Patrick Schlegel

Natural Killer Cell Mediated Antibody-Dependent Cellular Cytotoxicity in Tumor Immunotherapy with Therapeutic Antibodies

In the last decade several therapeutic antibodies have been Federal Drug Administration (FDA) and European Medicines Agency (EMEA) approved. Although their mechanisms of action in vivo is not fully elucidated, antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is presumed to be a key effector function. A substantial role of ADCC has been demonstrated in vitro and in mouse tumor models. However, a direct in vivo effect of ADCC in tumor reactivity in humans remains to be shown. Several studies revealed a predictive value of FcγRIIIa-V158F polymorphism in monoclonal antibody treatment, indicating a potential effect of ADCC on outcome for certain indications. Furthermore, the use of therapeutic antibodies after allogeneic hematopoietic stem cell transplantation is an interesting option. Studying the role of the FcγRIIIa-V158F polymorphism and the influence of Killer-cell Immunoglobuline-like Receptor (KIR) receptor ligand incompatibility on ADCC in this approach may contribute to future transplantation strategies. Despite the success of approved second-generation antibodies in the treatment of several malignancies, efforts are made to further augment ADCC in vivo by antibody engineering. Here, we review currently used therapeutic antibodies for which ADCC has been suggested as effector function.

Immune reconstitution and strategies for rebuilding the immune system after haploidentical stem cell transplantation

Haploidentical hematopoietic stem cell transplantation is a curative alternative option for patients without an otherwise suitable stem cell donor. In order to prevent graft‐versus‐host disease (GvHD), different in vitro and in vivo T cell–depletion strategies have been developed. A delayed immune reconstitution is common to all these strategies, and an impaired immune function after haploidentical transplantation with subsequent infections is a major cause of deaths in these patients. In addition to in vitro and in vivo T cell–depletion methods, posttransplant strategies to rapidly rebuild the immune system have been introduced in order to improve the outcome. Advances in in vitro and in vivo T cell–depletion methods, and adoptive transfer of immune cells of the innate and specific immune system, will contribute to reduce the risk of GvHD, lethal infections, and the risk of relapse of the underlying malignant disease.

Reduction of Minimal Residual Disease in Pediatric B-lineage Acute Lymphoblastic Leukemia by an Fc-optimized CD19 Antibody

Prognosis of primary refractory and relapsed pediatric B-lineage acute lymphoblastic leukemia (ALL) is very poor. Relapse rates significantly correlate with persistent minimal residual disease (MRD). In MRD, favorable effector-target ratios prevail and thus this situation might be optimally suited for immunotherapy with antibodies recruiting immunological effector cells. We here report on the generation, preclinical characterization and first clinical application in B-lineage ALL of an Fc-optimized CD19 antibody. This third-generation antibody (4G7SDIE) mediated enhanced antibody-dependent cellular cytotoxicity (ADCC) against leukemic blasts with effector cells from healthy volunteers and B-lineage ALL patients. The antibody was produced in a university-owned production unit and was applied on a compassionate use basis to 14 pediatric patients with refractory and relapsed B-lineage ALL at the stage of MRD. In 10/14 patients, MRD was reduced by ≥ 1 log or below the patient-individual detection limit, and 5/14 patients have achieved ongoing complete molecular remission with a median leukemia-free survival of 428 days. Two additional patients died in complete molecular remission due to complications not related to antibody therapy. Besides profound in vivo B-cell depletion, side effects were negligible. A clinical phase 1/2 study to further assess the therapeutic activity of 4G7SDIE is in preparation.

Favorable NK cell activity after haploidentical hematopoietic stem cell transplantation in stage IV relapsed Ewing’s sarcoma patients

Natural killer (NK) cell activity has been shown to have potential activity against Ewing’s sarcoma (EWS) especially in tumors with low HLA I expression and high NKG2D expression. Two patients with metastatic relapsed and primary metastatic stage IV EWS who had received two courses of high dose chemotherapy with autologous stem cell rescue were transplanted from a haploidentical parental stem cell donor. Patients are alive in ongoing CR for 10.2 and 3.4 years now. Post transplant local second and first relapses were treated successfully in both patients. In vivo IL-2 stimulation not only increased the number and activity of effector cells in one patient but was also associated with severe GvHD. In vitro studies demonstrated high NK cell activity against K562 and relevant activity against EWS cell line A673 post transplant. NK activity was enhanced by cytokine prestimulation as well as by EWS targeting anti-GD2 Ab. Haploidentical hematopoietic stem cell transplantation (HSCT) might contribute to long-term survival by NK cell-mediated effect exerted by donor-derived NK cells. Local tumor recurrence was manageable in both high-risk patients indicating systemic immune control preventing subsequent metastasizing. The efficacy of haploidentical HSCT, cytokine application and tumor targeting antibodies for the use of Ab-dependent cellular cytotoxicity needs evaluation in clinical trials.

