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1.
Blood ; 128(17): 2165-2174, 2016 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-27531680

RESUMO

Although double umbilical cord blood transplantation (dUCBT) in adult patients may be associated with less graft failure compared with single UCBT, hematopoietic recovery generally originates from a single cord blood unit (CBU). CBU predominance is still incompletely understood. We recently showed that blood CD4+ T-cell numbers rapidly increase after dUCBT, and early CD4+ T-cell chimerism predicts for graft predominance. Given the frequent HLA class II allele mismatches between CBUs in dUCBT, we hypothesized that alloreactive HLA class II-specific CD4+ T cells from the "winning" CBU may contribute to rejection of the "loser" CBU. We evaluated whether CD4+ T cells originating from the predominant (PD)-CBU would recognize HLA class II allele mismatches, expressed by the nonengrafting (NE)-CBU. Alloreactive effector CD4+ T cells toward 1 or more mismatched HLA class II alleles of the NE-CBU were detected in 11 of 11 patients, with reactivity toward 29 of 33 (88%) tested mismatches, and the strongest reactivity toward DR and DQ alleles early after dUCBT. Mismatched HLA class II allele-specific CD4+ T cells recognized primary leukemic cells when the mismatched HLA class II allele was shared between NE-CBU and patient. Our results suggest that cytotoxicity exerted by CD4+ T cells from the PD-CBU drives the rapid rejection of the NE-CBU, whose alloreactive effect might also contribute to graft-versus-leukemia.


Assuntos
Aloenxertos/imunologia , Linfócitos T CD4-Positivos/imunologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Efeito Enxerto vs Leucemia/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Adulto , Alelos , Anemia Aplástica/terapia , Animais , Quimerismo , Feminino , Citometria de Fluxo , Humanos , Leucemia/terapia , Linfoma não Hodgkin/terapia , Masculino , Pessoa de Meia-Idade
2.
Clin Immunol ; 169: 107-113, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27377533

RESUMO

Autologous T-cells genetically modified to express a chimeric antigen receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to twelve patients with CAIX-positive metastatic renal cell carcinoma. Here, we questioned whether plasma cytokine levels following treatment or in vitro cytokine production from the T-cell infusion products could serve as predictors for peripheral T-cell persistence or in vivo T-cell activity. We demonstrated that CAR surface as well as gene expression are down-regulated following T-cell infusion, and that peripheral numbers of CAR T-cells are best captured by flow cytometry and not by qPCR. Numbers of CAR T-cells in blood correlated with plasma levels of IFN-γ and IL-6, but not with any of the other cytokines tested. Plasma IFN-γ or IL-6 levels did not correlate with liver enzyme values. Thus, out of 27 cytokines tested, IFN-γ and IL-6 levels in plasma are potential surrogate markers for CAR T-cell persistence in solid tumors.


Assuntos
Carcinoma de Células Renais/imunologia , Interferon gama/imunologia , Interleucina-6/imunologia , Neoplasias Renais/imunologia , Linfócitos T/imunologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/terapia , Citocinas/sangue , Citocinas/imunologia , Citometria de Fluxo , Humanos , Imunoterapia Adotiva/métodos , Interferon gama/sangue , Interleucina-6/sangue , Neoplasias Renais/metabolismo , Neoplasias Renais/terapia , Contagem de Linfócitos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Linfócitos T/transplante , Fatores de Tempo
3.
Biochem Soc Trans ; 44(3): 951-9, 2016 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-27284065

