Towards a novel immune therapy platform with an. innate allogeneic haematopoietic stem cell transplantation

Transcription

1 Towards a novel immune therapy platform with an innate allogeneic haematopoietic stem cell transplantation in patients with haematological malignancies. Protocol: Innate allo-sct; Version number 6; date 13 August

2 1. Scheme of study Protocol: Innate allo-sct; Version number 6; date 13 August

3 Investigators and study administrative structure Responsibility Name Affiliation Address Sponsor UMC Utrecht Heidelberglaan CX Utrecht Principal investigator J. Kuball, MD UMC Utrecht KC Professor Department of Hematology Department of immunology Lundlaan EA Utrecht t: +31(0) f: +31(0) Co investigators M.A. de Witte, MD, PhD UMC Utrecht L. van de Wagen, MD Processing of I.C.M. Slaper-Cortenbach UMC Utrecht cellular products Cell Therapy Facility Protocol: Innate allo-sct; Version number 6; date 13 August

4 TABLE OF CONTENTS 1. Scheme of study Synopsis Introduction and rationale T cell depleted versus T cell repleted allo-sct The (potential) role of NK cells after allo-sct The (potential) role of γδt-cells after allo-sct Experience in HLA-haploidentical hematopoietic stem cell transplantation (haplo-hsct) Experience in patients with related and unrelated donors applied after non-myeloablative regimens Comparison of different transplantation regimens Considerations ATG Study objectives Primary endpoint: Secondary Endpoints: Study Design Study Population Inclusion criteria Exclusion criteria Treatment of subjects Time to transplant Conditioning regimen before HLA-identical Sibling or matched unrelated donor stem cell transplantation Donor selection Stem cell collection Protocol: Innate allo-sct; Version number 6; date 13 August

5 7.5 HPC(A) products Management of graft failure GVHD prophylaxis Treatment of GVHD Infection prophylaxis Required clinical investigations Required clinical investigations at entry (pre-transplant evaluation) and post transplantation Study procedures Follow-up of primary end points Follow-up of secondary end points Transplant related mortality / Progression free survival/ Overall survival Graft failure: Engraftment Levels of ATG prior and post SCT Clinical parameters Immune reconstitution Donor cells after αβ T cell depletion End of protocol treatments Withdrawal of individual subjects Replacement of individual subjects after withdrawal Other reasons for going off protocol Premature termination/alteration of the study: Safety reporting Section 10 WMO event Protocol: Innate allo-sct; Version number 6; date 13 August

6 10.2 Adverse and serious adverse events Follow-up of adverse events Data Safety and Monitoring Board Data collection CRF s Monitoring Statistical considerations Patient numbers and power considerations Interim analysis ETHICAL CONSIDERATIONS Regulation statement Recruitment and consent Benefits and risks assessment Burden: Risks: Benefits: Compensation for injury ADMINISTRATIVE ASPECTS AND PUBLICATION Handling and storage of data and documents Storage of samples Amendments Annual progress report End of study report Public disclosure and publication policy REFERENCE LIST Protocol: Innate allo-sct; Version number 6; date 13 August

7 APPENDIX A. ZUBROD-ECOG-WHO Performance Status Scale B. NYHA* scoring list C. Definitions of recovery, engraftment, chimerism and relapse D. Grading of GVHD E. Toxicity criteria F G. Product information Cell Therapy Product Protocol: Innate allo-sct; Version number 6; date 13 August

8 Protocol ID Innate allo-sct 2 Short title Principal investigator(s) (in Dutch: hoofdonderzoeker/uitvoerder) Independent physician(s) Laboratory sites Immunological monitoring Innate allo-sct Prof. Dr. J. Kuball Head of Dept. of Hematology, UMC Utrecht Dept. of Hematology, UMC Utrecht Heidelberglaan CX Utrecht Dr. P. Witteveen Dept. of Oncology, UMC Utrecht Heidelberglaan CX Utrecht Prof. Dr. J. Kuball Laboratory of Translational immunology Dept. of Hematology, UMC Utrecht Heidelberglaan CX Utrecht PK analyses Cell processing: Dr. J.J. Boelens & Dr. S. Nierkens Laboratory of Translational immunology WKZ & DLA, UMC Utrecht Heidelberglaan CX Utrecht I.C.M. Slaper-Cortenbach, PhD Head of the Cell Therapy Facility (CTF) UMC Utrecht Department of Pharmacology Heidelberglaan CX Utrecht Protocol: Innate allo-sct; Version number 6; date 13 August

9 PROTOCOL SIGNATURE SHEET Name Signature Date Sponsor or legal representative: Prof. Dr. J. Kuball hematologist Head of Dept. of Hematology, UMC Utrecht Coordinating Co- Investigators: Dr. M.A. de Witte Drs. L. van de Wagen Dept. of Hematology, UMC Utrecht Protocol: Innate allo-sct; Version number 6; date 13 August

10 LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS AE ATG agvhd Allo-SCT ALL AML CCMO CLL CML CMV CV cgvhd DLI DSMB EBMT EBV GVHD GVL HL HPC (A) IC IMP KIR MDS MM NHL NK cells NFU OS PD PK PLT PMN SAE Sponsor Adverse Event Anti-thymocyte globulin Acute graft versus host disease Allogeneic hematopoietic stem cell transplantation Acute lymphoblastic leukemia Acute myeloid leukemia Central Committee on Research Involving Human Subjects Chronic lymphoid leukemia Chronic myeloid leukemia Cytomegalovirus Curriculum Vitae Chronic graft versus host disease Donor lymphocyte infusion Data Safety Monitoring Board European group for Blood and Bone Marrow Transplantation Epstein Barr virus Graft versus host disease Graft versus leukemia effect Hodgkin lymphoma Hematopoietic progenitor cell apheresis Informed Consent Investigational Medicinal Product Killing inhibitor receptor Myelodysplastic Syndrome Multiple myeloma Non Hodgkin Lymphoma Natural Killer cells Nederlandse Federatie van Universitair Medische Centra Overall survival Pharmacodynamic Pharmacokinetic Platelets Polymorphonuclear neutrophils Serious Adverse Event The sponsor is the party that commissions the organization or performance of the research, for example a pharmaceutical company, academic hospital, scientific organization or investigator. A party that provides funding for a study but does not commission it is not regarded as the sponsor, but referred to as a subsidizing party. Protocol: Innate allo-sct; Version number 6; date 13 August

