RESUMO
Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.
Assuntos
Células Matadoras Induzidas por Citocinas , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Animais , Proliferação de Células , Citotoxicidade Imunológica , Células Matadoras Naturais , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Linfócitos TRESUMO
BACKGROUND: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19+ malignancies. METHODS: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs. RESULTS: CB-CIKs, like PB-CIKs, were mostly CD3+ T cells with mean 45% CD3+CD56+ and expressing mostly TCR(T cell receptor)αß with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4+ cells, mostly CD56-, as well as a greater proportion of naïve CCR7+CD45RA+ and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8+ cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19+ tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph+ CD19+ acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8â¯×â¯106 CIK/kg. DISCUSSION: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/transplante , Sangue Fetal/citologia , Neoplasias/terapia , Animais , Antígenos CD19/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/transplante , Terapia Combinada , Células Matadoras Induzidas por Citocinas/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/fisiologia , Feminino , Sangue Fetal/imunologia , Humanos , Imunoterapia Adotiva/métodos , Recém-Nascido , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/transplante , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Resultado do TratamentoRESUMO
BACKGROUND: We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs). METHODS: For sterility tests 14-day cultures were performed in culture media detecting aerobic and anaerobic microorganisms. RESULTS: In this study, 22/1643 (1.3%) of apheretic products for autologous or allogeneic HSCT were contaminated, whereas 14/73 bone marrow (BM) harvests (17.8%) were positive. In 22 cases, the contaminated HSCs were infused to patients, but there was no evidence of any adverse impact of contamination on the hematologic engraftment or on infections. Indeed none of the five positive hemocultures detected in patients following infusion could be linked to the contaminated stem cell product. Our Cell Factory also generated 286 ATMPs in good manufacturing practice (GMP) conditions since 2007 and all final products were sterile. In three cases of mesenchymal stromal cell expansions, the starting BM harvests were contaminated, but the cell products at the end of expansion were sterile, presumably thanks to the presence of an antibiotic in the culture medium. DISCUSSION: The decreased rate of contamination of cell harvests observed with time suggests that routine sterility testing and communication of the results to the collecting centers may improve clinical practices. Furthermore, we recommend the use of antibiotics in the medium for ATMP expansion, to decrease the likelihood of expanding microorganisms within clean rooms. Finally we discuss the costs of sterility testing of ATMPs by GMP-approved external laboratories.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Esterilização/métodos , Remoção de Componentes Sanguíneos , Meios de Cultura , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Esterilização/economia , Fatores de TempoRESUMO
Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.
Assuntos
Células Matadoras Induzidas por Citocinas/transplante , Transplante de Células-Tronco Hematopoéticas/métodos , Transfusão de Linfócitos/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Humanos , Transfusão de Linfócitos/efeitos adversos , Masculino , Pessoa de Meia-Idade , Prognóstico , Recidiva , Indução de Remissão , Análise de Sobrevida , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
Ofatumumab (OFA) is a human anti-CD20 Ab approved for treatment of fludarabine-refractory B chronic lymphocytic leukemia (B-CLL). The efficacy of different immunotherapeutic strategies is best investigated in conditions that are as physiologic as possible. We have therefore compared the activity OFA and rituximab (RTX), alone or in combination with chemotherapeutic agents in unmanipulated whole blood assays, using flow cytometry. OFA (10-100 µg/ml) lysed B-CLL targets in whole blood more efficiently and with faster kinetics than RTX, with a mean 56% lysis at 24 h compared with 16%. This activity of OFA was fully complement dependent, as shown by >99% inhibition by anti-C5 Ab eculizumab and a lack of NK cell activation in whole blood. OFA-mediated NK cell activation was blocked by complement. OFA-mediated lysis could be increased an additional 15% by blocking CD55 and CD59 complement inhibitors. Interestingly, OFA-mediated lysis correlated significantly with CD20 expression levels (r(2) = 0.79). OFA showed overlapping dose response curves similar to those for RTX in phagocytosis assays using either human macrophages or neutrophils. However, phagocytosis was inhibited in the presence of serum or whole blood. Finally, combined treatment with mafosfamide and fludarabine showed that these therapeutic drugs are synergistic in B-CLL whole blood assays and show superior activity when combined with OFA compared with RTX. These results confirm in B-CLL samples and in physiologic conditions the superior complement mediated cytotoxicity induced by OFA alone compared with RTX, the lack of NK cell activation, and phagocytosis in these conditions and suggest effective chemoimmunotherapy strategies using this new generation anti-CD20 Ab.