RESUMO

We studied safety and proof of concept of a phase I/II trial with chimeric antigen receptor (CAR) T-cells in patients with metastatic renal cell carcinoma (mRCC). The CAR was based on the G250 mAb that recognized an epitope of carboxy-anhydrase-IX (CAIX). Twelve patients with CAIX+ mRCC were treated in three cohorts with a maximum of 10 daily infusions of 2×10(7) to 2×10(9) CAR T-cells. Circulating CAR T-cells were transiently detectable in all patients and maintained antigen-specific immune functions following their isolation post-treatment. Blood cytokine profiles mirrored CAR T-cell presence and in vivo activity. Unfortunately, patients developed anti-CAR T-cell antibodies and cellular immune responses. Moreover, CAR T-cell infusions induced liver enzyme disturbances reaching CTC grades 2-4, which necessitated cessation of treatment in four out of eight patients (cohort 1+2). Examination of liver biopsies revealed T-cell infiltration around bile ducts and CAIX expression on bile duct epithelium, adding to the notion of on-target toxicity. No such toxicities were observed in four patients that were pretreated with G250 mAb (cohort 3). The study was stopped due to the advent of competing treatments before reaching therapeutic or maximum tolerated dose in cohort 3. No clinical responses have been recorded. Despite that, from this trial numerous recommendations for future trials and their immune monitoring could be formulated, such as choice of the target antigen, format and immunogenicity of receptor and how the latter relates to peripheral T-cell persistence.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/secundário , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Receptores de Antígenos de Linfócitos T/uso terapêutico , Linfócitos T , Carcinoma de Células Renais/imunologia , Citocinas/sangue , Humanos , Neoplasias Renais/imunologia , Proteínas Mutantes Quiméricas/uso terapêutico , Resultado do Tratamento
5.
Blood ; 117(1): 72-82, 2011 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-20889925

RESUMO

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted with a chimeric antibody receptor (CAR) directed toward carbonic anhydrase IX (CAIX), an antigen highly expressed in renal cell carcinoma. In the majority of patients, we observed distinct humoral and/or cellular anti-CAIX-CAR T-cell immune responses in combination with a limited peripheral persistence of transferred CAIX-CAR T cells in the majority of patients. Humoral immune responses were anti-idiotypic in nature and neutralized CAIX-CAR-mediated T-cell function. Cellular anti-CAIX-CAR immune responses were directed to the complementarity-determining and framework regions of the CAR variable domains. In addition, 2 patients developed immunity directed against presumed retroviral vector epitopes. Here, we document the novel feature that therapeutic cells, which were ex vivo engineered by means of transduction with a minimal γ-retroviral vector, do express immunogenic vector-encoded epitopes, which might compromise persistence of these cells. These observations may constitute a critical concern for clinical ex vivo γ-retroviral gene transduction in general and CAR-retargeted T-cell therapy in particular, and underscore the need to attenuate the immunogenicity of both transgene and vector.


Assuntos
Antígenos de Neoplasias/imunologia , Anidrases Carbônicas/imunologia , Carcinoma de Células Renais/terapia , Engenharia Genética , Vetores Genéticos/imunologia , Neoplasias Renais/terapia , Linfócitos T/imunologia , Transgenes/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Carcinoma de Células Renais/imunologia , Estudos de Coortes , Humanos , Neoplasias Renais/imunologia , Ativação Linfocitária , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Retroviridae/genética , Transgenes/fisiologia
6.
Cytotherapy ; 15(5): 620-6, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23388583

RESUMO

BACKGROUND AIMS: The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan). METHODS: Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time. RESULTS: We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years. CONCLUSIONS: High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies.


Assuntos
Carcinoma de Células Renais/terapia , Terapia Baseada em Transplante de Células e Tecidos , Neoplasias Renais/terapia , Receptores de Antígenos de Linfócitos T , Linfócitos T/imunologia , Adulto , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Anidrases Carbônicas/imunologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Engenharia Celular , Linhagem Celular , Fibronectinas/administração & dosagem , Humanos , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Masculino , Muromonab-CD3/administração & dosagem , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes/administração & dosagem , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/efeitos dos fármacos
7.
Cancer ; 118(23): 6005-11, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22736424

RESUMO

BACKGROUND: Inflammation may underlie cancer-related fatigue; however, there are no studies that assess the relation between fatigue and cytokines in patients with advanced disease versus patients without disease activity. Furthermore, the relation between cytokines and the separate dimensions of fatigue is unknown. Here, association of plasma levels of inflammatory markers with physical fatigue and mental fatigue was explored in advanced cancer patients and cancer survivors. METHODS: A total of 45 advanced cancer patients and 47 cancer survivors completed the subscales Physical Fatigue and Mental Fatigue of the Multidimensional Fatigue Inventory. Plasma concentrations of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1-ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and neopterin were measured. Nonparametric tests were used to assess differences in fatigue intensity and levels of inflammatory markers and to determine correlation coefficients between the fatigue dimensions and inflammatory markers. RESULTS: Compared with cancer survivors, patients with advanced cancer had higher levels of physical fatigue (median 16 vs 9, P < .001) and mental fatigue (median 11 vs 6, P = .01). They also had higher levels of all cytokines (P < .01). In advanced cancer, CRP (r = 0.49, P = .001), IL-6 (r = 0.43, P = .003), IL-1-ra (r = 0.32, P = .03), and neopterin (r = 0.25, P = .10) were correlated with physical but not with mental fatigue. In cancer survivors, only IL-1-ra was related to both physical fatigue (r = 0.24, P = .10) and mental fatigue (r = 0.35, P = .02). CONCLUSIONS: In advanced cancer, inflammation seems to be associated with physical fatigue, but not to mental fatigue. In cancer survivors, there was no convincing evidence that inflammation plays a major role in fatigue.


Assuntos
Fadiga/etiologia , Inflamação/complicações , Neoplasias/complicações , Neoplasias/patologia , Sobreviventes , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/análise , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-6/sangue , Interleucina-8/sangue , Masculino , Pessoa de Meia-Idade , Neoplasias/classificação
8.
Clin Cancer Res ; 28(8): 1572-1585, 2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35176144

RESUMO

PURPOSE: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma. PATIENTS AND METHODS: Dose-escalation phase I study with 3+3 cohorts, dosing 107 to 1 × 1011 viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding. RESULTS: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2-associated cytokine levels and numbers of CD3+ T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+ and CD8+ T cells. No evidence of viral shedding in excreta was observed. CONCLUSIONS: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses.


Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Adenoviridae/genética , Convecção , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Terapia Viral Oncolítica/efeitos adversos , Vírus Oncolíticos/genética
9.
J Immunother Cancer ; 8(2)2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33427690

RESUMO

BACKGROUND: Many cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may predict clinical outcome. METHODS: Here a total of 160 patients with advanced melanoma or non-small-cell lung cancer (NSCLC), treated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed cell death 1 (anti-PD-1), were divided into five discovery and cross-validation cohorts. The kinase activity profile was generated by analyzing phosphorylation of peripheral blood mononuclear cell lysates in a microarray comprising of 144 peptides derived from sites that are substrates for protein tyrosine kinases. Binary grouping into patients with or without clinical benefit was based on Response Evaluation Criteria in Solid Tumors V.1.1. Predictive models were trained using partial least square discriminant analysis (PLS-DA), performance of the models was evaluated by estimating the correct classification rate (CCR) using cross-validation. RESULTS: The kinase phosphorylation signatures segregated responders from non-responders by differences in canonical pathways governing T-cell migration, infiltration and co-stimulation. PLS-DA resulted in a CCR of 100% and 93% in the anti-CTLA-4 and anti-PD1 melanoma discovery cohorts, respectively. Cross-validation cohorts to estimate the accuracy of the predictive models showed CCRs of 83% for anti-CTLA-4 and 78% or 68% for anti-PD-1 in melanoma or NSCLC, respectively. CONCLUSION: Blood-based kinase activity profiling for response prediction to immune checkpoint inhibitors in melanoma and NSCLC revealed increased kinase activity in pathways associated with T-cell function and led to a classification model with a highly accurate classification rate in cross-validation groups. The predictive value of kinase activity profiling is prospectively verified in an ongoing trial.


Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Adulto , Idoso , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/patologia
10.
J Immunother Cancer ; 7(1): 149, 2019 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176366

RESUMO

BACKGROUND: Checkpoint inhibitors have become standard care of treatment for non-small cell lung cancer (NSCLC), yet only a limited fraction of patients experiences durable clinical benefit, highlighting the need for markers to stratify patient populations. METHODS: To prospectively identify patients showing response to therapy, we have stained peripheral blood samples of NSCLC patients treated with 2nd line nivolumab (n = 71), as well as healthy controls, with multiplex flow cytometry. By doing so, we enumerated 18 immune cell subsets and assessed expression for 28 T cell markers, which was followed by dimensionality reduction as well as rationale-based analyses. RESULTS: In patients with a partial response (PR), representing best overall response (BOR) according to RECIST v1.1, the number of CD8 T cells at baseline and during treatment is similar to those of healthy controls, but 2-fold higher than in patients with progressive and stable disease (PD and SD). CD8 T cell populations in PR patients show enhanced frequencies of T effector memory re-expressing CD45RA (TEMRA) cells, as well as T cells that express markers of terminal differentiation (CD95+) and egression from tumor tissue (CD69-). In PR patients, the fraction of CD8 T cells that lacks co-stimulatory receptors (CD28, ICOS, CD40L, 4-1BB, OX40) correlates significantly with the total numbers and differentiated phenotype of CD8 T cells. CONCLUSIONS: This study demonstrates that high numbers of peripheral CD8 T cells expressing differentiation markers and lacking co-stimulatory receptors at baseline are associated with response to nivolumab in NSCLC patients.


Assuntos
Antígenos CD28/genética , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Antígenos Comuns de Leucócito/genética , Neoplasias Pulmonares/tratamento farmacológico , Nivolumabe/uso terapêutico , Receptores CCR7/genética , Idoso , Antineoplásicos Imunológicos , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Nivolumabe/farmacologia , Estudos Prospectivos
11.
Front Immunol ; 9: 2034, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30245692

RESUMO

Introduction: Malignant pleural mesothelioma (MPM) is a malignancy with a very poor prognosis for which new treatment options are urgently needed. We have previously shown that dendritic cell (DC) immunotherapy provides a clinically feasible treatment option. In the current study, we set out to assess the immunological changes induced by DC immunotherapy in peripheral blood of MPM patients. Methods: Peripheral blood was collected from nine patients enrolled in a phase I dose escalation study, before and after treatment with DCs that were pulsed with an allogeneic tumor lysate preparation consisting of a mixture of five cultured mesothelioma cell lines. We used immune profiling by multiplex flow cytometry to characterize different populations of immune cells. In particular, we determined frequencies of T cell subsets that showed single and combinatorial expression of multiple markers that signify T cell activation, maturation and inhibition. Therapy-induced T cell reactivity was assessed in peptide/MHC multimer stainings using mesothelin as a prototypic target antigen with confirmed expression in the clinical tumor lysate preparation. T cell receptor (TCR) diversity was evaluated by TCRB gene PCR assays. Results: We observed an increase in the numbers of B cells, CD4 and CD8 T cells, but not NK cells at 6 weeks post-treatment. The increases in B and T lymphocytes were not accompanied by major changes in T cell reactivity toward mesothelin nor in TCRB diversity. Notably, we did observe enhanced proportions of CD4 T cells expressing HLA-DR, PD-1 (at 2 weeks after onset of treatment) and ICOS (6 weeks) and a CD8 T cell population expressing LAG3 (2 weeks). Discussion: DC immunotherapy using allogeneic tumor lysate resulted in enhanced frequencies of B cells and T cells in blood. We did not detect a skewed antigen-reactivity of peripheral CD8 T cells. Interestingly, frequencies of CD4 T cells expressing activation markers and PD-1 were increased. These findings indicate a systemic activation of the adaptive immune response and may guide future immune monitoring studies of DC therapies.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos HLA-DR/genética , Imunoterapia Adotiva , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/terapia , Mesotelioma/etiologia , Mesotelioma/terapia , Receptor de Morte Celular Programada 1/genética , Antígenos de Neoplasias/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/imunologia , Expressão Gênica , Antígenos HLA-DR/metabolismo , Humanos , Imunofenotipagem , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mesotelina , Mesotelioma/metabolismo , Mesotelioma/patologia , Mesotelioma Maligno , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
12.
Stem Cell Investig ; 4: 47, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28607921

