RESUMEN
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.
Asunto(s)
Médula Ósea/patología , Leucemia Mieloide Aguda/patología , Nicho de Células Madre , Andamios del Tejido/química , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Autorrenovación de las Células , Separación Celular , Células Clonales , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , Leucemia Mieloide Aguda/genética , Células Madre Mesenquimatosas/citología , Ratones , Fenotipo , Células del Estroma/patologíaRESUMEN
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.
Asunto(s)
ADP-Ribosil Ciclasa 1/química , ADP-Ribosil Ciclasa 1/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Afinidad de Anticuerpos/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunoterapia Adoptiva , Activación de Linfocitos/inmunología , Ratones , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia , Unión Proteica/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
ADP-Ribosil Ciclasa 1/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Adulto , Antineoplásicos/farmacología , Femenino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Antígenos CD/fisiología , Macrófagos/efectos de la radiación , Microvasos/efectos de la radiación , Traumatismos por Radiación/etiología , Receptores de Superficie Celular/fisiología , Piel/irrigación sanguínea , Factor de Crecimiento Transformador beta/fisiología , Adulto , Anciano , Biopsia , Western Blotting , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Endoglina , Femenino , Humanos , Inmunohistoquímica , Sistema Linfático/efectos de la radiación , Persona de Mediana Edad , Fosforilación/efectos de la radiación , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Piel/efectos de la radiación , Factor de Crecimiento Transformador beta/efectos de la radiaciónRESUMEN
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.