RESUMO
To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches.
Assuntos
Medula Óssea/patologia , Leucemia Mieloide Aguda/patologia , Nicho de Células-Tronco , Alicerces Teciduais/química , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Autorrenovação Celular , Separação Celular , Células Clonais , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia Mieloide Aguda/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Fenótipo , Células Estromais/patologiaRESUMO
Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using "light-chain exchange" technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated â¼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of â¼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD382+ MM cells, but spared CD38+ healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs.
Assuntos
ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão , ADP-Ribosil Ciclase 1/metabolismo , Animais , Afinidade de Anticorpos/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/terapia , Ligação Proteica/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Adulto , Antineoplásicos/farmacologia , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Resultado do TratamentoRESUMO
BACKGROUND AND PURPOSE: Microvascular damage is an important component of late radiation-induced morbidity. In our pre-clinical models, we demonstrated that repair of vessel injury is dependent on proper endoglin-mediated transforming growth factor-beta (TGF-ß) signalling and that it can be affected by infiltrating macrophages. We now wanted to extend these findings in irradiated patients, using skin as a model system, and assess whether bisphosphonates could modulate the response. MATERIALS AND METHODS: Paired skin biopsies from irradiated and non-irradiated sites were obtained from 48 breast cancer patients. In 8 patients, biopsies were repeated after 4months of bisphosphonate treatment. Immunohistochemistry was used to assess vascular alterations and leucocyte infiltration. Western Blot and qPCR were used to assess expression of growth factors and their receptors. RESULTS: Decreased blood vessel numbers at early time points were followed by increased endoglin expression and restoration of vessel number. Loss of small lymphatic vessels was associated with increased TGF-ß levels, whereas dilation of lymphatic vessels correlated with increased macrophage infiltration. Bisphosphonate treatment reduced leucocyte infiltration, but also prevented restoration of blood vessel numbers after irradiation. CONCLUSION: Radiation injury of the microvasculature is mediated through TGF-ß, whereas repair is modulated by the co-receptor endoglin and promoted by macrophages.
Assuntos
Antígenos CD/fisiologia , Macrófagos/efeitos da radiação , Microvasos/efeitos da radiação , Lesões por Radiação/etiologia , Receptores de Superfície Celular/fisiologia , Pele/irrigação sanguínea , Fator de Crescimento Transformador beta/fisiologia , Adulto , Idoso , Biópsia , Western Blotting , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Endoglina , Feminino , Humanos , Imuno-Histoquímica , Sistema Linfático/efeitos da radiação , Pessoa de Meia-Idade , Fosforilação/efeitos da radiação , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Pele/efeitos da radiação , Fator de Crescimento Transformador beta/efeitos da radiaçãoRESUMO
PURPOSE: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide- and bortezomib-refractory patients. EXPERIMENTAL DESIGN: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide- and bortezomib-refractory multiple myeloma patients. RESULTS: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide- and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumab-mediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide- and bortezomib-refractory patient. CONCLUSIONS: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide- and bortezomib-refractory patients.