Treatment of graft failure with TNI-based reconditioning and haploidentical stem cells in paediatric patients

Graft failure is a life‐threatening complication after allogeneic haematopoietic stem cell transplantation (HSCT). We report a cohort of 19 consecutive patients (median age: 8·5 years) with acute leukaemias (n = 14) and non‐malignant diseases (n = 5) who experienced graft failure after previous HSCT from matched (n = 3) or haploidentical donors (n = 16) between 2003 and 2012. After total nodal irradiation (TNI)‐based reconditioning combined with fludarabine, thiotepa and anti‐T cell serotherapy, all patients received T cell‐depleted peripheral blood stem cell grafts from a second, haploidentical donor. Median time between graft failure and retransplantation was 14 d (range 7–40). Sustained engraftment (median: 10 d, range 9–32) and complete donor chimerism was observed in all evaluable patients. 5 patients additionally received donor lymphocyte infusions. Graft‐versus‐host disease (GvHD) grade II and III occurred in 1 patient each (22%); no GvHD grade IV was observed. 2 patients had transient chronic GvHD. The regimen was well tolerated with transient interstitial pneumonitis in one patient. Treatment‐related mortality after one year was 11%. Event‐free survival and overall survival 3 years after retransplantation were 63% and 68%. Thus, a TNI‐based reconditioning regimen followed by transplantation of haploidentical stem cells is an option to rescue patients with graft failure within a short time span and with low toxicity.

Allogeneic Stem Cell Transplantation

Allogeneic stem cell transplantation in malignant diseases has evolved as a treatment option for patients with otherwise incurable diseases. The principle of this treatment is a significant tumor reduction by pretransplant chemotherapy or irradiation and immunological consolidation by various effector cells of the adaptive as well as of the innate immune system. The role of Graft versus Host disease (GvHD) and the concomitant Graft versus Tumor (GvT) effects is discussed and therapeutic approaches exploiting alloreactive T cell responses and ways to separate GvHD from GvT are shown. Antitumor response mechanisms that are also induced by alloreactive Natural Killer (NK) cells and γδ + T-cells belonging to the innate immune system are described. The innate donor-derived immune system might significantly contribute to the anti-tumor effects of allogeneic transplantation and the selection of donors will extend beyond classical high resolution typing of HLA alleles and finding the best matched HLA identical donor. The Killer Inhibitory Immunoglobuline-like Receptor (KIR) system is almost as polymorphic but independent from the HLA system and allows the selection of the optimal donor for certain malignant diseases. Especially in haploidentical transplantion, the KIR system plays an important role and new donor selection strategies might also apply in the future for the treatment of refractory solid tumors.

Haploidentical Transplants for Nonmalignant Diseases in Children

Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative treatment strategy to a variety of nonmalignant hematological and immunological disorders as well as inborn errors of metabolism. Successful allo-HCT is able to restore functional hematopoiesis and immune function and can substitute disabled enzymatic activity. Nevertheless, allo-HCT can also be associated with serious risks for transplantation-related morbidities or even mortalities like graft-versus-host disease (GvHD) or life-threatening infectious complications. Especially in nonmalignant disorders (NMD), risks and benefits have to be carefully balanced on an individual patient basis. Up to now, human leukocyte antigen (HLA)-matched siblings are the preferred source of hematopoietic graft. Only about one third of patients have HLA-matched sibling donor (MSD) and the number further decreases in patients with inherited disorders as siblings might be carriers or affected as well. HLA-matched unrelated donors (MUD) have become an important alternative. Chances of finding a HLA-MUD are particularly dismal for individuals belonging to certain ethnic groups, with often less than 10% compared to approximately 75% in the Caucasian population. Therefore, alternative donor sources have to been taken into account, especially when the clinical condition of the patients does not allow a further delay of the allo-HCT. This chapter focuses on HLA-haploidentical hematopoietic cell transplant (haplo-HCT) in NMD. It will highlight recent developments in graft manipulation utilizing safe application of haplo-HCT grafts and discuss important advantages which might lift haplo-HCT to a standard therapy in NMDs in the near future.

CD34+ selected stem cell boosts can improve poor graft function after paediatric allogeneic stem cell transplantation

Poor graft function (PGF) is a severe complication of haematopoietic stem cell transplantation (HSCT) and administration of donor stem cell boosts (SCBs) represents a therapeutic option. We report 50 paediatric patients with PGF who received 61 boosts with CD34+ selected peripheral blood stem cells (PBSC) after transplantation from matched unrelated (n = 25) or mismatched related (n = 25) donors. Within 8 weeks, a significant increase in median neutrophil counts (0·6 vs. 1·516 × 109/l, P < 0·05) and a decrease in red blood cell and platelet transfusion requirement (median frequencies 1 and 7 vs. 0, P < 0·0001 and <0·001), were observed, and 78·8% of patients resolved one or two of their cytopenias. 36·5% had a complete haematological response. Median lymphocyte counts for CD3+, CD3+CD4+, CD19+ and CD56+ increased 8·3‐, 14·2‐, 22.‐ and 1·6‐fold. The rate of de novo acute graft‐versus‐host disease (GvHD) grade I–III was only 6% and resolved completely. No GvHD grade IV or chronic GvHD occurred. Patients who responded to SCB displayed a trend toward better overall survival (OS) (P = 0·07). Thus, administration of CD34+ selected SCBs from alternative donors is safe and effective. Further studies are warranted to clarify the impact on immune reconstitution and survival.

