Redirecting T-cell Activity with Anti-BCMA/Anti-CD3 Bispecific Antibodies in Chronic Lymphocytic Leukemia and Other B-cell Lymphomas.

T-cell redirecting bispecific antibodies hold high promise for treatment of B-cell malignancies. B-cell maturation antigen (BCMA) exhibits high expression on normal and malignant mature B cells including plasma cells, which can be enhanced by inhibition of γ-secretase. BCMA is considered a validated target in multiple myeloma but whether mature B-cell lymphomas can be targeted by the BCMAxCD3 T-cell redirector teclistamab is currently unknown. BCMA expression on B-cell non-Hodgkin lymphoma and primary chronic lymphocytic leukemia (CLL) cells was assessed by flow cytometry and/or IHC. To assess teclistamab efficacy, cells were treated with teclistamab in presence of effector cells with/without γ-secretase inhibition. BCMA could be detected on all tested mature B-cell malignancy cell lines, while expression levels varied per tumor type. γ-secretase inhibition universally increased BCMA surface expression. These data were corroborated in primary samples from patients with Waldenstrom's macroglobulinemia, CLL, and diffuse large B-cell lymphoma. Functional studies with the B-cell lymphoma cell lines revealed teclistamab-mediated T-cell activation, proliferation, and cytotoxicity. This was independent of the level of BCMA expression, but generally lower in mature B-cell malignancies compared with multiple myeloma. Despite low BCMA levels, healthy donor T cells and CLL-derived T cells induced lysis of (autologous) CLL cells upon addition of teclistamab. These data show that BCMA is expressed on various B-cell malignancies and that lymphoma cell lines and primary CLL can be targeted using teclistamab. Further studies to understand the determinants of response to teclistamab are required to identify which other diseases might be suitable for teclistamab targeting. Significance: Besides reported BCMA expression on multiple myeloma, we demonstrate BCMA can be detected and enhanced using γ-secretase inhibition on cell lines and primary material of various B-cell malignancies. Furthermore, using CLL we demonstrate that low BCMA-expressing tumors can be targeted efficiently using the BCMAxCD3 DuoBody teclistamab.

A Bispecific Single-Domain Antibody Boosts Autologous Vγ9Vδ2-T Cell Responses Toward CD1d in Chronic Lymphocytic Leukemia.

PURPOSE: Although considerable progress has been made with autologous T cell-based therapy in B-cell malignancies, application in chronic lymphocytic leukemia (CLL) lags behind due to disappointing response rates as well as substantial toxicity that is of particular concern in the elderly CLL population. Vγ9Vδ2-T cells form a conserved T-cell subset with strong intrinsic immunotherapeutic potential, largely because of their capacity to be triggered by phosphoantigens that can be overproduced by CLL and other malignant cells. Specific activation of Vγ9Vδ2-T cells by a bispecific antibody may improve the efficacy and toxicity of autologous T-cell-based therapy in CLL. EXPERIMENTAL DESIGN: We evaluated CD1d expression in a cohort of 78 untreated patients with CLL and generated and functionally characterized a CD1d-specific Vγ9Vδ2-T cell engager based on single-domain antibodies (VHH). RESULTS: CD1d was expressed by CLL in the majority of patients, particularly in patients with advanced disease. The CD1d-specific Vγ9Vδ2-T cell engager induced robust activation and degranulation of Vγ9Vδ2-T cells, enabling Vγ9Vδ2-T cells from patients with CLL to lyse autologous leukemic cells at low effector-to-target ratios. Expression of CD1d on CLL cells is upregulated by all-trans retinoic acid, and sensitizes the malignant cells to bispecific VHH-induced lysis. Furthermore, we provide evidence that the Vγ9Vδ2-T cell receptor retains responsiveness to phosphoantigens when the bispecific VHH is bound, and aminobisphosphonates can therefore enhance bispecific Vγ9Vδ2-T cell engager-mediated tumor-specific killing. CONCLUSIONS: Collectively, our data demonstrate the immunotherapeutic potential of this novel CD1d-specific Vγ9Vδ2-T cell engager in CLL.

