Torsten Tonn

Clinical Outcome 2 Years After Intracoronary Administration of Bone Marrow–Derived Progenitor Cells in Acute Myocardial Infarction

Background—The aim of this study was to investigate the clinical outcome 2 years after intracoronary administration of autologous progenitor cells in patients with acute myocardial infarction (AMI). Methods and Results—Using a double-blind, placebo-controlled, multicenter trial design, we randomized 204 patients with successfully reperfused AMI to receive intracoronary infusion of bone marrow–derived progenitor cells (BMC) or placebo medium into the infarct artery 3 to 7 days after successful infarct reperfusion therapy. At 2 years, the cumulative end point of death, myocardial infarction, or necessity for revascularization was significantly reduced in the BMC group compared with placebo (hazard ratio, 0.58; 95% CI, 0.36 to 0.94; P=0.025). Likewise, the combined end point death and recurrence of myocardial infarction and rehospitalization for heart failure, reflecting progression toward heart failure, was significantly reduced in the BMC group (hazard ratio, 0.26; 95% CI, 0.085 to 0.77; P=0.015). Intracoronary administration of BMC remained a significant predictor of a favorable clinical outcome by Cox regression analysis when adjusted for classical predictors of poor outcome after AMI. There was no evidence of increased restenosis or atherosclerotic disease progression after BMC therapy nor any evidence of increased ventricular arrhythmias or neoplasms. In addition, regional left ventricular contractility of infarcted segments, as assessed by MRI in a subgroup of patients at 2-year follow-up, was significantly higher in the BMC group compared with the placebo group (P<0.001). Conclusions—Intracoronary administration of BMC is associated with a significant reduction of the occurrence of major adverse cardiovascular events maintained for 2 years after AMI. Moreover, functional improvements after BMC therapy may persist for at least 2 years. Larger studies focusing on clinical event rates are warranted to confirm the effects of BMC administration on mortality and progression of heart failure in patients with AMIs. Clinical Trial Registration—clinicaltrials.gov. Identifier: NCT00279175.

Transcoronary Transplantation of Functionally Competent BMCs Is Associated With a Decrease in Natriuretic Peptide Serum Levels and Improved Survival of Patients With Chronic Postinfarction Heart Failure: Results of the TOPCARE-CHD Registry

Although intracoronary administration of bone marrow–derived mononuclear progenitor cells (BMCs) may be associated with improved cardiac function in patients with chronic postinfarction heart failure, the impact on prognosis and clinical outcome of these patients is unknown. To identify potential predictors for a favorable clinical outcome, we assessed natriuretic peptide serum levels as objective markers of heart failure and the occurrence of cardiac death in relation to functional capacity of the infused cells in a consecutive series of 121 patients with chronic ischemic heart disease treated with intracoronary infusion of BMCs. Our analyses show that both N-terminal pro–brain natriuretic peptide (NT-proBNP) and N-terminal pro–atrial natriuretic peptide (NT-proANP) serum levels were significantly reduced in patients with established postinfarction heart failure 3 months after transcoronary progenitor cell administration. NT-proBNP serum levels greater than or equal to median (735 pg/mL) at baseline and a high number of infused progenitor cells with colony-forming capacity were the only independent predictors of a favorable response 3 months after intracoronary administration of BMCs. During extended clinical follow-up (577±442 days), a total of 14 deaths occurred in the overall patient population. Kaplan–Meier curves for both all cause and cardiac mortality showed that patients receiving a higher number of colony-forming cells were significantly less likely to die than those patients receiving low numbers of colony-forming cells (P=0.01). Most importantly, infusion of a high number of cells with colony-forming capacity was associated with a complete abrogation of increased mortality in patients with elevated NT-proBNP serum levels (≥735 pg/mL; median) at baseline (P<0.001). Taken together, our results show that patients with objective evidence of postinfarction heart failure demonstrate a significant reduction of both NT-proBNP and NT-proANP serum levels within 3 months following intracoronary infusion of BMCs. Importantly, infusion of progenitor cells with a high functional capacity is associated with a significantly lower mortality during further follow-up.

Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation

BACKGROUND: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal.

Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92.

