Oncohematology Research Group

Pamplona, Spain

Oncohematology Research Group

Pamplona, Spain
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Viudez A.,Complejo Hospitalario Of Navarra | Viudez A.,The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Medical Institutions | Ramirez N.,Oncohematology Research Group | Hernandez-Garcia I.,Complejo Hospitalario Of Navarra | And 2 more authors.
Critical Reviews in Oncology/Hematology | Year: 2014

The application of nanotechnology in oncology has increased the efficacy and efficiency of some cytotoxic agents. The paradigm in this field is nab-paclitaxel, a soluble form of paclitaxel that is linked to albumin nanoparticles. The development of nanotechnology as a delivery system for paclitaxel has provided better pharmacokinetic and pharmacodynamic characteristics, neutralizing its hydrophobicity. This procedure significantly improves the treatment of metastatic breast cancer compared to conventional paclitaxel-based therapies, including other type of cancers such as metastatic pancreatic cancer, stage IIIB-IV non-small cell lung cancer (NSCLC) and metastatic melanoma. In these last cases, significant differences were found in primary end-points for patients treated with nab-paclitaxel-based chemotherapy compared to those treated with conventional treatments. The application of nanotechnology in cancer treatment may also improve the efficacy of other known drugs, as a result of improved pharmacokinetic and pharmacodynamic profiles, similarly to paclitaxel. © 2014 Elsevier Ireland Ltd.


Beloki L.,Oncohematology Research Group | Ramirez N.,Oncohematology Research Group | Olavarria E.,Oncohematology Research Group | Olavarria E.,Complejo Hospitalario Of Navarra | And 2 more authors.
Cytotherapy | Year: 2014

Background aims: Cytomegalovirus (CMV) reactivation remains an important risk after hematopoietic stem cell transplantation, which can be effectively controlled through adoptive transfer of donor-derived CMV-specific T cells (CMV-T). CMV-T are usually obtained from donor peripheral blood mononuclear cells (PBMCs) collected before G-CSF mobilization. Despite previous studies that showed impaired T-cell function after granulocyte colony-stimulating factor (G-CSF) mobilization, recent publications suggest that G-CSF-primed PBMCs retain anti-viral function and are a suitable starting material for CMV-T manufacturing. The objective of this study was to assess the feasibility of generating CMV-T from G-CSF-mobilized donors by use of the activation marker CD137 in comparison with conventional non-primed PBMCs. Methods: CMV-T were isolated from G-CSF-mobilized and non-mobilized donor PBMCs on the basis of CMVpp65 activation-induced CD137 expression and expanded during 3 weeks. Functional assays were performed to assess antigen-specific activation, cytokine release, cytotoxic activity and proliferation after anti-genic re-stimulation. Results: We successfully manufactured highly specific, functional and cytotoxic CMV-T from G-CSF-mobilized donor PBMCs. Their anti-viral function was equivalent to non-mobilized CMV-T, and memory phenotype would suggest their long-term maintenance after adoptive transfer. Conclusions: We confirm that the use of an aliquot from G-CSF-mobilized donor samples is suitable for the manufacturing of CMV cellular therapies and thereby abrogates the need for successive donations and ensures the availability for patients with unrelated donors. © 2014 International Society for Cellular Therapy.


Beloki L.,Oncohematology Research Group | Ciaurriz M.,Oncohematology Research Group | Mansilla C.,Oncohematology Research Group | Bandres E.,Complejo Hospitalario Of Navarra | And 4 more authors.
Journal of Immunological Methods | Year: 2013

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8+ T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes.Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8+ T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy. © 2013 Elsevier B.V.


PubMed | Oncohematology Research Group and Complejo Hospitalario Of Navarra
Type: Comparative Study | Journal: Journal of clinical apheresis | Year: 2016

Peripheral blood progenitor cells (PBPCs) have become the major source of hematopoietic progenitor cells for allogeneic transplantation. In February 2008, Zarzio was approved by the European Medicine Agency for PBPCs mobilization, but this authorization was not based in trials analyzing safety and efficacy for PBPCs mobilization. Since August 2011, Zarzio has been used at our institution for PBPCs mobilization. In total 36 healthy family donors underwent PBPCs mobilization, 18 with Neupogen and 18 with Zarzio. Donor characteristics were equivalent between groups, and no severe adverse effects were registered in the Zarzio group. The number of CD34 cells collected/Kg recipient body weight was 6.7 10(6) (3.8-11.1) in the Zarzio group versus 8.4 10(6) (5.6-16.6) in the Neupogen group (P = 0.04). We collected the minimal target cell dose (2 10(6) /kg) in all donors from each group and no significant differences were found in the collection of the optimal cell dose (5 10(6) /kg) between groups, although 3/18 (16.6%) donors that received Zarzio failed to mobilize the optimal cell dose compared with 0% in the Neupogen group. A total of 35 patients proceeded to transplantation (17 in the Zarzio and 18 in the Neupogen groups, respectively). Platelet and neutrophil median time to engraftment was comparable between the two groups. Our retrospective study supports the conclusion that Zarzio mobilization of PBPCs in healthy donors is safe but perhaps not as effective as the reference Neupogen. However, more prospective trials are required to definitively asses the safety and efficacy of G-CSF biosimilars for PBPCs mobilization in healthy donors.


