Biomedical subjects

Giuseppe Gaipa

Publications and source records attributed to Giuseppe Gaipa.

9 recordsLinked to original sources

PubMed2025

Optimized GMP-grade production of non-viral Sleeping Beauty-generated CARCIK cells for enhanced fitness and clinical scalability.

BACKGROUND: Strict adherence to GMP guidelines and regulatory compliance is crucial when transitioning from research to clinical-grade production of ATMPs like CAR T cells. The success of CAR T cell therapy in treating hematological malignancies highlights the need for closed or automated systems to ensure quality and efficacy. Recent evidence also suggests that ex vivo culture conditions can significantly impact CAR T cell functionality. METHODS: We present our optimized methodology for expanding Sleeping Beauty transposon-engineered Chimeric Antigen Receptor-Cytokine-Induced Killer (CARCIK) cells using G-Rex devices and evaluate its impact on CARCIK cell phenotype and T cell fitness. RESULTS: Building on our previously validated protocol, we introduced key simplifications to optimize the CARCIK differentiation process. Delaying the nucleofection step eliminated the need for feeder cells while maintaining efficient CAR expression and high cell viability. Transitioning from T-flasks to G-Rex bioreactors reduced operator hands-on time from 21 to 28 days to 14-17 days and resulted in a less differentiated CARCIK cell product. Metabolic and transcriptional analyses showed that the novel protocol improves CARCIK cell fitness and in vivo efficacy against B-cell lymphoma. The novel method was validated in Good Manufacturing Practices (GMP) conditions at our two Cell Factories and yielded enough numbers of CARCIK-CD19 cells for clinical use. CONCLUSIONS: Optimizing non-viral CARCIK cell production using G-Rex bioreactors and refined timing adjustments has streamlined the workflow, enhanced cell fitness, and resulted in a highly effective therapeutic product with demonstrated in vivo efficacy in mice. These improvements reduced manipulation and contamination risks, while optimizing logistics and space efficiency, facilitating allogeneic CARCIK generation for a current phase I/II clinical trial (NCT05869279) in patients with R/R CD19 + non-Hodgkin Lymphoma (B-cell NHL) and Chronic Lymphocytic Leukemia (CLL), confirming the approach's scalability and clinical potential.

Humans↗

PubMed2006

Monitoring of minimal residual disease in leukemia, advantages and pitfalls.

The term 'minimal residual disease' (MRD) defines the level of disease detectable in patients in clinical remission during therapy, below the detection limit of conventional methods. Very sensitive methods can be used, able to identify one leukemic cell out of 10,000 normal lymphocytes. In vivo measurements of leukemia cytoreduction reflect the combined effect of clinical and biological variables, thus providing direct information on the effectiveness of treatment in each patient. Thus, these methods can potentially be used for tailoring treatment and personalize the cure. Although MRD studies are becoming an integral part of the modern management of patients with leukemia, several parameters are critical for the application and interpretation of MRD studies, including therapeutic context, timing of sampling, target genes and sensitivity of the polymerase chain reaction (PCR) assay, inter-laboratory standardization (particularly relevant in multicenter studies), selection of patients, retrospective or prospective nature of the study. Methodologies and pitfalls as well as results of clinical uses of MRD will be reviewed in this article by selecting significant examples of its clinical impact in the management of patients with leukemia.

Flow Cytometry↗

PubMed2005

CD34+ stem cell recovery after positive selection of "overloaded" immunomagnetic columns.

Techniques for CD34+ cell enrichment of hematopoietic progenitor cells in grafts destined for transplantation of certain patients with the aim of lowering the amount of infused T lymphocytes and subsequently decreasing the risk of graft versus host disease (GVHD) have been well developed. Adaptations of these techniques should be useful for isolation of other phenotypically defined stem cells. However, a major limitation of techniques now available consists of the number of total nucleated cells or phenotypically defined stem cells that can be processed in a single procedure. Here, we show that recommended levels are much lower than the levels of cells that can be effectively processed by immunomagnetic sorting. Twenty-nine procedures were performed using the Clini- MACS (Miltenyi Biotec) device, which is recommended for processing <6x10(10 )total nucleated cells or <6x10(8) CD34+ cells. Procedures were divided in groups according to their total cellular or CD34+ cell content. We achieved a median CD34+ cell recovery of 68.60% with a median purity of 98.56%, regardless of the loading dose when samples possessing 2-10x10(10) total nucleated cells and 0.8-12.5x10(8) CD34+ cells were applied to a single column. The median levels of CD3+ cells and CD19+ cells in the final product were depleted by 5 logs and 3.8 logs, respectively; no differences were noted when the initial loading dose was increased. Moreover, we found no correlation between the total number nucleated or CD34+ cells loaded and the resultant CD34+ cell recovery. In conclusion, levels of both total nucleated cells and CD34+ can be processed in a single procedure with satisfactory and similar CD34+ cell recovery when these columns are loaded with up to two times as many cells as recommended.