For years activated natural killer (A-NK) cells have been explored with respect to their efficacy in anticancer therapy, but, except for some anectdotal reports, no clear clinical benefit has been shown. However, as the understanding about the interactions of NK cells and tumor cells advances, the use of A-NK cells might be revisited with more sophisticated approaches that pay tribute to mechanisms which allow tumor cells to escape immune surveillance. Here the highly cytotoxic NK cell line NK-92 seems to be an attractive alternative for use in adoptive immunotherapy, because it was shown to exhibit substantial antitumor activity against a wide range of malignancies in vitro as well as in xenografted SCID mice. NK-92 cells are characterized by an almost complete lack of killer cell immunglobulin-like receptors (KIRs) yet conserved ability to perforin and granzyme B-mediated cytolytic activity, which make them unique among the few established NK and T cell-like cell lines. NK-92 is the only natural killer cell line that has entered clinical trials. Here we discuss the current status of development of this cell line for adoptive immunotherapy (AIT) of malignancies and review our first clinical experience in patients with advanced cancer who have received repeated transfusions of irradiated NK-92 in a phase I/II trial. Also we discuss issues that address safety aspects of immunotherapy with clonal cell lines and describe further manipulations, which hold the potential of significantly improving the clinical outcome of AIT with NK-92.

Intraarterial Administration of Bone Marrow Mononuclear Cells in Patients With Critical Limb Ischemia A Randomized-Start, Placebo-Controlled Pilot Trial (PROVASA)

Background—Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia, but double-blind, randomized trials are lacking. Methods and Results—Forty patients with critical limb ischemia were included in a multicenter, phase II, double-blind, randomized-start trial to receive either intraarterial administration of BM-MNC or placebo followed by active treatment with BM-MNC (open label) after 3 months. Intraarterial administration of BM-MNC did not significantly increase ankle-brachial index and, thus, the trial missed its primary end point. However, cell therapy was associated with significantly improved ulcer healing (ulcer area, 3.2±4.7 cm2 to 1.89±3.5 cm2 [P=0.014] versus placebo, 2.92±3.5 cm2 to 2.89±4.1 cm2 [P=0.5]) and reduced rest pain (5.2±1.8 to 2.2±1.3 [P=0.009] versus placebo, 4.5±2.4 to 3.9±2.6 [P=0.3]) within 3 months. Limb salvage and amputation-free survival rates did not differ between the groups.Repeated BM-MNC administration and higher BM-MNC numbers and functionality were the only independent predictors of improved ulcer healing. Ulcer healing induced by repeated BM-MNC administration significantly correlated with limb salvage (r=0.8; P<0.001). Conclusions—Intraarterial administration of BM-MNC is safe and feasible and accelerates wound healing in patients without extensive gangrene and impending amputation. These exploratory findings of this pilot trial need to be confirmed in a larger randomized trial in patients with critical limb ischemia and stable ulcers. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00282646.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy

NK-92 is a highly cytotoxic natural killer (NK) tumor cell line that possesses properties that make it an excellent candidate for adoptive cellular immunotherapy. However, the cytotoxicity of NK cells is dependent on cytokines such as interleukin 2 (IL-2). Although NK-92 cells maintain cytotoxicity for a time after withdrawal of IL-2, clinical use will probably require prolonged treatment with fully activated cells to eliminate disease effectively. The ability to support cytotoxic cells with exogenously administered IL-2 is limited by associated toxicity. Therefore, we describe the transfection of the IL-2-dependent NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer to create two IL-2-independent variants, NK-92MI and NK-92 CI, and describe their characterization and comparison with parental cells. Both variants were shown to contain, express, and synthesize the hIL-2 cDNA. IL-2 synthesis was higher in NK-92MI cells compared with NK-92CI cells, with no expression in parental cells. Functionally, the cytotoxicity of all three cell lines was similar and coincubation with IL-2-independent variants did not affect hematopoietic progenitor cells. NK-92MI and NK-92CI cells were more radiosensitive than NK-92 cells, with proliferation inhibited at lower radiation doses and increased morality and decreased cytotoxicity compared with parental cells. Data presented here show that we have created by particle-mediated gene transfer two IL-2-independent variants of NK-92 that are identical to parental cells in virtually all respects, including high cytotoxic activity. The nonviral transfection of these cells makes them suitable for clinical applications. These IL-2-independent cells should allow prolonged treatment with fully active natural killer cells without the need for exogenous IL-2 support.