Human CXCR5+ PD-1+ CD8 T cells in healthy individuals and patients with hematologic malignancies.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy, but varying response rates illustrate the need for biomarkers of response. Studies in mice have identified a subset of CD8 T cells that is essential for response to PD-1 ICB. These CD8 T cells co-express CXCR5, PD-1 and Tcf1, and provide effector T cells upon PD-1 ICB. It is unknown whether similar T cells play a role in PD-1 ICB in humans. We studied human peripheral blood and lymph nodes (LNs) for the frequency, phenotype, and functionality of CXCR5+ PD-1+ CD8 T cells. We find that CXCR5+ PD-1+ CD8 T cells are memory-like cells, express Tcf1, and lack expression of effector molecules. CXCR5+ PD-1+ CD8 T cells produce cytokines upon stimulation, but have limited proliferative capacity. We studied patients with hematologic malignancies with varying response rates to PD-1 ICB. Specifically in chronic lymphocytic leukemia, in which PD-1 ICB does not induce clinical responses, CXCR5+ PD-1+ CD8 T cells show loss of the memory phenotype and increased effector differentiation. In conclusion, we identified CXCR5+ PD-1+ CD8 T cells in human peripheral blood and LN, which could play a similar role during PD-1 ICB. Future studies should analyze CXCR5+ PD-1+ CD8 T cells during PD-1 ICB and their importance for therapeutic response.

A Bispecific Antibody Antagonizes Prosurvival CD40 Signaling and Promotes Vγ9Vδ2 T cell-Mediated Antitumor Responses in Human B-cell Malignancies.

Novel T cell-based therapies for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are thought to have strong potential. Progress, however, has been hampered by low efficacy and high toxicity. Tumor targeting by Vγ9Vδ2 T cells, a conserved T-cell subset with potent intrinsic antitumor properties, mediated by a bispecific antibody represents a novel approach promising high efficacy with limited toxicity. Here, we describe the generation of a bispecific Vγ9Vδ2 T-cell engager directed against CD40, which, due to its overexpression and biological footprint in malignant B cells, represents an attractive target. The CD40-targeting moiety of the bispecific antibody was selected because it can prevent CD40L-induced prosurvival signaling and reduce CD40-mediated resistance of CLL cells to venetoclax. Selective activation of Vγ9Vδ2 T cells in the presence of CD40+ tumor cells induced potent Vγ9Vδ2 T-cell degranulation, cytotoxicity against CLL and MM cells in vitro, and in vivo control of MM in a xenograft model. The CD40-bispecific γδ T-cell engager demonstrated lysis of leukemic cells by autologous Vγ9Vδ2 T cells present in patient-derived samples. Taken together, our CD40 bispecific γδ T-cell engager increased the sensitivity of leukemic cells to apoptosis and induced a potent Vγ9Vδ2 T cell-dependent antileukemic response. It may, therefore, represent a potential candidate for the development of novel treatments for B-cell malignancies.

HA-1H T-Cell Receptor Gene Transfer to Redirect Virus-Specific T Cells for Treatment of Hematological Malignancies After Allogeneic Stem Cell Transplantation: A Phase 1 Clinical Study.

Graft-vs.-leukemia (GVL) reactivity after HLA-matched allogeneic stem cell transplantation (alloSCT) is mainly mediated by donor T cells recognizing minor histocompatibility antigens (MiHA). If MiHA are targeted that are exclusively expressed on hematopoietic cells of recipient origin, selective GVL reactivity without severe graft-vs.-host-disease (GVHD) may occur. In this phase I study we explored HA-1H TCR gene transfer into T cells harvested from the HA-1H negative stem-cell donor to treat HA-1H positive HLA-A*02:01 positive patients with high-risk leukemia after alloSCT. HA-1H is a hematopoiesis-restricted MiHA presented in HLA-A*02:01. Since we previously demonstrated that donor-derived virus-specific T-cell infusions did not result in GVHD, we used donor-derived EBV and/or CMV-specific T-cells to be redirected by HA-1H TCR. EBV and/or CMV-specific T-cells were purified, retrovirally transduced with HA-1H TCR, and expanded. Validation experiments illustrated dual recognition of viral antigens and HA-1H by HA-1H TCR-engineered virus-specific T-cells. Release criteria included products containing more than 60% antigen-specific T-cells. Patients with high risk leukemia following T-cell depleted alloSCT in complete or partial remission were eligible. HA-1H TCR T-cells were infused 8 and 14 weeks after alloSCT without additional pre-conditioning chemotherapy. For 4/9 included patients no appropriate products could be made. Their donors were all CMV-negative, thereby restricting the production process to EBV-specific T-cells. For 5 patients a total of 10 products could be made meeting the release criteria containing 3-280 × 106 virus and/or HA-1H TCR T-cells. No infusion-related toxicity, delayed toxicity or GVHD occurred. One patient with relapsed AML at time of infusions died due to rapidly progressing disease. Four patients were in remission at time of infusion. Two patients died of infections during follow-up, not likely related to the infusion. Two patients are alive and well without GVHD. In 2 patients persistence of HA-1H TCR T-cells could be illustrated correlating with viral reactivation, but no overt in-vivo expansion of infused T-cells was observed. In conclusion, HA-1H TCR-redirected virus-specific T-cells could be made and safely infused in 5 patients with high-risk AML, but overall feasibility and efficacy was too low to warrant further clinical development using this strategy. New strategies will be explored using patient-derived donor T-cells isolated after transplantation transduced with HA-1H-specific TCR to be infused following immune conditioning.

Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up.

Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50% of treated patients, have been observed across hospitals, including a substantial fraction of durable, complete responses. PURPOSE: Execute a phase I/II feasibility study with TIL therapy in metastatic melanoma at the Netherlands Cancer Institute, with the goal to assess feasibility and potential value of a randomized phase III trial. EXPERIMENTAL: Ten patients were treated with TIL therapy. Infusion products and peripheral blood samples were phenotypically characterized and neoantigen reactivity was assessed. Here, we present long-term clinical outcome and translational data on neoantigen reactivity of the T cell products. RESULTS: Five out of 10 patients, who were all anti-PD-1 naïve at time of treatment, showed an objective clinical response, including two patients with a complete response that are both ongoing for more than 7 years. Immune monitoring demonstrated that neoantigen-specific T cells were detectable in TIL infusion products from three out of three patients analyzed. For six out of the nine neoantigen-specific T cell responses detected in these TIL products, T cell response magnitude increased significantly in the peripheral blood compartment after therapy, and neoantigen-specific T cells were detectable for up to 3 years after TIL infusion. CONCLUSION: The clinical results from this study confirm the robustness of TIL therapy in metastatic melanoma and the potential role of neoantigen-specific T cell reactivity. In addition, the data from this study supported the rationale to initiate an ongoing multicenter phase III TIL trial.

Distinct immune composition in lymph node and peripheral blood of CLL patients is reshaped during venetoclax treatment.

Morbidity and mortality due to immunosuppression remain among the foremost clinical challenges in chronic lymphocytic leukemia (CLL). Although immunosuppression is considered to originate within the lymph node (LN) microenvironment, alterations in T and natural killer (NK) cells have almost exclusively been studied in peripheral blood (PB). Whereas chemoimmunotherapy further deteriorates immune function, novel targeted agents like the B-cell lymphoma 2 inhibitor venetoclax potentially spare nonmalignant lymphocytes; however, the effects of venetoclax on nonleukemic cells have not been explored. We address these unresolved issues using a comprehensive analysis of nonmalignant lymphocytes in paired LN and PB samples from untreated CLL patients, and by analyzing the effects of venetoclax-based treatment regimens on the immune system in PB samples from previously untreated and relapsed/refractory patients. CLL-derived LNs contained twice the amount of suppressive regulatory T cells (Tregs) and CLL supportive follicular T helper (Tfh) cells compared with PB. This was accompanied by a low frequency of cytotoxic lymphocytes. The expression of PD-1 by CD8+ T cells was significantly higher in LN compared with PB. Venetoclax-based treatment led to deep responses in the majority of patients, but also to decreased absolute numbers of B, T, and NK cells. Tfh cell, Treg, and PD-1+ CD8+ T cell numbers were reduced more than fivefold after venetoclax-based therapy, and overproduction of inflammatory cytokines was reduced. Furthermore, we observed restoration of NK cell function. These data support the notion that the immunosuppressive state in CLL is more prominent within the LN. Venetoclax-based regimens reduced the immunosuppressive footprint of CLL, suggesting immune recovery after the elimination of leukemic cells.

Dissection of the Effects of JAK and BTK Inhibitors on the Functionality of Healthy and Malignant Lymphocytes.

Despite the emergence of small molecule inhibitors, current treatment strategies for chronic lymphocytic leukemia (CLL) are not curative, and the search for new therapeutic modalities continues. Prosurvival signaling derived from the microenvironment is often mediated via JAK signaling. However, whether JAK inhibitors are useful in CLL therapy has not been studied extensively. JAK inhibitors are valuable therapeutic agents in myelofibrosis and show promising results in graft-versus-host-disease. However, JAK inhibition is associated with an increased infection risk, presumably because of the effect on other immune cells, a feature shared with other kinase inhibitors used for CLL treatment, such as the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib. We compared functional effects of the JAK1/2 inhibitors momelotinib and ruxolitinib, the BTK inhibitors ibrutinib and tirabrutinib, and PI3Kδ inhibitor idelalisib on malignant CLL cells but also on healthy human T, B, and NK lymphocytes. We found several interesting differences among the inhibitors, apart from expected and well-known effects. Momelotinib but not ruxolitinib blocked cytokine-induced proliferation of CLL cells. Momelotinib also reduced BCR signaling, in contrast to ruxolitinib, indicating that these JAK inhibitors in fact have a distinct target spectrum. In contrast to tirabrutinib, ibrutinib had inhibitory effects on T cell activation, probably because of ITK inhibition. Remarkably, both BTK inhibitors stimulated IFN-γ production in a mixed lymphocyte reaction. Collectively, our results demonstrate that kinase inhibitors directed at identical targets may have differential effects on lymphocyte function. Their unique profile could be strategically employed to balance desired versus unwanted lymphocyte inhibition.

Small-scale GMP production of plasmid DNA using a simplified and fully disposable production method.

In the past years, the demand for small batches of clinical grade plasmid DNA has been growing. For that purpose, we designed and qualified a scaled-down Good Manufacturing Practices (GMP) production method, able to produce small batches (1-4 mg) of plasmid. The developed method does not require any complex production equipment and utilizes only disposable production materials, which makes it easy to implement and simplifies line-clearance. We have successfully used this method to produce several small batches of two different plasmids. The produced plasmids, both formulated in an Electroporation Buffer, are mixed and filled into small, single-use, aliquots. Quality control confirmed the robustness of the developed method and a stability study showed that the final formulation is stable for at least two years. The final patient formulation will be subsequently used in a phase I/II clinical trial in which retina cells of patients with Age Related Macular Degeneration, are transfected. The presented production method can be generically used for other plasmid constructs and final formulation designs.

Natural Killer Cell Hypo-responsiveness in Chronic Lymphocytic Leukemia can be Circumvented In Vitro by Adequate Activating Signaling.

Chronic lymphocytic leukemia (CLL) is characterized by an acquired immune dysfunction, which may underlie the hampered efficacy of cellular immunotherapy. Most data on dampened immune responses in CLL come from studies investigating CLL and T cell interactions. Natural killer (NK) cells may be an attractive alternative source of effector cells in immunotherapy in CLL, provided that functionality is retained within the CLL micro-environment. Despite their important role in anti-tumor responses, NK cells are not extensively characterized in CLL. Here, we studied the expression of activating and inhibitory receptors on CLL-derived and healthy control (HC) NK cells, and their functional response towards several stimuli. NK cells from CLL patients have an increased maturation stage, with an expansion of NKG2C+ NK cells in CMV seropositive individuals. The cytotoxicity receptor NKG2D is downregulated, and the killing capacity through this receptor was markedly reduced in CLL-derived NK cells. In contrast, activation via CD16 (FCγRIII) led to adequate activation and functional responses in CLL-derived NK cells. These findings indicate that NK cells in CLL are not intrinsically defect and still perform effector functions upon adequate activating signaling. Clinical relevance of this finding was shown by treatment with novel nanobody-Fc constructs, which induced cytotoxic responses in both CLL- and HC-derived NK cells via CD16. Our results show that NK cells, in contrast to the T cell compartment, retain their function within the CLL micro-environment, provided that they receive an adequate activating signal. These findings warrant future studies on NK cell mediated immunotherapeutic strategies in CLL.

Pulmonary immune responses against Aspergillus fumigatus are characterized by high frequencies of IL-17 producing T-cells.

In healthy individuals and in patients with invasive aspergillosis, Aspergillus-specific T-cells in peripheral blood display mainly a Thelper1 phenotype. Although in other fungal infections Thelper17 immunity is important, it was suggested that in aspergillus infection Thelper17 cells do not play a role or may even be detrimental. OBJECTIVES: To compare the cytokine profiles of Aspergillus-specific CD4+ T-cells in peripheral blood and in the lung. To investigate the Thelper phenotype at the primary location of A. fumigatus exposure. METHODS: Lung-derived T-cells and peripheral blood T-cells from COPD-patients were stimulated with overlapping peptides of 6 A. fumigatus proteins. Aspergillus-specific T-cells were identified on the basis of the activation marker CD154 and production of TNFα. In addition, production of the cytokines IFNγ, IL-17, IL-4 and IL-5 by the Aspergillus-specific T-cells was measured. RESULTS: The majority of lung-derived Aspergillus-specific T-cells displayed a Thelper17 phenotype, and only low percentages of cells produced IFNγ. In contrast, in the peripheral blood of COPD patients Aspergillus-specific T-cells displayed a Thelper1 phenotype, similar as peripheral blood-derived Aspergillus-specific T-cells from healthy individuals. CONCLUSIONS: These data demonstrate that in A. fumigatus infection, similar as in other fungal infections, Thelper17 cells may play a more important role in the immune response than was appreciated until now.

