Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.
The primary aim of this clinical trial was to determine the feasibility of delivering first-generation CAR T cell therapy to patients with advanced, CEACAM5+ malignancy. Secondary aims were to assess clinical efficacy, immune effector function and optimal dose of CAR T cells. Three cohorts of patients received increasing doses of CEACAM5+-specific CAR T cells after fludarabine pre-conditioning plus systemic IL2 support post T cell infusion. Patients in cohort 4 received increased intensity pre-conditioning (cyclophosphamide and fludarabine), systemic IL2 support and CAR T cells. No objective clinical responses were observed. CAR T cell engraftment in patients within cohort 4 was significantly higher. However, engraftment was short-lived with a rapid decline of systemic CAR T cells within 14 days. Patients in cohort 4 had transient, acute respiratory toxicity which, in combination with lack of prolonged CAR T cell persistence, resulted in the premature closure of the trial. Elevated levels of systemic IFNγ and IL-6 implied that the CEACAM5-specific T cells had undergone immune activation in vivo but only in patients receiving high-intensity pre-conditioning. Expression of CEACAM5 on lung epithelium may have resulted in this transient toxicity. Raised levels of serum cytokines including IL-6 in these patients implicate cytokine release as one of several potential factors exacerbating the observed respiratory toxicity. Whilst improved CAR designs and T cell production methods could improve the systemic persistence and activity, methods to control CAR T ‘on-target, off-tissue’ toxicity are required to enable a clinical impact of this approach in solid malignancies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-017-2034-7) contains supplementary material, which is available to authorized users.
Antigen-specific T lymphocytes are attractive as potential anticancer agents. The generation of large numbers of antigen-specific T cells is possible through the use of gene therapy to express targeting receptors on the T lymphocyte. Activated T lymphocytes were transduced to express carcino-embryonic antigen or neural cell adhesion molecule targeted CD3zeta chimeric immune receptors. The chimeric receptors were expressed as homodimers and also as heterodimers with the native CD3zeta. T lymphocyte populations were expanded in the absence of selection for the modified cells and were shown to produce cytokines when cultured in the presence of immobilized purified protein antigen. These lymphocytes also responded by cytokine production and cytolytic activity when challenged with tumor-cell lines expressing the antigen recognized by the chimeric immune receptor. The cytolytic activity appears to be largely perforin mediated. Furthermore, soluble carcino-embryonic antigen did not interfere with the functional activity of the carcino-embryonic antigen-targeted lymphocytes. Long-term (5-day) stimulation of the modified lymphocytes by protein antigen resulted in reduced viability similar to that induced by anti-CD3 antibodies alone. Viability was improved by a costimulatory signal indicating that such signals may be vital in the maintenance of long-term functional activity of receptor modified T lymphocytes.
We have generated murine T cells expressing chimeric immune receptors (CR) against human 5T4 oncofetal Ag (h5T4) and evaluated their tumor therapeutic efficacy alone and in combination with immunization using a replication-defective adenovirus encoding h5T4 (Rad.h5T4) and bone marrow-derived dendritic cells (BMDC). The h5T4-specific engineered T cells demonstrated Ag-specific, non-MHC-restricted cytolysis of h5T4-positive B16 and CT26 tumor cells in vitro by cytotoxicity assay and antitumor activity in vivo using a Winn assay. In the s.c. injected B16h5T4 melanoma model, early local but not systemic i.v. administration of syngeneic h5T4-specific CR T cells significantly increased mice survival. This improvement was further enhanced when combined with immunization with Rad.h5T4, followed by post-CR T cell treatment with BMDC in the active therapy model, possibly through mechanisms of enhancing Ag-specific cellular immune responses. This synergistic effect was lost without delivery of the BMDC. Our findings suggest that combining engineered T cells with specific vaccination strategies can improve the active tumor therapy.
Adoptive cell therapy employing gene-modified T-cells expressing chimeric antigen receptors (CARs) has shown promising preclinical activity in a range of model systems and is now being tested in the clinical setting. The manufacture of CAR T-cells requires compliance with national and European regulations for the production of medicinal products. We established such a compliant process to produce T-cells armed with a first-generation CAR specific for carcinoembryonic antigen (CEA). CAR T-cells were successfully generated for 14 patients with advanced CEA(+) malignancy. Of note, in the majority of patients, the defined procedure generated predominantly CD4(+) CAR T-cells with the general T-cell population bearing an effector-memory phenotype and high in vitro effector function. Thus, improving the process to generate less-differentiated T-cells would be more desirable in the future for effective adoptive gene-modified T-cell therapy. However, these results confirm that CAR T-cells can be generated in a manner compliant with regulations governing medicinal products in the European Union.
