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Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase I/II trial, we prophylactically administered multi-virus-specific T-cell products to protect recipients of T-cell depleted allogeneic stem-cell grafts against viral reactivations. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem-cell graft-derived T cells that survived T-cell depletion during conditioning or stem-cell graft manipulation. Using mRNA sequencing of the TCRβ-chains of the individual virus-specific T-cell populations within these T-cell products, we were now able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem-cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. Additionally, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivations. This study demonstrates that virus-specific T cells from prophylactically infused multi-antigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivations.

Alemtuzumab (ALM) is used for T cell depletion in the context of allogeneic hematopoietic stem cell transplantation (alloSCT) to prevent acute graft-versus-host disease and graft rejection. Following ALM-based T cell–depleted alloSCT, relatively rapid recovery of circulating T cells has been described, including T cells that lack membrane expression of the GPI-anchored ALM target Ag CD52. We show, in a cohort of 89 human recipients of an ALM-based T cell–depleted alloSCT graft, that early lymphocyte reconstitution always coincided with the presence of large populations of T cells lacking CD52 membrane expression. In contrast, loss of CD52 expression was not overt within B cells or NK cells. We show that loss of CD52 expression from the T cell membrane resulted from loss of GPI anchor expression caused by a highly polyclonal mutational landscape in the PIGA gene. This polyclonal mutational landscape in the PIGA gene was also found in CD52− T cells present at a low frequency in peripheral blood of healthy donors. Finally, we demonstrate that the GPI−/CD52− T cell populations that arise after ALM-based T cell–depleted alloSCT contain functional T cells directed against multiple viral targets that can play an important role in immune protection early after ALM-based T cell–depleted transplantation.

To optimally utilize therapeutic monoclonal antibodies in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) understanding their mechanisms of action and the factors influencing these mechanisms is required. We show strong correlations between target antigen expression levels and sensitivity to complement-dependent cytotoxicity (CDC) induced by rituximab, ofatumumab, or alemtuzumab in a panel of cell lines derived from primary B-ALL cells and in primary B-ALL samples. Simultaneous loss of expression of membrane-bound complement regulatory proteins (mCRP) CD55 and CD59 due to glycophosphatidylinositol-anchor deficiency, significantly increased sensitivity to CDC. Accordingly, induced increase in CD55 or CD59 expression protected cells against CDC. The extent of protection co-depended on antigen expression and antibody concentration. In contrast, natural variation in mCRP expression could not be used as a single factor to predict sensitivity to CDC. In conclusion, sensitivity of B-ALL cells to CDC was predominantly determined by antibody concentration and target antigen expression.

Alemtuzumab (ALM) is used for T cell depletion in the context of allogeneic hematopoietic stem cell transplantation (alloSCT) to prevent acute graft-versus-host disease and graft rejection. Following ALM-based T cell-depleted alloSCT, relatively rapid recovery of circulating T cells has been described, including T cells that lack membrane expression of the GPI-anchored ALM target Ag CD52. We show, in a cohort of 89 human recipients of an ALM-based T cell-depleted alloSCT graft, that early lymphocyte reconstitution always coincided with the presence of large populations of T cells lacking CD52 membrane expression. In contrast, loss of CD52 expression was not overt within B cells or NK cells. We show that loss of CD52 expression from the T cell membrane resulted from loss of GPI anchor expression caused by a highly polyclonal mutational landscape in the