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Testes Imunológicos de Citotoxicidade/métodos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/sangue , Antígenos CD20/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/sangue , Morte Celular/imunologia , Ativação do Complemento/imunologia , Relação Dose-Resposta Imunológica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Rituximab , Células Tumorais CultivadasRESUMO
BACKGROUND AIMS: Hematopoietic stem cell cryopreservation significantly contributed to autologous stem cell transplantation (ASCT). Cryopreserved stem cell units (SCU) are expected to be used soon after harvesting for most purposes, but, in a number of cases, they remain stored for some time, creating an increasing load for SCU depositories. Disposal policies vary widely in each center, and the existing guidelines are insufficient. METHODS: We conducted a survey of seven Gruppo Italiano Trapianto di Midollo Osseo centers to investigate the outcome of SCU harvested from January 2005 to December 2009 for ASCT. The data from 1603 collections were gathered, for a total of 5822 SCU. RESULTS: In our cohort, 79% of patients collected >5 × 106 CD34+ cells/kg, and 3.4% collected <2 × 106 CD34+ cells/kg. Up to 21% of all the patients and 42% of those with acute leukemia did not undergo reinfusion, and 37% of the cryopreserved SCU were excess, resulting from patients not reinfusing or partially reinfusing. Less than one-third of the excess SCU was disposed, and the major causes of disposal were death and, in a minority of cases, withdrawal of the indication for ASCT. In our analysis, very few first reinfusions occurred after 2 years, and those after 5 years were exceptional. Through the use of a multivariate analysis, we sought to identify the risk factors for collection non-use, independent of the centers' policies. Non-use of SCU was significantly associated with patients with acute leukemia, collections of <2 × 106 CD34/kg and lower age groups. CONCLUSIONS: These data serve as a valid basis to support rational recommendations for cost-effective storage and disposal of SCU.
Assuntos
Criopreservação , Células-Tronco Hematopoéticas/citologia , Transplante de Células-Tronco/métodos , Autoenxertos/citologia , Autoenxertos/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Hematopoéticas/metabolismo , HumanosRESUMO
We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 µg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Antígenos CD20/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Engenharia de Proteínas/métodos , Alemtuzumab , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/sangue , Anticorpos Monoclonais Murinos/uso terapêutico , Anticorpos Antineoplásicos/sangue , Anticorpos Antineoplásicos/uso terapêutico , Proteínas do Sistema Complemento/fisiologia , Testes Imunológicos de Citotoxicidade/métodos , Relação Dose-Resposta Imunológica , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Rituximab , Células Tumorais CultivadasRESUMO
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have recently been identified as a therapeutic option in several clinical conditions. Whereas bone marrow (BM) is considered the main source of MSC (BM-MSC), the invasive technique required for collection and the decline in allogeneic donations call for alternative sources. Human umbilical cord (UC) represents an easily available source of MSC (UC-MSC). METHODS: Sections of full-term UC were transferred to cell culture flasks and cultured in 5% human platelet lysate (PL)-enriched medium. Neither enzymatic digestion nor blood vessel removal was performed. After 2 weeks, the adherent cells were harvested (P1), replated at low density and expanded for two consecutive rounds (P2 and P3). RESULTS: We isolated and expanded MSC from 9/9 UC. UC-MSC expanded with a mean fold increase (FI) of 42 735 ± 16 195 from P1 to P3 in a mean of 29 ± 2 days. By processing the entire cord unit, we theoretically could have reached a median of 9.5 × 10(10) cells (ranging from 1.0 × 10(10) to 29.0 × 10(10)). UC-MSC expressed standard surface markers; they contained more colony-forming unit (CFU)-fibroblast (F) and seemed less committed towards osteogenic, chondrogenic and adipogenic lineages than BM-MSC. They showed immunosuppressive properties both in vitro and in an in vivo chronic Graft versus Host disease (cGvHD) mouse model. Both array-Comparative Genomic Hybridization (CGH) analysis and karyotyping revealed no chromosome alterations at the end of the expansion. Animal studies revealed no tumorigenicity in vivo. CONCLUSIONS: UC constitute a convenient and very rich source of MSC for the production of third-party 'clinical doses' of cells under good manufacturing practice (GMP) conditions.