Adenosine is generated from adenosine triphosphate, which is released by stressed and damaged cells. Adenosine levels are significantly increased in patients with bronchial asthma (BA) and mediate mast cell degranulation and bronchoconstriction. Over the last decade, increasing evidence has shown that adenosine can modulate the innate immune response during monocytes differentiation towards mature myeloid cells. These adenosine-differentiated myeloid cells, characterized by co-expression of monocytes/macrophages and dendritic cell markers such as CD14 and CD209, produce high levels of pro-inflammatory cytokines, thus contributing to the pathogenesis of BA and chronic obstructive pulmonary disease. We found that expression of ADORA2A and ADORA2B are increased in monocytes obtained from patients with BA, and are associated with the generation of CD14posCD209pos pro-inflammatory cells. A positive correlation between expression of ADORA2B and IL-6 was identified in human monocytes and may explain the increased expression of IL-6 mRNA in asthmatics. Taken together, our results suggest that monocyte-specific expression of A2 adenosine receptors plays an important role in pro-inflammatory activation of human monocytes, thus contributing to the progression of asthma.
Одними из наиболее распространенных хронических заболеваний бронхолегочной системы среди взрослого населения, приводящих к существенному снижению ка-чества жизни, обусловливающих раннюю инвалидизацию и высокую смертность больных являются хроническая обструктивная болезнь легких (ХОБЛ) и бронхиальная астма (БА). Данные заболевания причисляются к соци-ально значимым, так как они сопряжены с существенным экономическим и социальным ущербом. ХОБЛ и БА ха-рактеризуются как высокими прямыми (медикаментозное обеспечение, экстренная медицинская помощь), так и косвенными (длительные периоды нетрудоспособности, выплаты по инвалидности) затратами ресурсов здравоох-ранения [1]. Этиология данных заболеваний на настоящий момент четко не установлена, патогенез же связывают с персистирующим легочным воспалением, которое при ХОБЛ связано с активацией преимущественно Th1-звена, а при БА -Th2. Клиническая картина обеих патологий ха-рактеризуется обструкцией дыхательных путей -лабиль-ной в случае БА и постоянной при ХОБЛ, полифенотипич-ностью [2], а также значительным влиянием обострений на состояние здоровья, качество жизни больных и прогноз. В настоящее время человеческий организм принято рассматривать в свете его симбиотических отношений с населяющими его микроорганизмами [3]. Микробиота представляет собой сложную систему взаимосвязанных сообществ микроорганизмов, таких как бактерии, виру-сы, археи, грибки и простейшие, населяющих различные биотопы организма человека -желудочно-кишечный тракт, кожу с ее придатками и легкие. В состав самого обширного биотопа организма -кишечника -входят более 1000 видов различных бактерий, совокупный геном которых превышает геном человека более чем в 150 раз [4]. Макроорганизм и его микробное население в нор-мальных условиях находятся в состоянии динамического равновесия, поэтому для микрофлоры каждой области тела человека характерно относительное постоянство, и имеются эволюционно сложившиеся закономерности в количественном и качественном (видовом) ее составе как на уровне организма в целом, так и отдельных его органов и систем. Изменения состояния макроорганизма выража-ются сменой микробного пейзажа всех участков тела.На протяжении долгого времени основным методом оценки бактериального разнообразия являлось класси-ческое культивирование бактерий на питательных сре-дах. Однако культуральные методики не дают полной
Молекулярные и фармакогенетические механизмы тяжелой бронхиальной астмы В обзоре обобщены результаты исследований по определению доминирующих механизмов формирования и персистенции воспаления при тяжелой бронхиальной астме и результаты фармакогенетических исследований детерминации ответа на лекарственные средства. Данные механизмы в перспективе могут быть использованы как в диагностических целях, так и стать новыми таргетными мишенями терапии бронхиальной астмы. Применение фармакогенетической информации сделает возможным использование персонифицированного подхода к терапии бронхиальной астмы, что позволит скорректировать технологии ведения пациента и повысить вероятность достижения контроля болезни. Ключевые слова: бронхиальная астма, терапия, тяжелая бронхиальная астма, терапевтическая резистентность, молекулярные механизмы.
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