A Pilot Trial to Assess Potential Effects of Selective Intracoronary Bone Marrow–Derived Progenitor Cell Infusion in Patients With Nonischemic Dilated Cardiomyopathy Final 1-Year Results of the Transplantation of Progenitor Cells and Functional Regeneration Enhancement Pilot Trial in Patients With Nonischemic Dilated Cardiomyopathy

Background— Intracoronary administration of bone marrow–derived progenitor cells (BMC) was shown to improve coronary microvascular function in ischemic heart disease. Because coronary microvascular dysfunction is implicated in the pathogenesis and prognosis of nonischemic dilated cardiomyopathy (DCM), we investigated the effects of intracoronary BMC administration in patients with DCM. Methods and Results— Intracoronary infusion of BMC was performed in 33 patients with DCM by using an over-the-wire balloon catheter. Left ventricular contractility at baseline and after 3 months was assessed by analysis of left ventricular angiograms. Coronary hemodynamics were determined by intracoronary Doppler wire measurements. After 3 months, regional wall motion of the target area (contractility from −1.08±0.39 to −0.97±0.47 SD/chord, P =0.029) and global left ventricular ejection fraction (from 30.2±10.9 to 33.4±11.5%, P <0.001) were improved. Increase of regional contractile function was directly related to the functionality of the infused cells as measured by their colony-forming capacity. Minimal vascular resistance index was significantly reduced in the BMC-treated vessel after 3 months (from 1.53±0.63 to 1.32±0.61 mm Hg · s/cm; P =0.002, n=24), whereas no changes were observed in the reference vessel (from 1.60±0.45 to 1.49±0.45 mm Hg · s/cm; P =0.133, n=13). Twelve months after BMC infusion, N-terminal prohormone brain natriuretic peptide (NT-proBNP) serum levels were decreased, suggesting a beneficial effect on left ventricular remodeling processes (from 1610±993 to 1473±1147 pg/mL; P =0.038 for logNT-proBNP, n=26). Conclusions— Intracoronary administration of BMC seems to be associated with improvements in cardiac contractile and microvascular function in patients with DCM. Thus, randomized blinded studies are warranted to evaluate potential clinical benefits of intracoronary BMC administration in patients with DCM. Received August 26, 2008; accepted June 24, 2009.Background—Intracoronary administration of bone marrow–derived progenitor cells (BMC) was shown to improve coronary microvascular function in ischemic heart disease. Because coronary microvascular dysfunction is implicated in the pathogenesis and prognosis of nonischemic dilated cardiomyopathy (DCM), we investigated the effects of intracoronary BMC administration in patients with DCM. Methods and Results—Intracoronary infusion of BMC was performed in 33 patients with DCM by using an over-the-wire balloon catheter. Left ventricular contractility at baseline and after 3 months was assessed by analysis of left ventricular angiograms. Coronary hemodynamics were determined by intracoronary Doppler wire measurements. After 3 months, regional wall motion of the target area (contractility from −1.08±0.39 to −0.97±0.47 SD/chord, P=0.029) and global left ventricular ejection fraction (from 30.2±10.9 to 33.4±11.5%, P<0.001) were improved. Increase of regional contractile function was directly related to the functionality of the infused cells as measured by their colony-forming capacity. Minimal vascular resistance index was significantly reduced in the BMC-treated vessel after 3 months (from 1.53±0.63 to 1.32±0.61 mm Hg · s/cm; P=0.002, n=24), whereas no changes were observed in the reference vessel (from 1.60±0.45 to 1.49±0.45 mm Hg · s/cm; P=0.133, n=13). Twelve months after BMC infusion, N-terminal prohormone brain natriuretic peptide (NT-proBNP) serum levels were decreased, suggesting a beneficial effect on left ventricular remodeling processes (from 1610±993 to 1473±1147 pg/mL; P=0.038 for logNT-proBNP, n=26). Conclusions—Intracoronary administration of BMC seems to be associated with improvements in cardiac contractile and microvascular function in patients with DCM. Thus, randomized blinded studies are warranted to evaluate potential clinical benefits of intracoronary BMC administration in patients with DCM.