11 SUSAR TCR TRM WMO Suspected Unexpected Serious Adverse Reaction T cell receptor Treatment related mortality Medical Research Involving Human Subjects Act. In Dutch Wet Medischwetenschappelijk Onderzoek met Mensen Protocol: Innate allo-sct; Version number 6; date 13 August

12 2. Synopsis Rationale: Allogeneic stem cell transplantation (allo-sct) is still the treatment of choice for many patients suffering from haematological malignancies which can only occasionally be cured with conventional chemotherapy. Donor T cells contribute strongly to the beneficial effect of allo-sct due to a potent graft versus leukaemia effect after transplantation, however they also cause severe and life-threatening GVHD. Also relapses are frequently observed after allo-sct. Recent reports have shown that the innate immune system can contribute to tumor control, whereas the development of GVHD appears to be lower as has been observed after conventional allo-sct. Depletion of T-cells prior to allo-sct may be a valuable tool of discarding the potentially harmful T cells, but leaving the potential beneficial innate immune system present within the graft. This would theoretically allow protecting patients from infections and relapse shortly after allo-sct without inducing GVHD. As such, more patients might become eligible to additional immune interventions after allo-sct. Primary Objective: To implement an T-cell depleted stem cell transplantation as low- GVHD platform in order to increase the number of patients eligible for further immune interventions from day 100. Secondary Objective: To address all other clinical transplantation-related parameters and immunological reconstitution after an T-cell depleted stem cell transplantation. Study design: Phase II study Study population: Patients with haematological malignancies eligible for allogeneic stem cell transplantation. Intervention: Reduced toxicity myeloablative conditioning regime, T-cell depleted stem cell graft. Main study parameters/endpoints: Primary Endpoint: Incidence of agvhd at day 100. Protocol: Innate allo-sct; Version number 6; date 13 August

13 Secondary Endpoint: Treatment related mortality (TRM) at day 100 Donor engraftment (chimerism > 95%) at day 100 Nature and extent of the burden and risks associated with participation, benefit and group relatedness: The protocol comprises a different processing of the donor stem cells source, which has been used successfully for haplo-identical allo-sct 1,2. The conditioning regimen is also different from the standard conditioning regimen, but has been extensively used by others 3,4. Patients need to use a limited dose of immunosuppressive agents post allo-sct. All other acts, measurements, follow-up and level of care are similar to off-study patients undergoing allo-sct. The burden of the therapy is associated with the allo-sct itself which is a necessary therapeutic intervention in all subjects. Possible increased risks for the recipient are an increased toxicity of the conditioning, an increased chance of impaired engraftment due to TCR depletion and an increased chance of infections. Possible benefits include a proposed lower chance of acute and chronic GvHD (cgvhd) because of T cell depletion and the lack of side effects of immunosuppressive drugs. Planned interim analysis and DSMB: An interim analysis will be performed after 20 patients have been registered and data are available for 100 days. A DSMB will be installed which will advise the investigators about (dis)continuation of the trial after the interim analysis. 3. Introduction and rationale Allogeneic stem cell transplantation (allo-sct) has so far been the most effective immunotherapy for hematological malignancies. However, the transplant related mortality caused by GVHD and infections is still reported up to 40 %. In addition depending on the underlying disease also relapses frequently occur. Protocol: Innate allo-sct; Version number 6; date 13 August

14 In order to systematically design studies to improve the outcome of allo-sct, the process of an allo-sct can be divided in 5 components (figure 1A). In this study we propose T cell depletion as a novel way of stem cell manipulation in either matched related or matched unrelated donors (see below) in order to guide more patients to early immune interventions shortly after allo-sct. The primary gain of changing this component within a transplantation regimen can therefore be assessed at day 100, by determining the incidence of agvhd > grade III. Patients with agvhd I and II are in principle considered as eligible for later immune interventions. The safety of this approach can be determined by the TRM, incidence of infections and numbers of graft failures at day 100. Figure 1: A: Component model: An allogeneic stem cell transplantation can be divided in 5 components, of which one or several can be modified and studied separately. B: Proposed endpoints when T cell depletion is chosen as modification to be studied. The rationale behind T cell depletion is that innate-like cells, which include natural killer (NK) cells and γδt-cells, could provide unique advantages to therapeutic interventions aimed at treating hematological malignancies, including early protection against tumor relapse and viral infections without causing harmful graft-versus-host disease (GVHD). Recent molecular and conceptual insights into these subpopulations have opened new avenues to exploit their Protocol: Innate allo-sct; Version number 6; date 13 August

