A randomized phase 2 trial of idiotype vaccination and adoptive autologous T-cell transfer in patients with multiple myeloma.

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)-keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial, patients received either control (KLH only) or Id-KLH vaccine, autologous transplantation, vaccine-specific costimulated T cells expanded ex vivo, and 2 booster doses of assigned vaccine. In 36 patients (KLH, n = 20; Id-KLH, n = 16), no dose-limiting toxicity was seen. At last evaluation, 6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms, respectively (P = .22), and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with patients receiving KLH only, there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically, upregulation of genes associated with activation, effector function induction, and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly, in responding patients across both arms, upregulation of genes associated with T-cell activation was seen. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion, in this combination immunotherapy approach, we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828.

[Development of allogeneic CAR T-cells]. / Développement des CAR-T allogéniques.

Chimeric antigen receptor (CAR) T-cell therapy represents a major breakthrough in the field of hematology. "Off-the-shelf" allogeneic CAR T-cells from donors have many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches, possible standardization of the cell product, time for multiple cell modifications, redosing and decreased cost. However, allogeneic T-cells possess foreign immunological identities that can lead to graft-versus-host disease (GvHD) and their rejection by the host immune system. In this review, we describe the different approaches to produce allogeneic CAR T-cells with limited potential for GvHD and that can persist in the recipient. The preliminary clinical results obtained with the first generation of allogeneic CAR T-cells are presented as well as the perspectives in hematological malignancies and solid tumors.

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy.

BACKGROUND: Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression. METHOD: We performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15. RESULTS: Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers. CONCLUSIONS: ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

First immunotherapeutic CAR-T cells against the immune checkpoint protein HLA-G.

BACKGROUND: CAR-T cells immunotherapy is a breakthrough in the treatment of hematological malignancies such as acute lymphoblastic leukemia (ALL) and B-cell malignancies. However, CAR-T therapies face major hurdles such as the lack of tumor-specific antigen (TSA), and immunosuppressive tumor microenvironment sometimes caused by the tumorous expression of immune checkpoints (ICPs) such as HLA-G. Indeed, HLA-G is remarkable because it is both a potent ICP and a TSA. HLA-G tumor expression causes immune escape by impairing innate and adaptive immune responses and by inducing a suppressive microenvironment. Yet, to date, no immunotherapy targets it. METHODS: We have developed two anti-HLA-G third-generation CARs based on new anti-HLA-G monoclonal antibodies. RESULTS: Anti-HLA-G CAR-T cells were specific for immunosuppressive HLA-G isoforms. HLA-G-activated CAR-T cells polarized toward T helper 1, and became cytotoxic against HLA-G+ tumor cells. In vivo, anti-HLA-G CAR-T cells were able to control and eliminate HLA-G+ tumor cells. The interaction of tumor-HLA-G with interleukin (IL)T2-expressing T cells is known to result in effector T cell functional inhibition, but anti-HLA-G CAR-T cells were insensitive to this inhibition and still exerted their function even when expressing ILT2. Lastly, we show that anti-HLA-G CAR-T cells differentiated into long-term memory effector cells, and seemed not to lose function even after repeated stimulation by HLA-G-expressing tumor cells. CONCLUSION: We report for the first time that HLA-G, which is both a TSA and an ICP, constitutes a valid target for CAR-T cell therapy to specifically target and eliminate both tumor cells and HLA-G+ suppressive cells.

Vitamin D3 combined with antibody agents suppresses alloreactive memory T-cell responses to induce heart allograft long-term survival.

BACKGROUND: The pre-stored memory T cells in organ transplant patient carry a high risk of allograft rejection. The current study aimed to determine whether the allogenic response of adoptively transferred memory T cells in mice was suppressed by vitamin D3 monotherapy alone or in combination with monoclonal antibody treatment. METHODS: Prior to vascularized heterotopic heart transplantation, naïve C57BL/6 mice were primed with memory T cells. Recipient mice were administered vitamin D3 alone or in combination with monoclonal antibodies (anti-CD40L/ anti-LFA-1). Memory T cells and CD4+ forkhead box P3+ T cells in recipient spleens were measured using flow cytometry. Additionally, the expression of cytokines was measured by ELISA and quantitative PCR. Inflammatory factors in the grafts were identified by hematoxylin and eosin staining. RESULTS: Vitamin D3 in conjunction with anti-CD40L/ anti-LFA-1 antibodies were administered according to the median survival time from 6.5 to 80 days. The results revealed that grafts were protected through the prevention of inflammatory cell infiltration. Combined treatment decreased the mRNA levels of IL-2, IFN-γ and IL-10 and increased the mRNA levels of IL-4, Foxp3 and TGF-ß in the allograft. Rejection was suppressed by a reduction of CD4+CD44high CD62L+ and CD8+ CD44high CD62L+ memory T cells, the induction of regulatory T cells in the recipient spleen and a reduction of serum IL-2, IFN-γ and IL-10 levels. CONCLUSION: Vitamin D3 efficiently protected allografts from memory T-cell allo-responses when combined with anti-CD40L/anti-LFA-1 antibodies therapy.