RESUMO

Several parameters are involved in graft predominance after double umbilical cord blood transplantation (dUCBT), of which T-cell alloreactivity between the grafts is now considered to be the major denominator. We recently showed that alloreactive CD4+ T-cells originating from the predominant CBU recognize HLA-class II allele mismatches and can readily be detected in the majority of patients. In addition, it was shown that HLA-class II allele-specific CD4+ T-cells were able to recognize primary leukemic cells when the mismatched HLA-class II allele was shared between the rejected CBU and the patient. These results further underscored the role of alloreactive T-cells, notably class II specific CD4+ T-cells, in graft-versus-graft reactions and in graft-versus-leukemia after dUCBT.

13.
Clin Cancer Res ; 23(20): 6012-6020, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28645940

RESUMO

Adoptive therapy with T-cell receptor (TCR)-engineered T cells has shown promising results in the treatment of patients with tumors, and the number of TCRs amenable for clinical testing is expanding rapidly. Notably, adoptive therapy with T cells is challenged by treatment-related side effects, which calls for cautious selection of target antigens and TCRs that goes beyond their mere ability to induce high T-cell reactivity. Here, we propose a sequence of in vitro assays to improve selection of TCRs and exemplify risk assessments of on-target as well as off-target toxicities using TCRs directed against cancer germline antigens. The proposed panel of assays covers parameters considered key to safety, such as expression of target antigen in healthy tissues, determination of a TCR's recognition motif toward its cognate peptide, and a TCR's cross-reactivity toward noncognate peptides. Clin Cancer Res; 23(20); 6012-20. ©2017 AACR.


Assuntos
Imunoterapia Adotiva , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Motivos de Aminoácidos , Animais , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais , Testes Imunológicos de Citotoxicidade/métodos , Citotoxicidade Imunológica , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Humanos , Imunoterapia Adotiva/métodos , Técnicas In Vitro , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética
14.
Front Immunol ; 7: 648, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28082983

RESUMO

Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naïve to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naïve CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion.

16.
Clin Cancer Res ; 9(1): 76-83, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12538454

RESUMO

PURPOSE: Repeated administrations of recombinant human interleukin-12 (rHuIL-12) to cancer patients are characterized by a reduction of side effects during treatment. Induction of IFN-gamma, considered a key mediator of antitumor effects of IL-12, is known to decline on repeated administrations. We studied whether other immunological effects of rHuIL-12 are tapered in the course of treatment. EXPERIMENTAL DESIGN: In a Phase I study of 26 patients with advanced renal cell cancer, rHuIL-12 was administered s.c. on day 1, followed by 7 days rest and six injections administered over a 2-week time period. Plasma concentrations of various cytokines were monitored, as well as absolute counts of circulating leukocyte and lymphocyte subsets. RESULTS: The first injection of IL-12 was accompanied by rapid, transient, and dose-dependent increments of plasma levels IFN-gamma, tumor necrosis factor-alpha, IL-10, IL-6, IL-8, but not IL-4, as well as rapid, transient, and dose-dependent reductions of lymphocyte, monocyte, and neutrophil counts. The major lymphocyte subsets, i.e., CD4+ and CD8+ T cells, B cells, and natural killer cells, followed this pattern. On repeated rHuIL-12 injections, IL-10 concentrations increased further, whereas the transient increments of IFN-gamma, tumor necrosis factor-alpha, IL-6, and IL-8 concentrations, as well as the fluctuations of the leukocyte subset counts, were tapered. Dose escalation of IL-12 within clinically tolerable margins did not reduce the decline of these immunological effects. CONCLUSIONS: Induction of pro-inflammatory cytokines and associated fluctuations in leukocyte subset counts decrease on repeated administrations of rHuIL-12. The steady increment of IL-10 plasma levels may mediate the observed down-regulation of clinical and immunological effects.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Interleucina-12/administração & dosagem , Adulto , Idoso , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Carcinoma de Células Renais/sangue , Citocinas/metabolismo , Humanos , Imunofenotipagem , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-6/sangue , Interleucina-8/sangue , Células Matadoras Naturais/metabolismo , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossíntese
17.
Immunotherapy ; 7(5): 513-33, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26065477

