Infusion of Donor Derived Cytokine Induced Killer (CIK) Cells in Hematological Patients Relapsed After Haploidentical Stem Cell Transplant

The haematological neoplasia relapse is the cause of higher mortality after allogeneic stem cell transplantation (HSCT). When transplantation fails the most common therapeutic strategy is to increase the antitumor activity of the donor's immune system through the infusion of donor Lymphocytes (DLI). The use of DLI may limit the relapse, but may induce transplantation disease against the host (GvHD), in 40-60% of patients. With advances in transplantation procedures, the use of non-compatible (HLA-mismatched) haploidentical (aplo) donor cells has become feasible and is increasing. However, strategies for immune control of relapse after HSCT from haploidentical donor are hampered by the absence of prospective data that can guide treatment and limit the induction of GvHD in the setting of the HLA difference between the donor and the recipient. Cytokine-induced Killer Cells (CIK) are T lymphocytes from haploidentical donor expressing CD56 (e.g., double positive cells at CD3 / CD56). CIK are a product of advanced cell therapy (Advanced Therapeutic Medicinal Product, ATMP) for somatic cell therapy and have a reduced histocompatibility (MHC) complex: are cytotoxic, anti-tumor cells, possess the characteristics of both T cells and Natural Killer (NK) and show in vivo a very strong cytolytic activity against leukemia, but a low reactivity against the host. Therefore, this study has as its primary objective to investigate the safety of CIK cells deriving from the donor, especially in terms of the onset of GvHD, used as a treatment for relapse after transplantation with haploidentical stem cells. The study will allow to evaluate the possibility of using CIK cells, at the indicated dose combination (5x10 \* 6 cells / kg, 5x10 \* 6 and 10x10 \* 6 cells / kg) as an effective and safe therapy in the context of haploidentical transplantation.

Disease relapse is a major cause of mortality following allogeneic hematopoietic stem cell transplantation (HSCT) for hematologic malignancies. When allogeneic transplant fails the most common therapeutic strategy is to increase the anti-tumor activity of donor immune system through infusion of donor lymphocytes (DLI). Use of DLI can effectively treat limited relapses, but can induce graft-versus-host disease (GvHD) in the range of 40-60% of the patients. With the advances in HSCT procedure, the use of HLA-mismatched (haplo) family donors became feasible and extended the opportunity for HSCT to almost all patients lacking an HLA-matched donor. Due to the promising results of the procedure and the easier access to the donor, the number of haplo-HSCT transplant is steeply increasing. However, strategies aiming at eliciting the immune control of the relapse after haplo-HSCT are hampered by absence of prospective data that guide treatment and the fear of inducing GvHD in this setting of profound HLA disparity between donor and recipient.

* Despite the use of DLI in haplo-HSCT is not a standard practice, DLI are occasionally empirically used, mainly due to the lack of alternative treatment.
* Cytokine Induced Killer (CIK) cells are T lymphocytes expressing the CD56 marker, i.e. CIK are CD3/CD56 double positive cells. CIK cells are produced in vitro by incubating blood leukocytes according to a specific expansion protocol which includes initial stimulation with Interferon-gamma and anti-CD3 antibody, followed by expansion with (Interleukin-2) IL-2. CIK cells are non MHC restricted, cytotoxic, anti-tumoral cells which share characteristics of both T and NK cells and show in vivo a very strong cytolytic activity against leukemia, graft versus leukemia, while being essentially devoid of graft versus host reactivity. They show a natural non-MHC restricted (T Cell Receptor)TCR independent cytotoxicity against malignant targets cells, involving perforin and granzyme release and NKG2D, as well as CD56 and other small target molecules. In the past 15 years the investigators have extensively studied the use of cytokine-induced killer (CIK) cells as alternative cellular therapy to treat disease relapse after HSCT. The key feature of CIK cell therapy in the context of haplo- HSCT is the lower risk of inducing GvHD compared to DLI, that could permit to avoid the devastating effect of acute and chronic GvHD in terms of decreased survival, quality of life, disability and need of additional care. Indeed, beyond the dramatic impact in the quality and quantity of life of patients, costs for the health system of the management of acute and chronic GvHD are exceedingly high. At this regard, CIK cells have been regarded as the cellular therapy that is associated with a more favourable therapeutic index compared to DLI.
* In an early Phase I study the investigators observed remissions in 3 of 11 patients who were treated with infusion of CIK cells at a time when malignant cells were detectable. In each of these 3 cases, the patient had been previously treated unsuccessfully with conventional DLI. Successful remission induction by infusion of CIK cells after unsuccessful DLI suggests that the (Graft Versus Leukemia) GVL activity of CIK cells was attributable to NK-T cells. In a subsequent phase I clinical trial was assessed the feasibility of using CIK cells from HLA-matched sibling donors. Using a dose escalation design, the investigators showed that CIK cells could be given at doses as high as 1x10\^8 per kg recipient body weight without causing acute infusion-related toxicity. The investigators recently reported the final results of a phase II multicenter pilot study, showing that CIK cell treatment for the relapse after HSCT is effective with a low toxicity in terms of GvHD compared to DLI. Importantly for the present proposal, in this study, 5 patients were treated in the context of haplo- HSCT. These did not show any different response or safety concern compared to other patients, suggesting the feasibility of this approach in a haploidentical context. Similarly, another study of CIK cellular therapy in HSCT including also patients with haploidentical donors confirmed the feasibility and safety of this approach. Considering that infused T cells are associated with risk of GvHD induction, especially in the haploidentical transplantation setting, novel cellular therapies manipulated to minimize GvHD while enhancing the graft-versus leukemia efficacy need to be tested in early phase clinical trials. Thus, the aim of the present proposal is to use CIK cells as a platform for a safe and effective cellular therapy in the context of haploidentical transplantation. As no dose limiting toxicity has been reached in phase I/II studies and no clear relationship among dose and both toxicity and response emerged so far, in the present study the investigators planned to use the final standard dose of our previous phase II study of 5x106 cells/kg, 5x10\^6 and 10x10\^6 cells/kg with no schedule modification . Three infusion of CIK cells will be administered every 3 weeks.