PubMed | Oncohematology Research Group, University College London and Complejo Hospitalario Of Navarra
Type: Journal Article | Journal: Cytotherapy | Year: 2015

Cytomegalovirus (CMV) reactivation remains an important risk after hematopoietic stem cell transplantation, which can be effectively controlled through adoptive transfer of donor-derived CMV-specific T cells (CMV-T). CMV-T are usually obtained from donor peripheral blood mononuclear cells (PBMCs) collected before G-CSF mobilization. Despite previous studies that showed impaired T-cell function after granulocyte colony-stimulating factor (G-CSF) mobilization, recent publications suggest that G-CSF-primed PBMCs retain anti-viral function and are a suitable starting material for CMV-T manufacturing. The objective of this study was to assess the feasibility of generating CMV-T from G-CSF-mobilized donors by use of the activation marker CD137 in comparison with conventional non-primed PBMCs.CMV-T were isolated from G-CSF-mobilized and non-mobilized donor PBMCs on the basis of CMVpp65 activation-induced CD137 expression and expanded during 3 weeks. Functional assays were performed to assess antigen-specific activation, cytokine release, cytotoxic activity and proliferation after anti-genic re-stimulation.We successfully manufactured highly specific, functional and cytotoxic CMV-T from G-CSF-mobilized donor PBMCs. Their anti-viral function was equivalent to non-mobilized CMV-T, and memory phenotype would suggest their long-term maintenance after adoptive transfer.We confirm that the use of an aliquot from G-CSF-mobilized donor samples is suitable for the manufacturing of CMV cellular therapies and thereby abrogates the need for successive donations and ensures the availability for patients with unrelated donors.


Samuel E.R.,University College London | Beloki L.,Oncohematology Research Group | Newton K.,University College London | Mackinnon S.,University College London | Lowdell M.W.,University College London
PLoS ONE | Year: 2014

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells. © 2014 Samuel et al.


Verfuerth S.,University College London | Sousa P.S.E.,University College London | Beloki L.,Oncohematology Research Group | Murray M.,University College London | And 6 more authors.
Bone Marrow Transplantation | Year: 2015

Pre-clinical studies of allogeneic stem cell transplantation suggest that depletion of naive T cells from donor lymphocytes will reduce the risk of GvHD but preserve immunity to infectious pathogens. In this study, we have established a clinical-grade protocol under good manufacturing practice conditions for purging CD62L + naive T cells from steady-state leukapheresis products using the CliniMACS system. The efficacy of immunomagnetic CD62L depletion was assessed by analysis of cell composition and functional immune responses. A median 2.9 log CD62L depletion was achieved with no evidence of CD62L shedding during the procedure and a mean T-cell yield of 47%. CD62L - cells comprised an equal mix of CD4 + and CD8 + T cells, with elimination of B cells but maintenance of regulatory T cells and natural killer cell populations. CD62L-depleted T cells were predominantly CD45RA - and CD45RA + effector memory (>90%) and contained the bulk of pentamer-staining antivirus-specific T cells. Functional assessment of CD62L - cells revealed the maintenance of antiviral T-cell reactivity and a reduction in the alloreactive immune response compared with unmanipulated cells. Clinical-grade depletion of naive T cells using immunomagnetic CD62L beads from steady-state leukapheresis products is highly efficient and generates cells suitable for adoptive transfer in the context of clinical trials. © 2015 Macmillan Publishers Limited.


PubMed | Oncohematology Research Group, Royal Free London NHS Foundation Trust and University College London
Type: Journal Article | Journal: Bone marrow transplantation | Year: 2015

Pre-clinical studies of allogeneic stem cell transplantation suggest that depletion of naive T cells from donor lymphocytes will reduce the risk of GvHD but preserve immunity to infectious pathogens. In this study, we have established a clinical-grade protocol under good manufacturing practice conditions for purging CD62L(+) naive T cells from steady-state leukapheresis products using the CliniMACS system. The efficacy of immunomagnetic CD62L depletion was assessed by analysis of cell composition and functional immune responses. A median 2.9 log CD62L depletion was achieved with no evidence of CD62L shedding during the procedure and a mean T-cell yield of 47%. CD62L(-) cells comprised an equal mix of CD4(+) and CD8(+) T cells, with elimination of B cells but maintenance of regulatory T cells and natural killer cell populations. CD62L-depleted T cells were predominantly CD45RA(-) and CD45RA(+) effector memory (>90%) and contained the bulk of pentamer-staining antivirus-specific T cells. Functional assessment of CD62L(-) cells revealed the maintenance of antiviral T-cell reactivity and a reduction in the alloreactive immune response compared with unmanipulated cells. Clinical-grade depletion of naive T cells using immunomagnetic CD62L beads from steady-state leukapheresis products is highly efficient and generates cells suitable for adoptive transfer in the context of clinical trials.


PubMed | Oncohematology Research Group and University College London
Type: | Journal: Journal of translational medicine | Year: 2015

Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product.In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples.After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved.G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.


Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8(+) T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8(+) T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.

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