Cytomegalovirus-Induced Expression of CD244 after Liver Transplantation Is Associated with CD8+ T Cell Hyporesponsiveness to Alloantigen.

The chronic presence of viral Ags can induce T cell exhaustion, which is characterized by upregulation of coinhibitory receptors and loss of T cell function. We studied whether a similar phenomenon occurs after liver transplantation (LTx), when there is continuous exposure to alloantigen. Expression of coinhibitory receptors on circulating CD4(+) and CD8(+) T cells was analyzed longitudinally in 19 patients until 6 mo after LTx and cross-sectionally in 38 patients late (1-12 y) after LTx. Expression of the coinhibitory receptors CD160 and CD244 on circulating CD8(+) T cells was already higher 6 mo after LTx compared with pre-LTx, and the elevated expression was sustained late after LTx, with CD244 showing the more prominent increase. The strongest upregulation of CD244 on circulating CD8(+) T cells was observed in patients who experienced CMV infection after LTx. CMV infection also was associated with reduced CD8(+) T cell proliferation and cytotoxic degranulation in response to alloantigen late after LTx. Purified CD244(+)CD8(+) T cells from LTx patients showed lower proliferative responses to alloantigen, as well as to polyclonal stimulation, than did their CD244(-) counterparts. In addition, the CD244(+)CD8(+) T cell population contained the majority of CMV peptide-loaded MHC class I tetramer-binding cells. In conclusion, CMV infection after LTx, rather than persistence of alloantigen, induces the accumulation of dysfunctional CD244(+)CD8(+) T cells in the circulation that persist long-term, resulting in reduced frequencies of circulating alloreactive CD8(+) T cells. These results suggest that CMV infection restrains CD8(+) T cell alloresponses after LTx.

The molecular bases of δ/αß T cell-mediated antigen recognition.

αß and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αß and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αß T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-ß chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αß T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αßTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αßTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αßTCR binding. Our findings highlight how components from αß and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity.

A Good Manufacturing Practice procedure to engineer donor virus-specific T cells into potent anti-leukemic effector cells.

A sequential, two-step procedure in which T-cell-depleted allogeneic stem cell transplantation is followed by treatment with donor lymphocyte infusion at 6 months can significantly reduce the risk and severity of graft-versus-host disease, with postponed induction of the beneficial graft-versus-leukemia effect. However, patients with high-risk leukemia have a substantial risk of relapse early after transplantation, at a time when administration of donor lymphocytes has a high likelihood of resulting in graft-versus-host disease, disturbing a favorable balance between the graft-versus-leukemia effect and graft-versus-host disease. New therapeutic modalities are, therefore, required to allow early administration of T cells capable of exerting a graft-versus-leukemia effect without causing graft-versus-host disease. Here we describe the isolation of virus-specific T cells using Streptamer-based isolation technology and subsequent transfer of the minor histocompatibility antigen HA-1-specific T-cell receptor using retroviral vectors. Isolation of virus-specific T cells and subsequent transduction with HA-1-T-cell receptor resulted in rapid in vitro generation of highly pure, dual-specific T cells with potent anti-leukemic reactivity. Due to the short production procedure of only 10-14 days and the defined specificity of the T cells, administration of virus-specific T cells transduced with the HA-1-T-cell receptor as early as 8 weeks after allogeneic stem cell transplantation is feasible. (This clinical trial is registered at www.clinicaltrialsregister.eu as EudraCT number 2010-024625-20).

Mixed functional characteristics correlating with TCR-ligand koff -rate of MHC-tetramer reactive T cells within the naive T-cell repertoire.