Eligibility Criteria:
Inclusion Criteria:

1. Male or female patients 18 years or older
2. Patients treated with haploidentical allogeneic transplantation for hematologic malignancies, excluding Chronic Myeloid Leukemia (CML), such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), multiple myeloma (MM), myelofibrosis (MF) and myelodysplastic syndrome (MDS)
3. To be enrolled to the safety run-in cohort, patients must have:

   - Evidence of relapsed disease after allogeneic transplantation, including molecular, cytogenetic or overt hematologic relapse

   To be enrolled to the phase II cohort, patients must have:
   * Evidence of relapsed disease after allogeneic transplantation, including, molecular, cytogenetic or overt hematologic relapse, or
   * Mixed chimerism after the day +90, defined as \<75% donor in unfractionated bone marrow and/or \<75% donor in unfractionated Peripheral blood (PB) and/or \<75% donor in fractionated CD3+ peripheral blood.
4. Availability of a donor willing to donate peripheral blood mononuclear cells
5. Withdrawn of immune suppression at least 3 weeks before the beginning of the cell therapy program
6. Written informed consent prior to any study procedures being performed
7. For female patients:

   1. being postmenopausal for at least 1 year before the screening visit,OR
   2. being surgically sterile, OR
   3. if they are of childbearing potential, must agree to practice highly effective method of contraception and one additional effective (barrier) method from the time of signing the informed consent until the end of study. Highly effective method of contraception includes: (i) combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation: oral, intravaginal, transdermal; (ii) progestogen-only hormonal contraception associated with inhibition of ovulation: oral, injectable, implantable (intrauterine device (IUD), intrauterine hormone-releasing system (IUS), bilateral tubal occlusion, vasectomized partner, sexual abstinence) OR
   4. must agree to practice true abstinence, when this is in line with the preferred and usual lifestyle of the subject from the time of signing the informed consent until the end of study. \[Periodic abstinence (eg, calendar, ovulation, symptothermal, postovulation methods), withdrawal, spermicides only, and lactational amenorrhea are not acceptable methods of contraception. Female and male condoms should not be used together.\]
8. For male patients, even if surgically sterilized (i.e., status postvasectomy): a) with female partners of childbearing potential: must agree to practice barrier contraception (condom with or without spermicide) from the time of signing the informed consent until the end of study and his female partner must agree to practice method of contraception including one of the following: estrogen and progestogen containing hormonal contraception; inhibition of ovulation: oral, intravaginal, transdermal; progestogen-only hormonal contraception associated with inhibition of ovulation: oral, injectable, implantable (intrauterine device (IUD), intrauterine hormone-releasing system (IUS), bilateral tubal occlusion) from the time of signing the informed consent until the end of study.b) must agree to practice true abstinence, when this is in line with the preferred and usual lifestyle of the subject from the time of signing the informed consent until the end of study. \[Periodic abstinence (eg, calendar, ovulation, symptothermal, postovulation methods), withdrawal, spermicides only, and lactational amenorrhea are not acceptable methods of contraception. Female and male condoms should not be used together.\] c) must agree to refrain from donating sperm

Exclusion Criteria:

The presence of any of the following will exclude a subject from study enrolment 1. Donors positive for HIV, (Hepatitis B virus) HBV, (Hepatitis C virus) HCV, Treponema or unfit to donate peripheral blood mononuclear cells 2. Patients with active grade 2 or more acute or moderate chronic GVHD at study entry or before CIK infusion 3. Patients with rapidly progressive disease or not controlled by palliative supportive treatments, including chemotherapy, and with life expectancy less than 8 weeks 4. Any serious medical or psychiatric illness, including drug or alcohol abuse, that could, in the investigator's opinion, potentially interfere with the completion of treatment according to this protocol

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