15 exciting features for the development of new compounds and to revisit current therapeutic standards in the treatment of hematological cancers. In this study, we aim to perform an allogeneic stem cell transplantation (allo-sct) by applying an T-cell depleted stem cell graft. Specific removal of -T cells maintains innate cells in the stem cell graft. By removal of T cells we expect to reduce the incidence of agvhd. By retaining innate cells, we might maintain some early control of malignant as well as virally infected cells. We aim to establish this strategy as a novel platform to allow immune interventions early after allo-sct, such as conventional or modified DLI, antigen presenting cell (APC) vaccination or transfer of genetically modified T cells. Requirements for such a platform is that it is effective (defined as low chance of agvhd > grade III) and that it is safe (e.g. low TRM and limited numbers of graft failures). All these aspects will be addressed as primary or secondary endpoints in this study. 3.1 T cell depleted versus T cell repleted allo-sct Since the first allo-sct was conducted more than half a century ago, allo-sct is still the preferred treatment option for many patients with poor-risk hematological malignancies 5,6. Even though to date allo-sct is the most successful form of adoptive immunotherapy in this category of patients, allo-sct is also potentially the most detrimental, as the outcome is still substantially hampered by potentially life-threatening GVHD and relapse of the tumor. In addition, an incomplete immune reconstitution early after transplantation and immunosuppressive therapy to prevent or control GVHD renders allo-sct patients susceptible to viral infections 5. Donor T cells are considered to be one of the key players in determining the outcome of allo- SCT, since on-target recognition of malignant cells will result in a Graft-versus-Leukemia (GVL) effect, whereas off-target recognition will result in GVHD 5. Immunotherapeutic concepts to exploit the GVL effect of allo-sct without inducing too much toxicity can be roughly divided in two fundamentally different approaches. Patients can receive a full Protocol: Innate allo-sct; Version number 6; date 13 August

16 allogeneic graft, hence including -T cells and innate T cells. Its immunological effect (GVHD and GVL) is modulated by the administration of (nonspecific) immunosuppressive drugs such as cyclosporine, mycofenolic acid, or high dose cyclophosphamide directly post transplantation. Alternatively, patients can receive a T-cell depleted graft. This aims to diminish toxicity early after allo-sct by specifically reducing harmful T-cells and to enhance the GVL effect of the SCT by DLI at later time points, once inflammation due to conditioning is ceased and a more profound immunological response is warranted. Within the past two decades, a considerable amount of studies have shown the feasibility of T-cell depleted SCT in combination with DLI 6,7. A major breakthrough has been achieved recently by Pasquine and colleagues who demonstrated in a prospective and randomized phase III clinical trial that T cell depletion via enrichment of the CD34 + cells lowers long-term morbidity as a result of a substantially reduced chronic GVHD without negatively impacting relapse rates in patients with acute myeloid leukemia 8. These and other studies led to a recent approval of the FDA to use CD34 enrichment procedure by using the Miltenyi Biotec CliniMACS system for prevention of GVHD as a routine procedure for the isolation of hematopoietic progenitor cells for allo-sct. However, acute GVHD has still been observed at a high rate despite a profound T-cell depletion. This may be due to the myeloablative conditioning regimen including approximately 12 Gy whole body irradiation. Nowadays, combinations of intravenous busulfan with cyclophosphamide or fludarabine replace such toxic regimens in many centers 3,9 11. Selective depletion of αβt-cells has been suggested as a potentially attractive alternative processing of a donor graft 2. This strategy maintains other important, most likely less harmful effectors in the graft, namely NK cells and γδt-cells. NK cells and γδt-cells are innate-like cells that combine characteristics of innate and adaptive immunity 12,13. These cells target alternative classes of antigens on a broad spectrum of tumors and virus-infected cells, while preserving selective recognition to avoid detrimental reactivity toward healthy tissue. Recent Protocol: Innate allo-sct; Version number 6; date 13 August

17 breakthroughs in the understanding of the molecular target recognition mechanisms of these cells, as well as the elucidation of novel links between the cross-recognition of virusinfected cells and cancer cells, 18,19 do not only solve clinical riddles such as an improved leukemia control in allo-sct patients with CMV-reactivations 20,21, but also substantially contribute to the development of novel innate-like immunotherapies to improve clinical outcome of patients with hematological cancers The (potential) role of NK cells after allo-sct The activation of NK cells depends on signaling of both activating and inhibitory receptors that concertedly discriminate healthy from diseased cells by sensing stress-induced cellular changes 22. Major activating receptors on NK cells, recognize pathogen-derived or self stress molecules on diseased target cells, including tumor cells. As an example, NKG2D, by far the best-studied of these receptors, recognizes the stress-induced self-proteins MHC class I related proteins, which surface expression is selectively increased on transformed cells from hematological and solid origin, allowing NK cells to distinguish healthy from aberrant cells through NKG2D 23. In patients treated for haematological malignancies, genetic polymorphisms within these ligands correlate with relapse-free survival 24, demonstrating the relevance of NKG2D ligands to clinical outcome. In addition to activating receptors, NK cells express inhibitory receptors that continuously sense the presence of MHC class I molecules constitutively expressed on virtually all healthy cells 25. Expression of class I MHC molecules may be down-regulated upon viral infection or malignant transformation to escape detection by conventional T-cells. NK cells are capable of sensing this loss of self via reduced signaling through their inhibitory receptors. As an example, inhibitory killer cell immunoglobulin-like receptors (KIRs) detect classical MHC class I molecules, also termed human leukocyte antigens (HLA) -A, -B, and C. The classical MHC molecules are highly polymorphic, and each of the seven identified inhibitory KIRs preferentially recognizes a distinct subset of HLA alleles. Importantly, each individual inherits Protocol: Innate allo-sct; Version number 6; date 13 August