RESUMO

Cancer immune therapy, in particular the use of checkpoint inhibitors and adoptive transfer of T cells has recently demonstrated significant clinical responses against several tumor types. Unfortunately, these therapies are frequently accompanied by severe toxicities, underscoring the need for markers that provide information on therapy response. Monitoring immune responses in the tumor microenvironment and peripheral blood prior to and during these therapies will provide better insight into the mechanisms underlying clinical activities, and will potentially enable the identification of such markers. In this review, we present an overview of adoptive T-cell trials conducted with a special focus on immune monitoring, and argue that accurate monitoring of T cells is pivotal to further development of immune therapies to treat cancer.


Assuntos
Imunoterapia Adotiva , Monitorização Fisiológica/métodos , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Linfócitos T/transplante , Animais , Humanos , Neoplasias/patologia , Linfócitos T/patologia , Microambiente Tumoral/imunologia
18.
Cancer Gene Ther ; 9(7): 613-23, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12082462

RESUMO

In preparation of a clinical phase I/II study in renal cell carcinoma (RCC) patients, we developed a clinically applicable protocol that meets good clinical practice (GCP) criteria regarding the gene transduction and expansion of primary human T lymphocytes. We previously designed a transgene that encodes a single chain (sc) FvG250 antibody chimeric receptor (ch-Rec), specific for a RCC tumor-associated antigen (TAA), and that genetically programs human T lymphocytes with RCC immune specificity. Here we describe the conditions for activation, gene transduction, and proliferation for primary human T lymphocytes to yield: (a) optimal functional expression of the transgene; (b) ch-Rec-mediated cytokine production, and (c) cytolysis of G250-TAA(POS) RCC by the T-lymphocyte transductants. Moreover, these parameters were tested at clinical scale, i.e., yielding up to 5-10 x 10(9) T-cell transductants, defined as the treatment dose according to our clinical protocol. The following parameters were, for the first time, tested in an interactive way: (1) media compositions for production of virus by the stable PG13 packaging cell; (2) T-lymphocyte activation conditions and reagents (anti-CD3 mAb; anti-CD3+anti-CD28 mAbs; and PHA); (3) kinetics of T-lymphocyte activation prior to gene transduction; (4) (i) T-lymphocyte density, and (ii) volume of virus-containing supernatant per surface unit during gene transduction; and (5) medium composition for T-lymphocyte maintenance (i) in-between gene transduction cycles, and (ii) during in vitro T-lymphocyte expansion. Critical to gene transduction of human T lymphocytes at clinical scale appeared to be the use of the fibronectin fragment CH-296 (Retronectin) as well as Lifecell) X-fold cell culture bags. In order to comply with GCP requirements, we used: (a) bovine serum-free human T-lymphocyte transduction system, i.e., media supplemented with autologous patients' plasma, and (b) a closed cell culture system for all lymphocyte processing. This clinical protocol routinely yields 30-65% scFvG250 ch-Rec(POS) T lymphocytes in both healthy donors and RCC patients.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Imunoterapia/métodos , Neoplasias/terapia , Linfócitos T/metabolismo , Transdução Genética , Anticorpos Monoclonais , Antígenos CD28/genética , Complexo CD3/genética , Antígenos CD4/genética , Carcinoma de Células Renais/terapia , Divisão Celular , Meios de Cultura Livres de Soro , Citometria de Fluxo , Humanos , Neoplasias Renais/terapia , Leucócitos Mononucleares/citologia , Retroviridae/genética , Espectrometria de Fluorescência , Fatores de Tempo , Transgenes , Células Tumorais Cultivadas
19.
Hum Gene Ther Methods ; 25(6): 345-57, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25423330