Effect of Shock Wave–Facilitated Intracoronary Cell Therapy on LVEF in Patients With Chronic Heart Failure: The CELLWAVE Randomized Clinical Trial

IMPORTANCE The modest effects of clinical studies using intracoronary administration of autologous bone marrow-derived mononuclear cells (BMCs) in patients with chronic postinfarction heart failure may be attributed to impaired homing of BMCs to the target area. Extracorporeal shock wave treatment has been experimentally shown to increase homing factors in the target tissue, resulting in enhanced retention of applied BMCs. OBJECTIVE To test the hypothesis that targeted cardiac shock wave pretreatment with subsequent application of BMCs improves recovery of left ventricular ejection fraction (LVEF) in patients with chronic heart failure. DESIGN, SETTING, AND PARTICIPANTS The CELLWAVE double-blind, randomized, placebo-controlled trial conducted among patients with chronic heart failure treated at Goethe University Frankfurt, Germany, between 2006 and 2011. INTERVENTIONS Single-blind low-dose (n = 42), high-dose (n = 40), or placebo (n = 21) shock wave pretreatment targeted to the left ventricular anterior wall. Twenty-four hours later, patients receiving shock wave pretreatment were randomized to receive double-blind intracoronary infusion of BMCs or placebo, and patients receiving placebo shock wave received intracoronary infusion of BMCs. MAIN OUTCOMES AND MEASURES Primary end point was change in LVEF from baseline to 4 months in the pooled groups shock wave + placebo infusion vs shock wave + BMCs; secondary end points included regional left ventricular function assessed by magnetic resonance imaging and clinical events. RESULTS The primary end point was significantly improved in the shock wave + BMCs group (absolute change in LVEF, 3.2% [95% CI, 2.0% to 4.4%]), compared with the shock wave + placebo infusion group (1.0% [95% CI, -0.3% to 2.2%]) (P = .02). Regional wall thickening improved significantly in the shock wave + BMCs group (3.6% [95% CI, 2.0% to 5.2%]) but not in the shock wave + placebo infusion group (0.5% [95% CI, -1.2% to 2.1%]) (P = .01). Overall occurrence of major adverse cardiac events was significantly less frequent in the shock wave + BMCs group (n = 32 events) compared with the placebo shock wave + BMCs (n = 18) and shock wave + placebo infusion (n = 61) groups (hazard ratio, 0.58 [95% CI, 0.40-0.85]; P = .02). CONCLUSIONS AND RELEVANCE Among patients with postinfarction chronic heart failure, shock wave-facilitated intracoronary administration of BMCs vs shock wave treatment alone resulted in a significant, albeit modest, improvement in LVEF at 4 months. Determining whether the increase in contractile function will translate into improved clinical outcomes requires confirmation in larger clinical end point trials. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00326989.

CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties

Background In vitro proliferative and differentiation potential of mesenchymal stromal cells generated from CD271+ bone marrow mononuclear cells (CD271-mesenchymal stromal cells) has been demonstrated in several earlier and recent reports. In the present study we focused, in addition to proliferative and differentiation potential, on in vitro and in vivo immunosuppressive and lymphohematopoietic engraftment-promoting potential of these mesenchymal stromal cells compared to bone marrow-derived mesenchymal stromal cells generated by plastic adherence (plastic adherence-mesenchymal stromal cells). Design and Methods We set up a series of experimental protocols in order to determine the phenotype of CD271-mesenchymal stromal cells, and their clonogenic, proliferative, differentiation and immunosuppressive potential. The potential of CD271-mesenchymal stromal cells to improve the engraftment of CD133+ hematopoietic stem cells at co-transplantation was evaluated in immunodeficient NOD/SCID-IL2Rγnull mice. Results In vitro studies demonstrated that CD271-mesenchymal stromal cells differentiate along adipogenic, osteogenic and chondrogenic lineages (trilineage potential), produce significantly higher levels of cytokines than plastic adherence-mesenchymal stromal cells, and significantly inhibit the proliferation of allogeneic T-lymphocytes in mixed lymphocyte reaction assays. Elevated levels of prostaglandin E2, but not nitric monoxide, mediated the majority of this immunosuppressive effect. In vivo studies showed that CD271-mesenchymal stromal cells promoted significantly greater lymphoid engraftment than did plastic adherence-mesenchymal stromal cells when co-transplanted with CD133+ hematopoietic stem cells at a ratio of 8:1 in immunodeficient NOD/SCID-IL2Rγnull mice. They induced a 10.4-fold increase in the number of T cells, a 2.5-fold increase in the number of NK cells, and a 3.6-fold increase in the number of B cells, indicating a major qualitative difference between these two mesenchymal stromal cell populations. Conclusions Our results indicate that CD271 antigen provides a versatile marker for prospective isolation and expansion of multipotent mesenchymal stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. The co-transplantation of such cells together with hematopoietic stem cells in patients with hematologic malignancies may prove valuable in the prevention of impaired/delayed T-cell recovery and graft-versus-host disease.