Antigens, CD34↗

PubMed2005

Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation.

We prospectively evaluated the reconstitution of lymphocyte subpopulations in nine children with lysosomal diseases who underwent 11 allogeneic haematopoietic cell transplants (HCTs) following CD34(+) immunomagnetic enrichment, limited T-cell addback and in vivo B-cell depletion. Absolute lymphocyte count recovery was slow to cross the 5th percentile, occurring at a median of 10 months after HCT in patients with full chimaerism. Natural killer cells represented up to 90% of the total lymphoid population during the first 3 months. CD4(+) lymphocyte recovery occurred 9-18 months after HCT. In most patients, CD8(+) lymphocyte recovery was slow and comparable with that of CD4(+) lymphocytes. The CD4(+)/CD8(+) ratio normalised by 3-7 months after HCT in 50% of the patients. CD8(+) lymphocyte recovery was enhanced in patients with viral reactivation. Reconstitution of B-lymphocytes was particularly delayed in patients treated with rituximab. Declining chimaerism, rejection and viral reactivation were the most common problems in our series. Because of the unique graft manipulation, the pace of lymphocyte reconstitution was particularly slow, suggesting that these patients are at a significantly increased risk of infections for up to 2 years after HCT.

CD4-Positive T-Lymphocytes↗

PubMed2003

Expression of CD58 in normal, regenerating and leukemic bone marrow B cells: implications for the detection of minimal residual disease in acute lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: CD58, a member of the Ig superfamily, is expressed by hematopoietic and non- hematopoietic cells. It has been demonstrated to be over-expressed in precursor-B acute lymphoblastic leukemia (ALL) blasts when compared to in their normal counterparts, suggesting its potential use in the detection of minimal residual disease (MRD) by flow cytometry (FC). To assess the reliability and accuracy of CD58 for this purpose, we studied its expression in a large series of normal and ALL bone marrow (BM) samples using quantitative FC. DESIGN AND METHODS: We studied 180 precursor-B ALL BM samples at diagnosis (8 pro-B, 164 early-B, 8 mature-B ALL) and 123 follow-up BM samples (n=54 at day +15 and n=69 at day +78), as well as 51 normal BM samples and 7 regenerating BM samples from patients with T-ALL at week 12. We used four-color quantitative FC, focusing analysis on CD58 expression. In follow-up samples from day +78, the MRD level was simultaneously evaluated by real time quantitative polymerase chain reaction (RQ-PCR) amplification of antigen receptor genes. RESULTS: CD58 expression was significantly higher in ALL blasts than in normal B lymphocytes, while no significant differences between regenerating and normal B lymphocytes were observed. CD58 was expressed in 99.4% of the precursor-B ALL cases and 93.5% of these showed over-expression compared to normal. No significant modulation of CD58 expression during remission induction therapy was noted. Finally, 66 (95.6%) of 69 BM samples simultaneously analyzed using both FC and RQ-PCR at day +78 showed concordant results regarding MRD. INTERPRETATION AND CONCLUSIONS: Our results confirm and further evidence the role of CD58 in the diagnosis and monitoring of precursor-B ALL. In particular, we demonstrated its stability and accuracy in MRD detection at clinically relevant time points. These findings indicate that CD58 is a powerful tool for MRD detection in precursor-B ALL.

Adolescent↗

PubMed2003

Minimal residual disease as a surrogate marker for risk assignment to ALL patients.