18 a KIR repertoire, or haplotype, with a subset of available KIR alleles, resulting in a wide variability between KIR haplotypes among individuals. Consequently, a hallmark report by Ciccone and colleagues demonstrated that NK cells kill allogeneic cells when their inhibitory KIRs are not engaged due to a mismatch in HLA alleles 26. The impact of NK cell alloreactivity on the outcome of an allogeneic transplantation has been demonstrated by the introduction of haploidentical transplantation protocols (i.e. donor and recipient share one HLA haplotype but are fully mismatched for the other). In haploidentical transplantation, T-cells are frequently depleted from the graft to prevent GVHD caused by the substantial lack of HLA matching 27. This results in a prominence of innate-like cells after allo- SCT which is usually not observed after non-t-cell depleted transplantations, and thus allows studying the impact of a juvenile innate-like immune system on the control of leukemia and infections. Remarkably, patients with a KIR/HLA-mismatched stem cell donor generally develop NK cells alloreactivity against host cells, including leukemic cells, while having a reduced risk of developing GVHD 28, The (potential) role of γδt-cells after allo-sct Similar to NK cells and in agreement with their innate-like character, γδt-cells are implicated in the rapid response to a variety of disease conditions, including malignant transformation, by lysing target cells and secreting high amounts of cytokines such as IFNγ 13. γδt-cells express, like NK cells, activating and inhibitory NK receptors that modulate their activation by aberrant cells 30, although the exact involvement of these receptors in γδt-cell activation remains puzzling. This is in part due to the expression of an additional unique activating immune receptor by γδt-cells, namely the somatically rearranged T-cell receptor (TCR). γδtcells express TCRs composed of a γ and a δ chain, and these γδtcrs can strongly contribute to γδt-cell activation alongside NK receptors. The definition of cognate antigens for γδtcrs is still extremely challenging. The general assumption has so far been that the γδtcr recognizes a variety of molecular stress signals Protocol: Innate allo-sct; Version number 6; date 13 August

19 on infected, transformed or otherwise stressed cells. Thus, in contrast to conventional αßtcrs, γδtcrs do not rely on antigen presentation by classical MHC molecules. Two distinct subsets of γδt-cells have been identified based on tissue localization and associated expression of defined TCRγ and TCRδ chains. In human peripheral blood the predominant γδt-cell subset carries Vγ9Vδ2+ TCRs and comprises 1-5% of circulating T-cells, while γδtcells located in epithelial tissues express mainly Vδ1+ or Vδ3+ chains paired with diverse Vγ chains and represent approximately 50% of all local T-cells 31. Both subsets have shown to react with antigens or ligands, of which an enhanced expression is observed in malignant cells. In addition, γδt-cells can either augment the function of dendritic cells (DCs) or function themselves as a DC 32,33. Important roles for γδt-cells have been described in immunity against viruses, including CMV 34. In both healthy individuals as well as immunocompromised patients including patients after allo-sct, CMV reactivation correlates with increased numbers of circulating Vδ2 neg γδt-cells 19,35. Importantly, subsets of CMV-reactive γδt-cells are capable of crossrecognizing solid 36 as well as hematological 19 cancers, which suggests that these cells could provide protection against both CMV disease and leukemic relapse. Indeed, recent studies in large allo-sct cohorts show a strong favorable association between CMV reactivation after allo-sct and a reduced risk of leukemic relapse 20,21,37, and CMV- and leukemia-crossreactive γδt-cells provide a likely explanation for these puzzling observations 38. In summary, the innate-like NK cell and γδt-cell immune populations are capable of responding rapidly to a wide variety of infections and solid and hematological malignancies, and they have been attributed direct and indirect roles in tumor immunosurveillance and disease. This is mediated by shared but also unique receptors that in part recognize related antigens, reflecting unifying tumor-sensing mechanisms of these innate-like immune cells. The emerging insights into cross-reactivity of innate-like cells to malignancies and viral infection combined with the lack of classical MHC-restriction in the process of antigen Protocol: Innate allo-sct; Version number 6; date 13 August

20 recognition, put NK cells, γδt-cells and their individual receptors in a new spotlight as attractive tools to overcome the obstacles associated with hematological cancers and allo- SCT Experience in HLA-haploidentical hematopoietic stem cell transplantation (haplo- HSCT) Considering the potential beneficial anti-tumor effects of NK and γδt-cells, selective depletion of αβ T-cells could be an attractive approach to dissect GVHD from GVL. The feasibility of such an approach has been showed in recently published 2 or presented 40,41 data in which patients were transplanted with αβ T cell depleted stem cells from haploidentical donors. In the study by Bettiana et al 2, 23 children with nonmalignant disorders received HLA-haploidentical hematopoietic stem cell transplantation (haplo-hsct) after ex vivo elimination of T cells and CD19 + B cells. The median number of transplanted αβt-cells was 4 x 10 4 /kg (range 1-9,5 x 10 4 /kg). No patient received post-transplantation immunosuppression. 4 out of 23 patients showed a graft failure, but could be rescued by a second allograft. No patients developed agvhd > grade III. Cumulative incidence of transplantation-related mortality was 9.3%. T cell depletion has also been applied in children with high risk leukaemia 40,42. The median number of infused αβ T-cells was 14 x 10 3 /kg. Also in these patients, no further post-transplant GVHD prophylaxis was given. In the first centre, all 10 patients reached complete donor chimerism. One patient experienced a transient stage 3 GVHD of the skin which required only topical treatment. No patient experienced chronic GVHD up to 12 months. Three patients relapsed after transplantation, 7 patients are in remission for 5 months (range 3-12). There was no transplant-related death so far. The second centre comprised 13 patients with ALL (9), AML (3) and NHL (1). All children but 1 had relapsed/refractory disease. In particular, 11 patients were transplanted in CR and 2 with active disease. Again, no further post-transplant GVHD prophylaxis was given. All patients engrafted. No patient experienced agvhd > grade II or chronic GVHD. Protocol: Innate allo-sct; Version number 6; date 13 August