Favorable immune recovery and low rate of GvHD in children transplanted with partially T cell-depleted PBSC grafts

Transplantation of peripheral blood stem cells (PBSC) from matched unrelated donors (MUD) is still associated with a significant risk for graft vs. host disease (GvHD), especially in pediatric patients receiving grafts from adult donors containing high amounts of T cells. Here, we present long-term follow-up results on 25 pediatric patients, (acute leukemia n = 15, NHL n = 3, CML n = 3, MDS n = 5), transplanted with CD34 or CD133 positively selected PBSC from MUDs supplemented with an add-back of 1 × 107/kg body weight (kgBW) unselected T cells resulting in a median T-cell depletion (TCD) of 1.97 log. A total of 24/25 (96%) patients had primary engraftment. Early T-cell recovery was significantly improved compared to patients receiving CD34-selected grafts without T-cell add-back and similar to patients receiving unmanipulated bone marrow. GvHD incidence was low with 8/4% aGvHD grade II/III, no grade IV and 13% limited cGvHD. In total, 16/25 (64%) patients are alive after a median follow-up of 10 years. Five-year event-free survival (EFS) was 68%, relapse probability 24% and transplantation-related mortality (TRM) 12%. Thus, in PBSC allotransplants from MUD, partial TCD with serotherapy and CSA/MTX prophylaxis, can effectively reduce GvHD without hampering engraftment and immune reconstitution.

Abstract B144: Reduction of minimal residual disease in pediatric B-precursor acute lymphoblastic leukemia by an Fc-optimized CD19 antibody

B-precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood cancer. Although this disease can be successfully treated in 80 % of patients by chemotherapy, prognosis for primary refractory or relapsed patients is very poor. Even after allogeneic stem cell transplantation (SCT), relapse rates are considerable and correlate significantly with persistent minimal residual disease (MRD) prior to or after SCT. In an MRD constellation favorable effector-target ratios prevail and thus it is well suited for immunotherapy with therapeutic antibodies. We developed a chimeric Fc-optimized third-generation CD19 antibody (4G7SDIE) and produced it in pharmaceutical quality. This antibody mediates markedly enhanced antibody dependent cellular cytotoxicity (ADCC) through its improved capability to recruit FcγRIIIa-bearing effector cells. In this study, 4G7SDIE was applied on a compassionate need basis to pediatric patients with relapsed or refractory BCP-ALL in an MRD situation (n=14) or in relapse (n=2) (initial refractory disease, n=3; 1st relapse, n=7; ≥ 2nd relapse, n=6; 12/16 patients had previous SCT). Side effects were negligible. In all patients complete CD20 + B-cell depletion was observed during therapy. After discontinuation of 4G7SDIE therapy B cell counts recovered rapidly to normal levels. Both patients in relapse did not respond to 4G7SDIE treatment. In 9/14 MRD-positive patients, MRD was reduced by ≥ 1 log or fell below MRD-detection threshold of 10 -4 over the course of treatment. 2/9 responders were receiving additional treatment. In CD107a assays primary NK cells and γδ T cells were identified as main effector cell populations. The FcγRIIIA-V158F polymorphism had no influence on ADCC mediated by 4G7SDIE. Cytotoxicity assays confirmed sustained functionality of patient effector cells over the course of 4G7SDIE treatment. In vitro cytotoxicity assays were performed using PBMC from transplanted patients obtained at different time points of 4G7SDIE treatment. Lysis of autologous leukemic blasts was increased when 4G7SDIE or autologous patient serum taken after antibody infusion was added. After infusion of 20 mg/m 2 4G7SDIE serum half-life was 34 ± 13 hours (n=3) and serum levels of 4G7SDIE remained above saturating concentrations of ≥ 700 ng/ml (EC 50 =65 ng/ml) at day 13 and following treatment cycle, respectively. Notably, in 3/3 analyzed patients under 4G7SDIE therapy, a transient down modulation of CD19 surface expression on the leukemic blasts was observed. In vitro antigenic shift assays on primary leukemic blasts showed considerable but very heterogeneous shift of CD19 surface expression. Furthermore, a positive correlation between CD19 surface expression levels and 4G7SDIE mediated lysis was observed. These observations hint at in vivo tumor escape mechanisms and furthermore indicate selective pressure exerted by immunotherapy with 4G7SDIE, underlining its therapeutic potential, but also delineating possible limitations. In conclusion, promising anti-leukemic effects of the 4G7SDIE antibody have been observed in vitro and in vivo . We are currently preparing a phase I/IIa trial. Citation Format: Ursula Joerdis Eva Seidel, Ludger Grosse-Hovest, Patrick Schlegel, Martin Hofmann, Friedhelm R. Schuster, Roland Meisel, Kai-Erik Witte, Steffen Aulwurm, Elwira Pyz, Hans-Georg Rammensee, Gundram Jung, Rupert Handgretinger, Peter Lang. Reduction of minimal residual disease in pediatric B-precursor acute lymphoblastic leukemia by an Fc-optimized CD19 antibody. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B144.