Background Donor lymphocyte infusion is an effective form of adoptive immunotherapy for hematologic malignancies after allogeneic stem cell transplantation. Graft-versus-host disease, however, often develops due to recognition of ubiquitously-expressed minor histocompatibility antigens. Transfer of T-cell receptors recognizing hematopoiesis-restricted minor histocompatibility antigens to virus-specific T cells may be a powerful anti-tumor therapy with a low risk of graft-versus-host disease. The purpose of this study was to develop an optimal T-cell receptors-encoding multi-cistronic retroviral vector and an efficient method for generating T-cell receptors-engineered virus-specific T cells.Design and Methods Retroviral vectors encoding the T-cell receptors for the hematopoiesis-restricted minor histocompatibility antigen HA-2 with and without selection markers were compared for T-cell receptors surface expression and HA-2-specific lysis. In addition, two different methods, i.e. peptide stimulation of CD8+ cells and Pro5® MHC pentamer-based isolation of antigen-specific T cells, were investigated for their efficiency to generate T-cell receptors-transduced virus-specific T cells.Results Bi-cistronic vectors without selection markers most efficiently mediated T-cell receptors surface expression and HA-2-specific lysis. Furthermore, both methods were useful for generating gene-modified cells, but the purity of virus-specific T cells was higher after pentamer isolation. Finally, the capacity of gene-modified cells to express the transgenic T-cell receptors at the cell surface markedly differed between virus-specific T cells and was correlated with lysis of relevant target cells.Conclusions Our data support T-cell receptors gene transfer to pentamer-isolated virus-specific T cells using bi-cistronic retroviral vectors and illustrate the relevance of selection of gene-modified T cells with appropriate transgenic T-cell receptors surface expression for clinical gene therapy.

To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8 + T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A * 0201-restricted and/ or HLA-B * 0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-γ)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-γ), and cultured with autologous feeders and low-dose interluekin-2. After 7―14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6-17 x 10 6 cells, comprising 54%―96% CMV pp65-specific CD8 + T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8 + T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8 + T-cell lines could be demonstrated.

Killer Ig-like receptors (KIR) are expressed by human NK cells and T cells. Although Ag-specific cytolytic activity and cytokine production of KIR+ T cells can be inhibited by KIR ligation, the effect of KIR on proliferation is unclear. KIR+ T cells have been reported to have a general proliferative defect. To investigate whether KIR+ T cells represent end-stage dysfunctional T cells, we characterized KIR+ CMV-specific T cells in allogeneic stem cell transplantation patients and healthy donors. In both patients and healthy donors, a significant percentage KIR+ T cells was detected at various time points. All stem cell transplantation patients studied showed KIR expression on CMV-specific T cells, while not all donors had KIR-expressing CMV-specific T cells. From two of the patients and one donor KIR+ CMV-specific T clones were isolated and analyzed functionally. T cells were detected that expressed KIR that could not encounter their corresponding KIR ligands in vivo, illustrating that KIR expression by these T cells was not based on functional selection but a random process. Our data demonstrate that KIR+ T cells are fully functional T cells that are only restricted in effector functions and proliferation upon KIR ligation. The level of KIR-mediated inhibition of the effector functions and proliferation depended on the strength of TCR stimulation. We observed no diminished general proliferative capacity and therefore we conclude that these T cells do not represent end-stage dysfunctional T cells.

Patients with relapsed hematological malignancies after allogeneic stem cell transplantation (alloSCT) can be successfully treated by donor lymphocyte infusions (DLI). Since DLI consists of a variety of T cells with different specificities, the benificial anti-leukemic effect of DLI is often accompanied by Graft-versus-Host Disease (GvHD). Genetic modification of T cells to express T cell receptors (TCR) with defined anti-tumor specificity would be an attractive strategy to specifically eradicate the malignant cells without induction of GvHD. We previously demonstrated that transfer of the minor histocompatibility antigen HA-2 specific TCR to CMV specific T cells led to the generation of T cells with dual specificity for CMV as well as HA-2. CMV and EBV specific T cells are ideal target cells for TCR gene transfer, since the majority of human individuals have high frequencies of these T cells due to latent persistence of CMV and EBV. In addition, based on their virus specificity, these T cells do not induce GvHD in an alloSCT setting, and we hypothesize that due to frequent encounter with viral antigens, TCR transferred virus specific T cells will survive for a prolonged period of time in vivo. The aim of this study is to develop a clinical grade method for the generation of TCR transduced virus specific T cells for cellular immunotherapy. CMV and EBV specific T cells were isolated from healthy individuals using pentamers in combination with clinical grade available anti-biotin magnetic beads. Isolation by pentamer-coated beads induced stimulation, expansion and efficient transduction of virus specific T cells, leading to the generation of cell lines with high frequencies of virus specific (>80%) and transduced (20–40%) T cells. T cells were transduced with multi-cistronic retroviral vectors encoding the α and β chains of the HA-2 TCR linked by an IRES or 2A-like sequence. No differences in transduction efficiency and TCR surface expression were observed between the IRES and 2A-like vectors. The transduced virus specific T cells were shown to exhibit dual specificity and tetramer staining of the introduced TCR correlated with specific lysis of target cells endogenously-expressing HA-2. Furthermore, variation in surface expression of the introduced TCR was observed between T cells with different virus specificities. T cells directed against the HLA-A1 epitope of CMV-pp50, for example, efficiently expressed the HA-2 TCR, whereas T cells specific for the HLA-B8 epitope of EBV-EBNA-3A did not express the introduced TCR. Functional analyses demonstrated that TCR-transduced pp50 specific T cells were dual specific, recognizing HA-2 as well as pp50 positive target cells, whereas TCR-engineered EBNA-3A specific T cells were primarily EBNA-3A specific. The efficiency of surface expression of the transferred TCR was shown to be determined by intrinsic properties of the TCRs, illustrating that for TCR gene transfer purposes TCRs need to be selected that exhibit high competition potential, whereas recipient T cells need to express endogenous TCRs with low competition potential. For clinical application, TCRs will be transferred to virus specific T cells selected for their capacity to efficiently express the introduced TCR without loss of virus specificity. The safety, clinical and immunological efficacy of TCR gene transfer to virus specific T cells as cellular anti-tumor immunotherapy after alloSCT will be investigated in a clinical phase I/II study.