21 With a median follow-up of 4 months (range 1-9) 10 patients are alive and disease-free; 2 patients relapsed (1 died) and 1 had fatal lung aspergillosis. Altogether, these data indicate that transplantation of αβtcr-depleted cells from a haploidentical donor is safe and feasible, with a low incidence of both acute and chronic GVHD. The anti-leukemic efficacy of this approach in comparison to other methods of T-cell depletion needs to be evaluated with a longer patient follow-up Experience in patients with related and unrelated donors applied after nonmyeloablative regimens To test whether depletion of αβ T-cells is also feasible for allo-sct from HLA matched related or unrelated donors in adults, we have performed an analyses of transplantations at our center in patients with very unfavorable leukemia s (Table 1). The data set includes a prospective cohort (patient 1-5) performed at a time point when the αβtcr depletion antibody was still not commercially available on the market, and a retrospective analyses from patients who were treated after the αβtcr depletion antibody became commercially available (patient 6-14). Pt no Age Sex Disease Risk profile I Leukemic relapse 2nd SCT* Survival Cause of death Overall survival (months) F AML EVI-1 no CR after first induction cycle 4 months no Death Relapse F MDS EVI-1 n.a. yes Death Infection F AML EVI-1 no CR after first induction cycle no Alive F AML CCA 26 months no Death Relapse M CML blast crise AML, t(9;22) yes Alive 35 II M t( AML) CCA,10% blasts before AlloSCT n.a. no Death MOF sepsis M AML CCA, no CR after first induction cycle 17 months yes Death Relapse M AML t(6;9) 6 months no Death Relapse M AML CCA 4 months no Alive 18 III F AML FLT-ITD+ no CR after first induction cycle 6 months no Death Relapse M B-ALL No CR after induction therapy 6 months no Death Relapse 12 Monosomy 7, no CR after first no Alive M AML induction M B-ALL t(9;22) no Alive 10 Monosomy 7, EVI-1, no CR after first 6 months no Death Relapse F AML induction Table 1: Patient characteristics (I/II/III refer to cohort; see below). CCA=complex cytogenetic Protocol: Innate allo-sct; Version number 6; date 13 August

22 abnormalities; CR = complete remission. MOF = multi organ failure; * 2 nd transplant because of rejection Cohort Conditioning Prophylaxis I Cyclophosphamide 4800mg/m2 ciclosporine Fludarabine 120mg/mg2 II Fludarabine 120mg/mg2 no Busilvex AUC=90 (4 days) III ATG 4mg/kg no Fludarabine 120mg/mg2 Busilvex AUC=90 (4 days) Table 2: Conditioning regimens used In the subsequent cohorts we intensified the conditioning regimen (table 2). The reason for intensifying the conditioning regimen is that with either cyclophosphamide/ fludarabine (cohort I) or fludarabine/busilvex (cohort II) too many graft failures (>20%) were observed (2 out of 5 patients in cohort I and 1 out of 4 patients in cohort II). This prompted us to exploit fludarabine/busilvex and ATG as conditioning regimen, which has been reported to be safe up to 70 years of age 43. Median Range Overall depletion + T cells (log) Transplanted + T cells (x10 3 /kg) Recovery CD34 + cells (%) Infusion CD34 + cells (x10 6 /kg) 6, Table 3 Efficiency depletion (N=14) Protocol: Innate allo-sct; Version number 6; date 13 August

23 As depicted in table 3, using anti-tcr antibodies in combination with magnetic microbeads as described in paragraph 7.5 resulted adequate depletion efficiency of αβ T- cells, while maintaining a sufficient recovery of CD34 + hematopoietic progenitor cells. Table 4 summarizes the outcome of the last cohort of patients. None of these patients experienced serious toxicity within the first 28 days. All patients engrafted and all patients reached a donor chimerism > 95% within 2 months, although patient #5 showed a decline in T cell chimerism. Subsequently in this specific patient a DLI of 1 x 10 6 T cells / kg was administered at 2 months, resulting in agvhd grade III of the intestine 6 weeks later. All other 4 patients showed no GVHD > grade II. Within the complete cohort 9 out of 14 died. Of these 9 patients, 7 died because of a relapse and 2 patients died because of an infection (Table 1). None of the patients experienced lifethreatening GVHD. This rate of mortality is not unexpected in this cohort of patients with very poor cytogenetic abnormalities and/or who were not in CR before transplantation. For instance, the average event-free-survival after 2 year after consolidation ranges from 6-24 % within this category of patients. These data emphasize that it is most likely essential to guide more patients to early and repetitive post transplantation immune interventions. Risk profile Poor (no CR1) Poor (no CR1) Very poor (monosomy 7, no CR1) Phil+ Very poor (monosomy 7, evi- 1, no CR1) Disease AML ALL AML ALL AML Time to neutrophil recovery (d) Time to platelet recovery (d) Chimerisme > 95% , but at 2 months; 40% T cell fraction; 95% non-t cell -> DLI 1 x 10E6 agvhd Grade I skin Grade I skin Grade III intestine (post DLI) Table 4 Clinical outcome Cohort III (ATG/fludarabine/busilvex) day 100 Protocol: Innate allo-sct; Version number 6; date 13 August

24 This small cohort shows that performing our protocol for a αβ T-cell depleted allo SCT with ATG, fludarabine and busulfan results in: - Reproducible depletion efficiencies of T-cells - Reproducible CD34 + recovery - Sufficiently high numbers of infused CD34 + T cells in all patients - Improved engraftment in all patients with an ATG / fludarabine / busilvex regimen as compared to less myeloablative regimens. - No serious toxicity of the ATG / fludarabine / busilvex conditioning regimen within the first 28 days. - No GVHD > grade II in the patients who did not receive a DLI before day Comparison of different transplantation regimens A potential risk benefit assessment for crucial endpoints such as agvhd, graft failure, and TRM 100 as compared to other transplantation regimens is indicated in table 5, based on currently available data. T cell depletion in haplo SCT in nonmalignant diseases 2 (prospective study N=24) T cell depletion in haplo SCT in malignant diseases 40,41. (retrospective study N=23) T cell depletion in MUD/MRD UMCU (retrospective cohort N=5) Non T-cell depleted MUD/MRD UMCU (retrospective cohort N=595) agvhd > grade III 0 % 0% 0% 20% Graft failure 20 % 0% 0% 3.2% TRM 100 days 0 % 4,3% 0% < 5% Table 5: Transplant-related parameters in various cohorts of patients 3.2 Considerations ATG In this current study we want to study the incidence of agvhd of the ATG/fludarabine/busulfan conditioning regimen combined an -depleted allograft in order to Protocol: Innate allo-sct; Version number 6; date 13 August