RESUMO

Therapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T cells specific for carbonic anhydrase IX (CAIX), we observed toxicities that (most likely) indicated in vivo function of CAR T cells as well as low T cell persistence and clinical response rates. The latter observations were confirmed by later clinical trials in other solid tumor types and other gene-modified T cells. To improve the efficacy of T cell therapy, we have redefined in vitro conditions to generate T cells with young phenotype, a key correlate with clinical outcome. For their impact on gene-modified T cell phenotype and function, we have tested various anti-CD3/CD28 mAb-based T cell activation and expansion conditions as well as several cytokines prior to and/or after gene transfer using two different receptors: CAIX CAR and MAGE-C2(ALK)/HLA-A2 TCR. In a total set of 16 healthy donors, we observed that T cell activation with soluble anti-CD3/CD28 mAbs in the presence of both IL15 and IL21 prior to TCR gene transfer resulted in enhanced proportions of gene-modified T cells with a preferred in vitro phenotype and better function. T cells generated according to these processing methods demonstrated enhanced binding of pMHC, and an enhanced proportion of CD8+, CD27+, CD62L+, CD45RA+T cells. These new conditions will be translated into a GMP protocol in preparation of a clinical adoptive therapy trial to treat patients with MAGE-C2-positive tumors.


Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Anidrase Carbônica IX , Anidrases Carbônicas/imunologia , Senescência Celular , Vetores Genéticos/genética , Antígeno HLA-A2/imunologia , Humanos , Imunofenotipagem , Imunoterapia Adotiva , Testes de Liberação de Interferon-gama , Interleucinas/farmacologia , Ativação Linfocitária , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/metabolismo , Linfócitos T/transplante , Transdução Genética
20.
Clin Cancer Res ; 20(18): 4776-83, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25024258

RESUMO

PURPOSE: RGB-286638 is a multitargeted inhibitor with targets comprising the family of cyclin-dependent kinases (CDK) and a range of other cancer-relevant tyrosine and serine/threonine kinases. The objectives of this first in human trial of RGB-286638, given i.v. on days 1 to 5 every 28 days, were to determine the maximum tolerated dose (MTD) and to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of this new drug. EXPERIMENTAL DESIGN: Sequential cohorts of 3 to 6 patients were treated per dose level. Blood, urine samples, and skin biopsies for full PK and/or PD analyses were collected. RESULTS: Twenty-six patients were enrolled in 6-dose levels from 10 to 160 mg/d. Four dose-limiting toxicities were observed in 2 of the 6 patients enrolled at the highest dose level. These toxicities were AST/ALT elevations in 1 patient, paroxysmal supraventricular tachycardias (SVTs), hypotension, and an increase in troponin T in another patient. The plasma PK of RGB-286638 was shown to be linear over the studied doses. The interpatient variability in clearance was moderate (variation coefficient 7%-36%). The PD analyses in peripheral blood mononuclear cells, serum (apoptosis induction) and skin biopsies (Rb, p-Rb, Ki-67, and p27(KIP1) expression) did not demonstrate a consistent modulation of mechanism-related biomarkers with the exception of lowered Ki-67 levels at the MTD level. The recommended MTD for phase II studies is 120 mg/d. CONCLUSIONS: RGB-286638 is tolerated when administered at 120 mg/d for 5 days every 28 days. Prolonged disease stabilization (range, 2-14 months) was seen across different dose levels.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Ureia/análogos & derivados , Adulto , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/efeitos adversos , Pirazóis/farmacocinética , Ureia/administração & dosagem , Ureia/efeitos adversos , Ureia/farmacocinética
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