Patients with hematological malignancies can be successfully treated by T cell-depleted allogeneic stem cell transplantation (alloSCT) followed by donor lymphocyte infusion (DLI). Failure of some relapsed hematological malignancies to respond to DLI is probably due to immune tolerance induced by regulatory and/or anergic T cells. We previously showed that functional T cells with redirected anti-leukemic reactivity can be generated by transfer of TCRs specific for minor histocompatibility antigens (mHags) to total peripheral blood mononuclear cells (PBMC) as well as CMV-specific T cells. By introducing TCRs into CMV or EBV specific T cells, T cells with proper memory/effector phenotypes are targeted, and due to virus persistence these T cells may show prolonged survival in vivo. The purpose of this study is to develop an efficient method for the isolation, retroviral transduction and expansion of TCR-transduced CMV- and EBV-specific T cells for cellular immunotherapy of patients with relapsed hematological malignancies after alloSCT. For clinical application, construction of single retroviral vectors coding for the α as well as β chains of mHag-specific TCRs is required. We used the MP71 retroviral vector for TCR gene transfer, since this vector contains Myeloproliferative Sarcoma Virus LTR sequences and a Mouse Embryonic Stem Cell Virus leader, which has been optimized for use in the clinic. The MP71 vector also contained a Woodchuck Hepatitis Response Element (WPRE). The WPRE, which is used as an element enhancing transgene expression at the post-transcriptional level, has recently been described to encode 60 amino acids of a protein with potential oncogenic activity. Therefore, we reconstructed the MP71 vector by introduction of a multiple cloning site (MCS) and, for safety issues, deleted the WPRE. The TCR α and β genes specific for the hematopoietic-restricted mHag HA-2 were linked by a 50-bp sequence encoding a “self-cleaving” 2A-like peptide and introduced into the MCS of the MP71 vector. Linkage of the TCR α and β genes by the 2A-like sequence allowed additional linkage of the low affinity nerve growth factor receptor (LNGFR) or human CD20 selection marker genes by an IRES sequence. The advantage of the human CD20 gene is that it can also function as suicide gene, allowing elimination of transduced cells in vivo if undesired side effects occur. Introduction of the single MP71 retroviral vector coding for the HA-2 TCR α and β chains as well as LNGFR into a TCR α- and β-deficient Jurkat T cell line led to high levels of TCR surface expression correlating with LNGFR marker gene expression. These data indicate proper cleavage and assembly of the transduced TCR α and β chains. Moreover, removal of the WPRE did not affect the surface expression level of the transduced TCR. Furthermore, CMV- and EBV-specific T cells were isolated from human individuals by the IFN-γ capture assay and subsequently transduced with a single retroviral vector coding for the HA-2-TCR α and β chains as well as LNGFR. CMV- and EBV-specific T cells from different human individuals could be successfully isolated to 60–90% purity and the TCR-transduced CMV- and EBV-specific T cells were shown to be fully functional, recognizing the viral peptides as well as the endogenously-processed HA-2 mHag.