Assuntos
Células-Tronco Mesenquimais , Cordão Umbilical/citologia , Adipogenia , Animais , Plaquetas/citologia , Testes de Carcinogenicidade , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Hibridização Genômica Comparativa , Meios de Cultura , Modelos Animais de Doenças , Fibroblastos/citologia , Doença Enxerto-Hospedeiro/imunologia , Humanos , Imunossupressores/farmacologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Células-TroncoRESUMO
Because macrophages have been implicated as major players in the mechanism of action of rituximab, we have investigated the factors that modulate their tumor cell killing potential. Human macrophages, differentiated in vitro from peripheral blood monocytes, were used in binding and phagocytosis assays using B-chronic lymphocytic leukemia or lymphoma target cells opsonized with rituximab. Phagocytosis was maximal at 0.1 microg/ml rituximab and was not significantly affected by CD20 expression levels or by CD16A polymorphism at position 158 (Val/Phe). The role of FcgammaRs was demonstrated by complete inhibition of phagocytosis by excess human Igs. Because macrophages can be differentiated to M1- or M2-type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by proinflammatory stimuli (IFN-gamma/LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analyzed the effect of these different polarization programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2- to 3-fold greater phagocytic capacity compared with GM-CSF-induced cells. Furthermore, addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF- and GM-CSF-differentiated macrophages. LPS/IFN-gamma had little effect. Expression of CD16, CD32, and CD64 in different macrophage populations correlated with phagocytic activity. Interestingly, several B lymphoma cell lines were observed to secrete 400-1300 pg/ml IL-10 in vitro, and coculture of human macrophages with lymphoma conditioned medium increased significantly their phagocytic capacity. Our data suggest that cytokines secreted by lymphoma cells can favor alternate activation of macrophages with a high phagocytic capacity toward rituximab-opsonized targets.
Assuntos
Anticorpos Monoclonais/imunologia , Transtornos Linfoproliferativos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Proteínas Opsonizantes/imunologia , Fagocitose/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Western Blotting , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Imunofluorescência , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Técnicas In Vitro , Ativação de Macrófagos/imunologia , Fator Estimulador de Colônias de Macrófagos , Macrófagos/metabolismo , Proteínas Opsonizantes/farmacologia , Fagocitose/efeitos dos fármacos , Polimorfismo Genético , Receptores de IgG/imunologia , RituximabRESUMO
BACKGROUND AIMS: Human multipotent mesenchymal stromal cells (hMSC) are considered good candidates for a growing spectrum of cell therapies. We have validated a protocol that makes use of the washouts of discarded collection sets, left over at the end of the filtration of bone marrow (BM) explants performed for hematopoietic stem cell (HSC) transplantation. METHODS: The method consists of direct plating of cells without density-gradient isolation followed by two detachment steps and expansion in 5% human platelet lysate (hPL). RESULTS: In a median of 26 days, 14 bags for adult patients and nine bags for pediatric patients for a standard dose of 1x10(6) hMSC/kg body weight could be prepared from the expansion of a fraction of the cells recovered from seven independent washouts. Moreover, 151 vials could be frozen from the remaining cells. The theoretical full expansion of all the frozen vials (validated by the expansion of two independent vials) could have allowed the production of 173 bags for adults and 348 bags for pediatric patients. CONCLUSIONS: The washouts of discarded bags and filters left over at the end of routine BM explants filtration are a very abundant source of hMSC precursors that can be easily utilized for clinical applications.
Assuntos
Células da Medula Óssea/citologia , Separação Celular/métodos , Filtração , Células-Tronco Mesenquimais/citologia , Manejo de Espécimes/métodos , Diferenciação Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Criopreservação , Humanos , Imunofenotipagem , Controle de QualidadeAssuntos
Anticorpos Biespecíficos , Leucemia Linfocítica Crônica de Células B , Humanos , Anticorpos Biespecíficos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Feminino , Linfócitos T/imunologia , Pessoa de Meia-Idade , Idoso , Reconstituição Imune , Adulto , Imunoterapia Adotiva/efeitos adversosRESUMO
Bone marrow-mesenchymal stem cells (BM-MSC) ameliorate renal dysfunction and repair tubular damage of acute kidney injury by locally releasing growth factors, including the insulin-like growth factor-1 (IGF-1). The restricted homing of BM-MSC at the site of injury led us to investigate a possible gene-based communication mechanism between BM-MSC and tubular cells. Human BM-MSC (hBM-MSC) released microparticles and exosomes (Exo) enriched in mRNAs. A selected pattern of transcripts was detected in Exo versus parental cells. Exo expressed the IGF-1 receptor (IGF-1R), but not IGF-1 mRNA, while hBM-MSC contained both mRNAs. R- cells lacking IGF-1R exposed to hBM-MSC-derived Exo acquired the human IGF-1R transcript that was translated in the corresponding protein. Transfer of IGF-1R mRNA from Exo to cisplatin-damaged proximal tubular cells (proximal tubular epithelial cell [PTEC]) increased PTEC proliferation. Coincubation of damaged PTEC with Exo and soluble IGF-1 further enhanced cell proliferation. These findings suggest that horizontal transfer of the mRNA for IGF-1R to tubular cells through Exo potentiates tubular cell sensitivity to locally produced IGF-1 providing a new mechanism underlying the powerful renoprotection of few BM-MSC observed in vivo.