25 guide more patients to early immune interventions after allo-sct. Although we did not observe in the last small cohort any graft failure, graft failures remain an issue in larger cohorts of T cell depleted transplantations 44. Therefore we aim to optimize host immune depletion without affecting cellular subsets of the graft. This may be achieved by treating patients with ATG on 4 consecutive days, with a daily 10-hour infusion of 1,5 mg/kg/day. This treatment regimen is supported by pharmacokinetic (PK) and pharmacodynamic (PD) analyses on ATG in children performed in our institute and LUMC 45,46. A PK model was developed and validated in 257 children undergoing hematopoietic cell transplantation with a weight-range of kg. Body weight and base line lymphocytes were included in the model as explanatory covariates. Adequate levels of ATG post transplantation associated with improved survival rates and immune reconstitution without increased incidence of graftversus-host disease (GvHD) 47. The results of both the PK and PD analyses in pediatric patients can, to a large extent, be extrapolated to the adult patient, as the pediatric database covers a weight range that is applicable in adults. The model proved to be stable and robust throughout this wide range of patients and can therefore serve as a basis to design the treatment regimen in adults. PK sampling in this study will provide further pharmacological information in the adult population. Based on the pediatric model, simulations were performed to test multiple treatment regimens, aiming at a post-hct AUC of approximately AU*day/ml. A treatment consisting of a dose of ATG on 4 consecutive days, with a daily 10-hour infusion of 1,5 mg/kg/day from day -12 to -9 seems adequate to achieve this exposure. 4. Study objectives 4.1 Primary endpoint: Incidence and grade of acute GVHD at day 100 Protocol: Innate allo-sct; Version number 6; date 13 August

26 4.2 Secondary Endpoints: TRM at day 100 Number of graft failures at day 100 Donor engraftment (chimerism > 95%) at day 100 Levels of ATG prior and post SCT Time to neutrophil engraftment Time to platelet engraftment Time to red blood cell transfusion independence Immune reconstitution including but not limited to total number of CD3 + T cells, CD4 + and CD8+ subtyping of T cells, CD3-CD16/56+ (NK cells), T-cells at 3, 6, 12 and 24 months after transplantation, assessment of NK and TCR repertoires at defined time points. Incidence of infections Incidence and grade of chronic GvHD Long term TRM Long term (secondary) graft failure Progression free survival (PFS: i.e. time from transplantation until progression/relapse or death from any cause, whichever comes first) Overall survival (OS) calculated from transplantation. Patients still alive or lost to follow up are censored at the date they were last known to be alive 5. Study Design This is a bi-centric prospective phase II study (UMCU in Utrecht and Johannes Gutenberg University in Mainz). Patients with haematological malignancies with a matched related donor or a matched unrelated donor, are treated with a T-cell depleted allo-sct. Conditioning regimen (see 7.1) Day 0 Infusion of T-cell depleted HPC(A) product Protocol: Innate allo-sct; Version number 6; date 13 August

27 Follow-up according to local protocol Day 100: end of protocol 6. Study Population The study population includes patients with haematological malignancies eligible for allo-sct and a suitable donor. Inclusion of 35 patients within 4 years should be feasible. 6.1 Inclusion criteria Adults (> 18 years) Haematological malignancies eligible for allo-sct according to the policy of the local centre WHO performance status 2 Written informed consent 6.2 Exclusion criteria Relapse of allo-sct within 5 months after allo-sct Bilirubin and/or transaminases > 2.5 x normal value Creatinine clearance < 40 ml/min Cardiac dysfunction as defined by: - Unstable angina - Unstable cardiac arrhythmias Active, uncontrolled infection 7. Treatment of subjects 7.1 Time to transplant Time to transplant is preferentially in patients with preceding chemotherapy directly after mucositis resolved. Protocol: Innate allo-sct; Version number 6; date 13 August

28 7.2 Conditioning regimen before HLA-identical Sibling or matched unrelated donor stem cell transplantation Agent Dose/day Route of administration Days ATG^ 1,5mg/kg i.v. -12 to -9 Fludarabine 40 mg/m 2 i.v. -5 to -2 Busilvex (AUC90)* i.v. -5 to -2 Table 6 Conditioning regimen ^ATG (THYMOGLOBULIN ;(Anti-thymocyte Globulin [Rabbit]) * Busilvex will be administered intravenously for 4 days in 180 minutes and will be prepared by the pharmacy. Therapeutic Drug Monitoring of busilvex will be performed and adjusted / targeted to a cumulative AUC of 80-90mg*h/L to reach a situation of myeloablation and limited toxicity. i.v. busilvex with AUC monitoring can be replaced by i.v. busilvex (3.2 mg/kg/day at day -5 to day -2 without required monitoring) or with oral busulfan (1 mg/kg a 6 hours day -5 to -2) according to the discretion of the local investigator. 7.3 Donor selection Either HLA matched siblings (MRD) or matched HLA matched unrelated donors (MUD) will be eligible (9/10 or 10/10). Donor selection will be performed in line with local guidelines. 7.4 Stem cell collection Donors will be treated with recombinant human granulocyte colony stimulating factor at a dose of 10 microgram/kg/daily subcutaneously divided in 2 doses for 5 days. Leukapheresis will be undertaken at day 5. The aimed cell number for collection is between 5-10 x 10 6 and infusion between 2-10 CD34 + cells/kg (see Table 3). Protocol: Innate allo-sct; Version number 6; date 13 August

