Michael Merker

Interleukin-15-activated cytokine-induced killer cells may sustain remission in leukemia patients after allogeneic stem cell transplantation: feasibility, safety and first insights on efficacy

Anecdotal clinical cases previously suggested the potential of cytokine-induced killer (CIK) cells as an anti-leukemic immunotherapy (IT). Here, we report the aggregate experience from three hematology-oncology centers regarding the feasibility, safety and efficacy of interleukin (IL)-15-activated

Pre-Emptive Immunotherapy for Clearance of Molecular Disease in Childhood Acute Lymphoblastic Leukemia after Transplantation

Monitoring of minimal residual disease (MRD) or chimerism may help guide pre-emptive immunotherapy (IT) with a view to preventing relapse in childhood acute lymphoblastic leukemia (ALL) after transplantation. Patients with ALL who consecutively underwent transplantation in Frankfurt/Main, Germany between January 1, 2005 and July 1, 2014 were included in this retrospective study. Chimerism monitoring was performed in all, and MRD assessment was performed in 58 of 89 patients. IT was guided in 19 of 24 patients with mixed chimerism (MC) and MRD and by MRD only in another 4 patients with complete chimerism (CC). The 3-year probabilities of event-free survival (EFS) were .69 ± .06 for the cohort without IT and .69 ± .10 for IT patients. Incidences of relapse (CIR) and treatment-related mortality (CITRM) were equally distributed between both cohorts (without IT: 3-year CIR, .21 ± .05, 3-year CITRM, .10 ± .04; IT patients: 3-year CIR, .18 ± .09, 3-year CITRM .13 ± .07). Accordingly, 3-year EFS and 3-year CIR were similar in CC and MC patients with IT, whereas MC patients without IT experienced relapse. IT was neither associated with an enhanced immune recovery nor an increased risk for acute graft-versus-host disease. Relapse prevention by IT in patients at risk may lead to the same favorable outcome as found in CC and MRD-negative-patients. This underlines the importance of excellent MRD and chimerism monitoring after transplantation as the basis for IT to improve survival in childhood ALL.

Cytomegalovirus-specific cytokine-induced killer cells: concurrent targeting of leukemia and cytomegalovirus

BACKGROUND AIMS Human cytomegalovirus (CMV) infection and reactivation is a leading complication of allogeneic hematopoietic stem cell transplantation (HSCT). In addition to drug treatment, the adoptive transfer of virus-specific T cells to restore cellular immunity has become a standard therapy after allogeneic HSCT. We recently demonstrated potent anti-leukemic activity of interleukin (IL)-15-activated cytokine-induced killer (CIK) cells. With the use of the same expansion protocol, we asked whether concurrent CMV antigen-pulsing might generate CIK cells with anti-leukemic and anti-CMV activity. METHODS CIK cells expanded in the presence of interferon-γ, IL-2, IL-15 and anti-CD3 antibody were pulsed once with CMV(pp65) peptide pool. CMV-specific CIK (CIK(pp65)) and conventional CIK cells were phenotypically and functionally characterized according to their cytokine secretion pattern, degranulation capacity and T-cell receptor (TCR)-mediated and NKG2D-mediated cytotoxicity. RESULTS We demonstrated that among CIK cells generated from CMV-seropositive donors, a single stimulation with CMV(pp65) protein co-expanded cytotoxic CMV-specific cells without sacrificing anti-tumor reactivity. Cells generated in this fashion lysed CMV(pp65)-loaded target cells and CMV-infected fibroblasts but also leukemic cells. Meanwhile, the alloreactive potential of CIK(pp65) cells remained low. Interestingly, CMV reactivity was TCR-mediated and CMV-specific cells could be found in CD3(+)CD8(+)CD56(+/-) cytotoxic T-cell subpopulations. CONCLUSIONS We provide an efficient method to generate CIK(pp65) cells that may represent a useful cell therapy approach for preemptive immunotherapy in patients who have both an apparent risk of CMV and impending leukemic relapse after allogeneic stem cell transplantation.

Treatment of relapse after allogeneic stem cell transplantation in children and adolescents with ALL: the Frankfurt experience

Therapy for post-transplant relapse of paediatric ALL is limited. Standardised curative approaches are not available. We hereby describe our local procedure in this life-threatening situation. A total of 101 ALL patients received their first allogeneic stem cell transplantation (SCT) in our institution. After relapse, our primary therapeutic goal was to cure the patient with high-dose chemotherapy or specific immunotherapy (HDCHT/SIT) followed by a second SCT from a haploidentical donor (transplant approach). If this was not feasible, low-dose chemotherapy and donor lymphocyte infusions (LDCHT+DLI) were offered (non-transplant approach). A total of 23 patients suffered a post-transplant relapse. Eight patients received HDCHT/SIT, followed by haploidentical SCT in 7/8. Ten received LDCHT+DLI. The eight patients treated with a second transplant and the ten treated with the non-transplant approach had a 4-year overall survival of 56% and 40%, respectively (P=0.232). Prerequisites for successful treatment of post-transplant relapse by either a second transplant or experimental non-transplant approaches are good clinical condition and the capacity to achieve haematological remission by the induction treatment element.

Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children

Pediatric patients with recurrent, refractory or advanced soft tissue sarcoma (STS) who are simultaneously showing signs of cumulative treatment toxicity are in need of novel therapies. In this preclinical analysis, we identified ErbB2 as a targetable antigen on STS cells and used cytokine-induced killer (CIK) cells transduced with the lentiviral 2nd-generation chimeric antigen receptor (CAR) vector pS-5.28.z-IEW to target ErbB2-positive tumors. Solely CIK cell subsets with the CD3+ T cell phenotype showed up to 85% cell surface expression of the respective CAR. A comparison of wildtype (WT), mock-vector and ErbB2-CAR-CIK cells showed, that engineered cells exhibited diminished in vitro expansion, retained WT CIK cell phenotype with higher percentages of differentiated effector memory/effector cells. Activating natural killer (NK) cell receptor NKG2D-restricted target cell recognition and killing of WT and ErbB2-CAR-CIK cells was maintained against ErbB2-negative tumors, while ErbB2-CAR-CIK cells demonstrated significantly increased cytotoxicity against ErbB2-positive targets, including primary tumors. ErbB2-CAR- but not WT CIK cells proliferated, infiltrated and efficiently lysed tumor cell monolayers as well as 3D tumor spheroids. Here, we demonstrate a potential cell therapeutic approach using ErbB2-CAR-CIK cells for the recognition and elimination of tumor cells expressing ErbB2, which we identified as a targetable antigen on high-risk STS cells.

Haploidentical allogeneic hematopoietic stem cell transplantation in patients with high-risk soft tissue sarcomas: results of a single-center prospective trial

The prognosis of patients with soft tissue sarcomas (STS) such as rhabdomyosarcoma (RMS), Ewing sarcoma (ES), or synovial sarcoma (SS) has improved dramatically in recent decades [1, 2]. However, patients with metastatic disease at the time of diagnosis or with certain histological subtypes (e.g., alveolar RMS (aRMS)) still face a poor prognosis [2–4]. Therefore, novel therapeutic strategies are required for these patients. Haploidentical allogeneic hematopoietic stem cell transplantation (HSCT) has been proposed as a potentially curative therapy for pediatric patients with refractory or relapsed solid malignancies due to its presumable graftversus-tumor (GvT) effect [5]. Indeed, various preliminary clinical studies indicated a potential GvT effect in patients with metastatic and relapsed ES [6], neuroblastoma [5, 7], and hepatoblastoma [8], accompanied by moderate treatment-related toxicity. Furthermore, rapid platelet and particularly rapid leukocyte recovery following haploidentical allogeneic HSCT suggest potent antitumor responses provided by the newly established donor-derived immune system [5]. However, retrospective analyses in pediatric STS patients found no benefit from non-haploidentical allogeneic HSCT in high-risk STS [9, 10]. Therefore, based on the promising preliminary data and limited treatment options of the case studies with haploidentical allogeneic HSCT, patients with refractory or relapsed STS in line with this prospective single-center trial (EudraCT number 2006-000393-76) were offered haploidentical allogeneic HSCT from a family donor. Safety and outcomes were analyzed. Patients (children and young adults) with the abovementioned diseases who received their first allogeneic HSCT in our center between January 1, 2005 and January 1, 2015 were included in this study. CD3/CD19depleted grafts were used for the transplantation of all patients. Written informed consent was provided by the patient or/ and their legal representative before starting the respective conditioning regimen. The study was registered at EudraCT (2006-000393-76) and approved by the local ethics committee (no. 87/06) and the German regulatory authorities (Paul-Ehrlich-Institut, PEI). A reduced-intensity conditioning regimen with T-cell depletion in the graft and the host, including fludarabine (5× 30 mg/m), thiotepa (2× 5 mg/kg), melphalan (2× 70 mg/m), and a monoclonal anti-CD3 antibody (Ab) (muromonab-CD3, OKT-3®) was employed until withdrawal of the latter. Since January 2011, anti-thymocyte globulin (ATG) Fresenius (3× 10 mg/kg) was administered instead of OKT-3® along with the aforementioned chemotherapy regimen. Michael Merker and Michael Torsten Meister contributed equally to this work

AlloHSCT in paediatric ALL and AML in complete remission: improvement over time impacted by accreditation?

Allogeneic hematopoietic stem cell transplantation (alloHSCT) has become a well-established treatment option for many patients suffering from malignant and non-malignant diseases. In the past decade, high-resolution HLA-typing, remission surveillance, pre-emptive immune intervention, and standardisation in supportive care measures have substantially improved transplant outcomes. This retrospective study evaluated transplant procedures in 162 paediatric patients with acute lymphoblastic leukaemia (n = 124) or acute myeloid leukaemia (n = 38) who received their first alloHSCT in our institution over an 11-year period. We observed a significant reduction in risk of non-relapse mortality (NRM) over time (HR = 0.34, 95% CI 0.12–0.98; P = 0.05), the 4-year NRM estimate decreased from 20% in 2005–2008 to 7% in 2012–2016 (P = 0.02) and an increase in survival after relapse. There was no significant difference in patients who received a graft from a sibling, haplo, or an unrelated donor with regard to their overall survival (P = 0.45), event-free survival (P = 0.61), and non-relapse mortality (P = 0.19). Our data suggest that a specific transplant infrastructure with a highly experienced team in an accredited transplant centre likely contributes to better transplant outcomes for acute leukaemia patients in complete remission regardless of donor type.