The use of minimal residual disease (MRD) measurement as a "surrogate" marker of molecular response to treatment, can potentially improve the evaluation of treatment response and enable estimates of the residual leukemic cell burden during clinical remission, thereby improving the selection of therapeutic strategies and, possibly, long-term clinical outcome. The most specific and sensitive methods for MRD monitoring currently available are polymerase chain reaction amplification of fusion transcripts and rearranged immunoglobulin or antigen-receptor genes, and flow cytometric detection of aberrant immunophenotypes. Several retrospective studies in childhood acute lymphoid leukemias (ALL) have used one of the different approaches for the detection of MRD. The strong association between MRD and risk of relapse was observed in children and adult patients irrespective of the methodology used. The promising results on the predictivity of MRD evaluation at the end of induction treatment has challenged the need for a new definition of remission. There is now urgent need to incorporate MRD data in clinical studies, properly designed to address treatment questions. In this context, several ongoing cooperative study groups have adopted a MRD-based risk group classification to explore whether a better tailored treatment would result in further improvement in cure rates for children with ALL.

Biomarkers, Tumor↗

PubMed2002

A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors.

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

B-Lymphocytes↗

PubMed2002

Commitment of juvenile myelo-monocytic (JMML) leukemic cells to spontaneously differentiate into dendritic cells.

Juvenile myelo-monocytic leukemia (JMML) is a severe malignant stem cell disorder of childhood. A proportion of cells from JMML mononuclear cells (MNC) spontaneously differentiate in vitro into dendritic cells (DC). We have studied MNC from 14 JMML patients, and characterized their functional activity as antigen presenting cells (APC). Large cells, differentiated after seven days of culture, expressed high levels of MHC II molecules and Mannose Receptor, variable levels of CD80 and CD86, and low levels of CD1a. Similar to immature DC, cells from JMML had high levels of dextran endocytosis, and were able to elicit proliferation of allogeneic T lymphocytes in mixed leukocyte reaction (MLR). CD40L-matured DC from JMML was associated with relevant increase of CD80, CD86 and CD83, increased APC activity, responded in chemotaxis assays to MIP-3beta and secreted increased amounts of macrophage derived chemokine (MDC). Immature DC and CD40L-matured DC from JMML produced very low amounts of IL-12, whereas the production of IL-10 was higher than normal DC. In line with these findings, they showed defective capacity to polarize naive T cells to differentiate into Th1 effectors. These results indicate that MNC from JMML are committed to spontaneously differentiate into DC with morphological and phenotypical characteristics similar to normal DC. The cytokine profile produced by these APC is likely to suppress and not to elicit a protective immune response.

Antigen Presentation↗

PubMed2002

Characterization of CD34+, CD13+, CD33- cells, a rare subset of immature human hematopoietic cells.

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cells that express CD34 are heterogeneous in their lineage affiliation and degree of maturation. Expression of CD13 and CD33 antigens indicates myeloid lineage association, but the precise sequence of expression of these two markers during differentiation is unclear. We noted the presence of CD34+ cells expressing CD13 but lacking CD33, a subset of cells not yet well characterized. In this report we describe the prevalence and the immunophenotype of this cell subset. DESIGN AND METHODS: We studied the immunophenotype of immature myeloid cells in human bone marrow samples from 11 healthy transplantation donors and in 4 cord blood samples. We used four-color flow cytometry and a large panel of monoclonal antibodies directed against lineage and differentiation-associated antigens. Three additional bone marrow samples were analyzed after immunomagnetic sorting of CD34+ cells. We focused our analysis on the subset of cells defined by the expression of CD34 and CD13 and the lack of CD33. RESULTS: We found CD34+, CD13+, CD33- cells in all 11 bone marrow and 4 cord blood samples studied. These cells represented 0.5 0.5% (mean SD) and 0.8 1.2% of mononucleated cells, respectively. CD34+, CD13+, CD33- cells appeared to be more immature than those expressing CD33 because of their light scatter characteristics (smaller size and lower granularity), the expression of markers associated with early hematopoietic cells (CD90, CD133 and CD117), and the absence of lineage-associated markers. INTERPRETATION AND CONCLUSIONS: These findings suggest that the expression of CD13 precedes that of CD33 during myeloid differentiation, and that CD34+, CD13+, CD33- cells are at an early stage of human myeloid cell differentiation.

Adolescent↗