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1.
J Transl Med ; 17(1): 82, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30871636

RESUMO

BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.


Assuntos
Interferon alfa-2/farmacologia , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Monócitos/metabolismo , Polietilenoglicóis/farmacologia , Animais , Contagem de Células , Morte Celular/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Criopreservação , Feminino , Humanos , Interferon alfa-2/toxicidade , Interferon-alfa/toxicidade , Interferon gama/toxicidade , Camundongos , Monócitos/efeitos dos fármacos , Polietilenoglicóis/toxicidade , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/toxicidade
2.
J Transl Med ; 16(1): 13, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29368612

RESUMO

BACKGROUND: Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. METHODS: TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. RESULTS: The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. CONCLUSION: Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.


Assuntos
Técnicas de Cultura de Células/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Transdução Genética , Proliferação de Células , Sobrevivência Celular , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/imunologia , Papillomaviridae/metabolismo , Fenótipo
3.
Blood ; 128(13): 1688-700, 2016 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-27412889

RESUMO

Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 10(6) CAR(+) T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient's MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967.


Assuntos
Antígeno de Maturação de Linfócitos B/imunologia , Imunoterapia Adotiva/métodos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Linfócitos T/imunologia , Antígeno de Maturação de Linfócitos B/sangue , Medula Óssea/imunologia , Medula Óssea/patologia , Citocinas/sangue , Humanos , Imunoterapia Adotiva/efeitos adversos , Leucopenia/etiologia , Mieloma Múltiplo/sangue , Proteínas do Mieloma/metabolismo , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/imunologia , Indução de Remissão , Trombocitopenia/etiologia , Carga Tumoral/imunologia
4.
Int J Oncol ; 24(4): 861-7, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15010823

RESUMO

Breast cancer cells pose a difficult target for detection due to their lack of tumor specific markers. Small numbers of metastatic cells circulate through the blood and reach target organs, but current methods are presently not sensitive enough to allow for early detection. Our goal is to develop a method for specific and sensitive detection for breast cancer tumor cells by using cell enrichment coupled to RT-PCR with selective primer pairs. Fifty million normal donor mononuclear cells (NDMNC) were spiked with serial dilutions of a breast cancer cell line, positively selected for surface expression of the epithelial cell adhesion molecule (EPCAM) with anti-Ber-EP4 mAb. Total RNA was isolated and the cDNA was transcribed and measured by quantitative PCR for cytokeratin 19 (CK19), epidermal growth factor receptor (EGF-R), and beta-2-microglobulin (beta2M) transcripts using specific hybridization probes with the LightCycler System. A sensitivity of 1 tumor cell in 5 million NDMNC was consistently achieved with a metastatic breast cancer cell line with target primer sets for CK19 and EGF-R. Less, but appreciable sensitivity was achieved by spiking NDMNC with other breast cancer cells. Detection of both gene targets ranged between 1 in 5,000 to 5 million cells. The method described, a cell enrichment procedure and RT-PCR using specific hybridization probes (spanning introns and hybridizing to areas that discriminate transcribed pseudogenes), results in increased sensitivity while decreasing false positives above those methods previously reported. This general approach may have wide applicability to increasing sensitivity of minimal residual disease detection in other cancers.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/química , DNA de Neoplasias/análise , Receptores ErbB/genética , Queratinas/genética , Neoplasias da Mama/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasia Residual , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Células Tumorais Cultivadas , Microglobulina beta-2/genética
5.
Blood ; 111(8): 4392-402, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17878399

RESUMO

Selective allodepletion is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were cocultured with responder cells from HLA-matched or -mismatched donors. Optimal results were achieved when cocultured cells were incubated with 7.5 muM TH9402, followed by dye extrusion and exposure to 5 Joule/cm(2) light energy at 5 x 10(6) cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100 000, while third-party responses remained higher than 1:10 000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Teste de Histocompatibilidade , Depleção Linfocítica/métodos , Rodaminas/farmacologia , Linfócitos T/citologia , Linfócitos T/imunologia , Doadores de Tecidos , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/efeitos da radiação , Antígenos CD4/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Ficoll , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunidade/efeitos dos fármacos , Imunidade/efeitos da radiação , Interleucina-2/farmacologia , Espaço Intracelular/efeitos dos fármacos , Luz , Muromonab-CD3/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/microbiologia , Linfócitos T/virologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos da radiação , Fatores de Tempo
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