RESUMEN
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.
Asunto(s)
Carcinoma de Células Renales/inmunología , Interferón gamma/inmunología , Interleucina-6/inmunología , Neoplasias Renales/inmunología , Linfocitos T/inmunología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/terapia , Citocinas/sangre , Citocinas/inmunología , Citometría de Flujo , Humanos , Inmunoterapia Adoptiva/métodos , Interferón gamma/sangre , Interleucina-6/sangre , Neoplasias Renales/metabolismo , Neoplasias Renales/terapia , Recuento de Linfocitos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Linfocitos T/trasplante , Factores de TiempoRESUMEN
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.
RESUMEN
Autologous T cells genetically modified to express a chimeric antibody receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to 12 patients with CAIX-expressing metastatic renal cell carcinoma (RCC). Patients were treated in three cohorts with a maximum of 10 infusions of a total of 0.2 to 2.1 × 10(9) CAR T cells. CTC grade 2-4 liver enzyme disturbances occurred at the lowest CAR T cell doses, necessitating cessation of treatment in four out of eight patients in cohorts 1 and 2. Examination of liver biopsies revealed CAIX expression on bile duct epithelium with infiltration of T cells, including CAR T cells. Subsequently four patients were pre-treated with CAIX monoclonal antibody (mAb) G250 to prevent CAR-specific toxicity and showed no liver toxicities and indications for enhanced peripheral T cell persistence. No clinical responses were recorded. This report shows that CAIX-targeting CAR T cells exerted antigen-specific effects in vivo and induced liver toxicity at the lowest dose of 0.2 × 10(9) T cells applied, illustrating the potency of receptor-modified T cells. We provide in-patient proof that the observed "on-target" toxicity is antigen-directed and can be prevented by blocking antigenic sites in off-tumor organs and allowing higher T cell doses.
Asunto(s)
Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/inmunología , Linfocitos T/inmunología , Anhidrasas Carbónicas/metabolismo , Citocinas/metabolismo , Citometría de Flujo , Dosificación de Gen/genética , Humanos , Inmunohistoquímica , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Carcinoma de Células Renales/terapia , Tratamiento Basado en Trasplante de Células y Tejidos , Neoplasias Renales/terapia , Receptores de Antígenos de Linfocitos T , Linfocitos T/inmunología , Adulto , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/inmunología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/inmunología , Ingeniería Celular , Línea Celular , Fibronectinas/administración & dosificación , Humanos , Neoplasias Renales/genética , Neoplasias Renales/inmunología , Masculino , Muromonab-CD3/administración & dosificación , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes/administración & dosificación , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/efectos de los fármacosRESUMEN
Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent "on-target" reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2(336-344)/HLA-A2 (MC2/A2) and MAGE-A3(243-258)/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαß genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vß28 and TCR-Vα38/Vß2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.