29 7.5 HPC(A) products T-cell reduction by depletion with anti-tcr antibodies in combination with magnetic microbeads will be performed, using the automated CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany). The cell depletion method is an established procedure in an allotransplant setting 48,49 and will be performed according to standard operating procedures. The number of T-cells and the T-cell subsets will be measured in the graft. The maximal contamination with T-cells for all dose levels is 5x10 5 /kg. This dose is chosen for 2 reasons. In patients who received an T-cell depleted haplo-sct, a maximum contamination of 1 x 10 5 T cells was well tolerated without subsequent immuunsuppresion 2 and we expect that donor T cells of a MRD of MUD will be less immunogenic. Secondly, in the first 14 patients who were treated in our pilot study in 3 patients the graft contained between 1 and 2 x 10 5 T cells / kg. These patients showed no GVHD > grade II. In case of a major ABO-incompatibility between patient and donor and in case when the number of red cells exceeds 200 x 10 9 in the end product, HPC(A) will be infused slowly according to the local guidelines. All HPC(A) products will be infused within 24 hours after the depletion procedure. Grafts will be infused at day Management of graft failure Primary or secondary graft failure as judged by neutrophil counts will be considered a treatment failure. Patients will be treated according to institutional guidelines at the investigator s discretion. Management of graft failure should be discussed with the national coordinating investigator or the respective leading investigator. 7.7 GVHD prophylaxis Mycophenolic acid (Cellcept) 15mg/kg 3 times a day (max 1000 mg 3 times a day) will be administered for 30 days as standard GHVD prophylaxis. If the contamination with T cells Protocol: Innate allo-sct; Version number 6; date 13 August

30 is above 1x10 6 T cells / kg, full immune prophylaxis will be administered according to the local guidelines. 7.8 Treatment of GVHD Treatment of GvHD will be performed according to the guidelines of the local investigator. 7.9 Infection prophylaxis Infections should be controlled before start of the conditioning regimen. Selective decontamination (SD) consisting e.g. of anti-bacterial agents and antimycotic agents will be administered according to local protocols. Surveillance cultures will be sampled according to local protocols. Monitoring, prophylaxis and treatment of viral infections (e.g. CMV and EBV) will be performed according to local protocols. Pneumocystis carinii and toxoplasmosis prophylaxis is administered according to local protocols. Vaccination (e.g. Pneumovax/DKTP/influenza) is administered according to local guidelines. 8. Required clinical investigations 8.1 Required clinical investigations at entry (pre-transplant evaluation) and post transplantation Patients will be evaluated before transplantation according to local protocol and international guidelines (JACIE), which includes blood and bone-marrow sampling. Required additional clinical investigations: 0-4 weeks prior to transplantation: lymphocyte subsets will be measured in peripheral blood (as in ) 0-4 weeks prior to transplantation: peripheral blood as well as bone marrow sampling for biological studies ATG levels will be retrospectively measured post the 2 nd and 4 th gift of ATG, at day 0 (day of stem cell infusion) and 7 days post infusion in 5 ml of serum (described in 50 ). Protocol: Innate allo-sct; Version number 6; date 13 August

31 Months after allo SCT WHO Performance x x x x x x x x 2 Physical examination x x x x x x x x 3 Scoring GVHD x x x x x x x x 4 Haematology x x x x x x x x 5 Chemistry x x x x x x x x 6 ABO x x x x x x x x 7 PCR CMV, EBV x x x x x x x 8 PB chimerism (T and non-t) x x x x x x* x* x* 9 Base line immune monitoring x x x x x x x x 10 Extended immune monitoring x x x x x x x x 11 BM morphology, immunophenotyping and MRD * x x x 12 Extended BM monitoring x x x 13 Disease evaluation x x x x Table 7: required investigations. * if MRD-marker is present 1. According to WHO classification, (appendix A). 2. Careful examination including weight, signs of toxicity, infection 3. According to appendix D 4. Hematology: complete blood cell counts (CBC) three times a week from day 0 until ANC>0.5 x 10 9 /l for 2 consecutive measurements and subsequently on each visit in the outpatient clinic. 5. Chemistry: electrolytes, albumin, glucose, creatinine, BUN, bilirubin, ALT/AST, alkaline phosphatase at least once weekly during admission and subsequently at each outpatient clinic visit. Protocol: Innate allo-sct; Version number 6; date 13 August

32 6. In case of ABO-incompatibility between recipient and any of the donors: ABO-blood group 7. During hospitalization 1x/week, thereafter every outpatient clinic visit for a year. This is part of standard care and will be performed according to the local guidelines. 8. Heparinized peripheral blood to perform chimerism analysis by DNA (VNTR). 10 ml (from haematopoietic recovery 6 ml) monthly starting from day 30 until 6 month after allo-sct. *Stop if chimerism is twice >95% at month 6. Prolonged measurement of chimerism if not >95% after 6 months, then monthly until twice >95%. Stop at 12 months. 9. Total (true) counts of B-, T-, CD4, CD8, NK and γδt-cells. From Leucocytes >0.4 x 10 9 /L. During clinical admission every two weeks, thereafter, every month. 10. For extended immune monitoring cryopreservation of 36 ml plasma ( natrium heparine ) en 9 ml serum ( volbloed ) is performed. These studies include but are not limited to extended analysis of the immune repertoires. 11. Bone marrow aspiration for morphology, immunophenotyping and MRD depending on presence of MRD markers. 12. For extend bone marrow monitoring 9 ml bone marrow ( natrium heparine ) is sampled. These studies include but are not limited to immune repertoires in the bone marrow, MRD monitoring, properties of residual blasts and the surrounding stromal cells. 13. Complete evaluation depending on underlying disease. 8.2 Study procedures The main intervention is the infusion of a T-cell depleted graft. All other procedures are standard for all allo-sct patients, except that patients within this study will receive no standard GVHD prophylaxis. Post transplantation, patients are seen weekly for clinical evaluation and blood examination. This is identical to patients who received a standard Protocol: Innate allo-sct; Version number 6; date 13 August

