliniMACS Plus). • OR alternatively, TCD of the stem cell product (PBSC or BM) will be performed by incubation with 1 mcg MabCampath per 106 cells for 30 minutes at room temperature under continuous agitation followed by washing.

18. GVHD prophylaxis • No pharmacological GVHD prophylaxis will be given other then TCD unless quality control of the graft(s) shows that T-cell content is above 105/kg and then IV 3 mg/kg CSA will be initiated. • Immediately upon the appearance of signs and symptoms of GVHD, i.v. methylprednisolone (2 mg/kg) and cyclosporine will be administered.

19. Donor • An attempt to identify and select a donor with killer immunoglobulin-like receptor (KIR) ligand incompatibility in the graft-versus-host direction will be done. • If possible/relevant the mother should be used for donation.

20. Follow-up • Physical exam, CBC and biochemistry, toxicity grading: day 0, 7, 14, 21, 30, 100. • Chimerism: days 30, 60, 100. • GVHD: days 14, 21, 30, then monthly until 100d. • Disease staging: day 100. • Immune reconstitution (by FACS): days 30, 100.