The low frequency of antigen-specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T-cell repertoire. Here, we combine the generation of naïve repertoire-derived antigen-specific T-cell lines based on MHC-tetramer staining and magnetic-bead enrichment with in-depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T-cell lines were generated from seronegative individuals. Generated T-cell lines consisted of a variety of immunodominant CMV-epitope-specific oligoclonal T-cell populations restricted to various HLA-molecules (HLA-A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV-specific T cells was studied. Although all CMV-specific T cells were isolated based on their reactivity toward a specific peptide-MHC complex, we observed a large variation in the functional avidity of the MHC-tetramer positive T-cell populations, which correlated with the structural avidity measured by the recently developed Streptamer koff -rate assay. Our data demonstrate that MHC-tetramer staining is not always predictive for specific T-cell reactivity, and challenge the sole use of MHC-tetramers as an indication of the peripheral T-cell repertoire, independent of the analysis of functional activity or structural avidity parameters.

Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells.

By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8(+) as well as CD4(+) T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4(+) T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αß needed for optimal co-receptor function in HLA-I-TCR transduced CD4(+) T-cells. For this purpose, we transduced human peripheral blood derived CD4(+) T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αß co-receptor in HLA-I-TCR transduced CD4(+) T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4(+) T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient.

PRAME-specific Allo-HLA-restricted T cells with potent antitumor reactivity useful for therapeutic T-cell receptor gene transfer.

PURPOSE: In human leukocyte antigen (HLA)-matched stem cell transplantation (SCT), it has been shown that beneficial immune response mediating graft-versus-tumor (GVT) responses can be separated from graft-versus-host disease (GVHD) immune responses. In this study, we investigated whether it would be possible to dissect the beneficial immune response of allo-HLA-reactive T cells with potent antitumor reactivity from GVHD-inducing T cells present in the detrimental immune response after HLA-mismatched SCT. EXPERIMENTAL DESIGN: The presence of specific tumor-reactive T cells in the allo-HLA repertoire was analyzed at the time of severe GVHD after HLA-mismatched SCT, using tetramers composed of different tumor-associated antigens (TAA). RESULTS: High-avidity allo-HLA-restricted T cells specific for the TAA preferentially expressed antigen on melanomas (PRAME) were identified that exerted highly single-peptide-specific reactivity. The T cells recognized multiple different tumor cell lines and leukemic cells, whereas no reactivity against a large panel of nonmalignant cells was observed. These T cells, however, also exerted low reactivity against mature dendritic cells (DC) and kidney epithelial cells, which was shown to be because of low PRAME expression. CONCLUSIONS: On the basis of potential beneficial specificity and high reactivity, the T-cell receptors of these PRAME-specific T cells may be effective tools for adoptive T-cell therapy. Clinical studies have to determine the significance of the reactivity observed against mature DCs and kidney epithelial cells.

Rapid re-expression of retrovirally introduced versus endogenous TCRs in engineered T cells after antigen-specific stimulation.

To broaden the applicability of cellular immunotherapy, adoptive transfer of T-cell receptor (TCR) transferred T cells may be an attractive strategy. Using this approach, high numbers of defined antigen-specific T cells can be engineered. As the introduced TCR has to compete for cell surface expression with the endogenous TCR, the introduced TCR chains are under control of a strong viral promotor, which, in contrast to the endogenous promotor, is constitutively active. We examined whether this difference in regulation would result in differences in TCR internalization and re-expression of the introduced and endogenous TCR on dual TCR engineered T cells and the antigen-responsiveness of both the TCRs. We showed comparable TCR downregulation of TCRs expressed under regulation of a retroviral promotor or the endogenous promotor. However, the introduced TCRs were rapidly re-expressed on the cell surface after TCR stimulation. Despite rapid re-expression of the introduced TCR, T cells exerted similar antigen-sensitivity compared with control T cells, showing that cell mechanisms other than TCR cell surface expression are involved in antigen-sensitivity directly after antigen-specific stimulation. These results showed that TCR transduced T cells are functionally not different from nontransduced T cells and can potentially be used as an effective treatment strategy.

Optimization of the HA-1-specific T-cell receptor for gene therapy of hematologic malignancies.

To broaden the applicability of adoptive T-cell therapy for the treatment of hematologic malignancies, we aim to start a clinical trial using HA-1-TCR transferred virus-specific T cells. TCRs directed against the minor histocompatibility antigen (MiHA) HA-1 are good candidates for TCR gene transfer to treat hematologic malignancies because of the hematopoiesis-restricted expression and favorable frequency of HA-1. For optimal anti-leukemic reactivity, high cell-surface expression of the introduced TCR is important. Previously, however, we have demonstrated that gene transferred HA-1-TCRs are poorly expressed at the cell-surface. In this study several strategies were explored to improve expression of transferred HA-1-TCRs.