33 allogeneic stem cell transplantation. In addition, at certain time points (table 7 and 8), additional material (blood and bone marrow) will be obtained for biological studies as indicated. These materials will be obtained during regular sampling. Pre transplantation During ATG Months post transplantation administration peripheral blood 45 ml 4 x 5 ml 45 ml 45 ml 45 ml 45 ml 45 ml 45 ml 45 ml 45 ml bone marrow 9 ml 9 ml 9 ml 9 ml Table 8: blood/bone marrow required for biological studies 8.3 Follow-up of primary end points The assessment of eventual occurrence of agvhd is a standard item of each outpatient visit of study and non-study patients. agvhd is diagnosed and rated on the basis of Gluckberg criteria (Appendix D). 8.4 Follow-up of secondary end points Transplant related mortality / Progression free survival/ Overall survival As defined by European group for Blood and Bone Marrow Transplantation (EBMT) Graft failure: Primary graft failure: failure to achieve an absolute ANC >500/μl at Day +28 Secondary graft failure: initial neutrophil engraftment followed by a decline in absolute neutrophil count (ANC) <500/μl and unresponsiveness to growth factor therapy Engraftment Neutrophils > 0.5 x 10 9 /L first day of 3 consecutive days Platelets > 50 x 10 9 /L : first day of at least 7 days without transfusions at day 180 Number of erythrocyte and platelet-transfusions Chimerism >95% Protocol: Innate allo-sct; Version number 6; date 13 August

34 8.4.4 Levels of ATG prior and post SCT Levels of ATG prior and post SCT will be used to perform PK and PD analyses as previously described Clinical parameters Incidence of infections Incidence and grade of chronic GvHD (appendix D) Immune reconstitution True counts of CD3 + T cells, CD4 + and CD8+ subtyping of T cells, CD3- CD16/56+ (NK cells), T-cells Extended analysis of immune repertoire, including assessment of, and NKcell repertoires 52, Donor cells after αβ T cell depletion The total amount of viable CD34 + cells and CD34 + cells/kg recipient will be determined, Total αβt-cell number (defined as γδtcr negative and CD3 positive) will be measured after αβ T cell depletion procedure. NK-cell number will be measured after αβ T cell depletion procedure. γδ T-cell number will be measured after the -αβ T cell depletion procedure. Within this population number of infused Vδ2+/ Vδ1 cells is also determined. Microbiological contamination will be determined. ABO-Rh blood group is determined from the pre harvest donor screening in peripheral blood Sex of the donor is known. Date and timing of the harvest is known. Protocol: Innate allo-sct; Version number 6; date 13 August

35 Before the αβt cell depletion of MUD transplants, part of the stem cell product, (containing unmodified T cells) will be cryopreserved for the later infusion of a potential DLI. Indicated are numbers of living cells after cryopreservation. 9. End of protocol treatments 9.1 Withdrawal of individual subjects Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject from the study for urgent medical reasons. 9.2 Replacement of individual subjects after withdrawal Individual subjects will be replaced after withdrawal if the transplantation procedure is cancelled as a result of relapse before allogeneic stem cell transplantation. 9.3 Other reasons for going off protocol Death Relapse after initial CR (i.e., before completion of treatment) Major protocol violation Graft failure Collection of information of patients who are withdrawn from the study treatment for other reasons then death or withdrawn of their consent will continue. Data obtained from patients who are withdrawn from the study treatment for other reasons withdrawn of their consent will be used for statistical analysis. 9.4 Premature termination/alteration of the study: The sponsor may decide to terminate the study prematurely based on the following criteria: There is evidence of an unacceptable risk for study patients (i.e. safety issue) Protocol: Innate allo-sct; Version number 6; date 13 August

36 The DSMB recommends ending the trial based on viable arguments (e.g. insufficient enrollment of patients). Statistical analyses for all decisions are available under section Safety reporting 10.1 Section 10 WMO event In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and the CCMO if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the CCMO, except insofar as suspension would jeopardize the subjects health. The investigator will take care that all subjects are kept informed Adverse and serious adverse events SAEs must be reported to the SCT facility within 24 hours by after the event was known to the investigator, using the SAE report form provided. This initial report should contain a minimum amount of information regarding the event, associated treatment and patient identification, as described in the detail in the instructions for the SAE report form. Complete detailed information should be provided in a follow-up report within a further 2 business days, if necessary. All SAEs will be reported to the coordinating investigator who will report them online on ToetsingOnline, within 15 days after the sponsor has first knowledge of the serious adverse reactions. Adverse events (AE) are defined as any undesirable experience occurring to a subject during the study, whether or not considered related to the allo-sct. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded. AEs will be reported on the CRF. Protocol: Innate allo-sct; Version number 6; date 13 August

37 A serious adverse event is any untoward medical occurrence or effect that at any dose: results in death; is life threatening (at the time of the event); requires hospitalization or prolongation of existing in-patients hospitalization; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; is a new event of the trial likely to affect the safety of the subjects, such as an unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the treatment of a life threatening disease, major safety finding from a newly completed animal study, etc. SAEs that result in death or are life threatening should be reported expedited. The expedited reporting will occur not later than 7 days after the responsible investigator has first knowledge of the adverse reaction. This is for a preliminary report with another eight days for completion of the report. The investigator will decide whether the serious adverse event is related to the treatment (i.e. unrelated, unlikely, possible, probable, definitely and not assessable) and the decision will be recorded on the serious adverse event form. The assessment of causality is made by the investigator using the following: Protocol: Innate allo-sct; Version number 6; date 13 August

38 10.3 Follow-up of adverse events All adverse events will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist. Follow-up reports on SAEs will be added to the reported SAEs on ToetsingOnline Data Safety and Monitoring Board The Data and Safety Monitoring Board will advise the Principal Investigator and the Coinvestigator(s) about the continuation of the study. The DSMB will evaluate the general progress and the feasibility of the study, the quality and completeness of the data, side effects and safety. The DSMB consists of at least 3 members, among whom (at least) one statistician and minimally two physicians. The members of the DSMB are invited on personal title on the basis of their expert knowledge of the disease involved or the research methodology. Members of the DSMB will have ample experience with randomized clinical trials. Protocol: Innate allo-sct; Version number 6; date 13 August