Universal Ready-to-Use Immunotherapeutic Approach for the Treatment of Cancer: Expanded and Activated Polyclonal γδ Memory T Cells.

In the last years, important progresses have been registered in the treatment of patients suffering from oncological/haematological malignancies, but more still needs to be done to reduce toxicity and side effects, improve outcome and offer new strategies for relapsed or refractory disease. A remarkable part of these clinical benefits is due to advances in immunotherapy. Here, we investigate the generation of a novel, universal and ready-to-use immunotherapeutic product based on γδ-T lymphocytes. These cells are part of the innate immune system, exerting potent natural cytotoxicity against bacteria, viruses and tumours. This ability, coupled with their negligible alloreactivity, makes them attractive for adoptive immunotherapy approaches. To achieve a cell product suitable for clinical use, we developed a strategy capable to generate polyclonal γδ-T cells with predominant memory-Vδ1 phenotype in good manufacturing practice (GMP) procedures with the additional possibility of gene-modification to improve their anti-tumour activity. Irradiated, engineered artificial antigen-presenting cells (aAPCs) expressing CD86/41BBL/CD40L and the cytomegalovirus (CMV)-antigen-pp65 were used. The presence of CMV-pp65 and CD40L proved to be crucial for expansion of the memory-Vδ1 subpopulation. To allow clinical translation and guarantee patient safety, aAPCs were stably transduced with an inducible suicide gene. Expanded γδ-T cells showed high expression of activation and memory markers, without signs of exhaustion; they maintained polyclonality and potent anti-tumour activity both in vitro (against immortalised and primary blasts) and in in vivo studies without displaying alloreactivity signals. The molecular characterisation (phophoproteomic and gene-expression) of these cell products underlines their unique properties. These cells can further be armed with chimeric antigen receptors (CAR) to improve anti-tumour capacity and persistence. We demonstrate the feasibility of establishing an allogeneic third-party, off-the-shelf and ready-to-use, γδ-T-cell bank. These γδ-T cells may represent an attractive therapeutic option endowed with broad clinical applications, including treatment of viral infections in highly immunocompromised patients, treatment of aggressive malignancies refractory to conventional approaches, bridging therapy to more targeted immunotherapeutic approaches and, ultimately, an innovative platform for the development of off-the-shelf CAR-T-cell products.

Donor/recipient mixed chimerism does not predict graft failure in children with beta-thalassemia given an allogeneic cord blood transplant from an HLA-identical sibling.

BACKGROUND: Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with beta-thalassemia given a bone marrow transplant. We investigated the relationship between the degree of mixed chimerism over time and clinical outcome of children undergoing cord blood transplantation for beta-thalassemia. DESIGN AND METHODS: Twenty-seven consecutive children given a cord blood transplant from a related donor were analyzed by short tandem repeat polymerase chain reaction and their chimerism results were compared with those of 79 consecutive patients who received a bone marrow transplant from either a relative (RD-BMT, n=42) or an unrelated donor (UD-BMT, n=37). Cord blood and bone marrow recipients received comparable preparative regimens. RESULTS: All cord blood recipients engrafted and displayed mixed chimerism early after transplantation; 13/27 converted to full donor chimerism over time, while 14 maintained stable mixed chimerism; all patients are alive and transfusion-independent. Twenty-four of the 79 bone marrow-recipients (12 UD- and 12 RD-BMT) exhibited full donor chimerism at all time points examined, 4/79 (2 UD- and 2 RD-BMT) did not engraft and 51/79 (23 UD- and 28 RD-BMT) displayed mixed chimerism at the time of hematologic reconstitution. Forty of 51 bone marrow recipients with mixed chimerism converted to full donor chimerism (17 UD- and 23 RD-BMT), 3/51 maintained stable mixed chimerism (1 UD- and 2 RD-BMT), while 8/51 (5 UD- and 3 RD-BMT) progressively lost the graft, and became transfusion-dependent again. CONCLUSIONS: Mixed chimerism is a frequent event and does not predict the occurrence of graft failure in children with beta-thalassemia given a cord blood transplant from a relative.

Ex vivo expansion of mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efficacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identification/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed.