Allorestricted T lymphocytes with a high avidity T‐cell receptor towards NY‐ESO‐1 have potent anti‐tumor activity

The cancer‐testis antigen NY‐ESO‐1 has been targeted as a tumor‐associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T‐cell‐based therapy is the induction of T cells capable of recognizing the NY‐ESO‐1‐expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY‐ESO‐1_157–165 epitope known to be naturally presented with HLA‐A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY‐ESO‐1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA‐A2‐positive, NY‐ESO‐1‐expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY‐ESO‐1‐specific T lymphocytes displayed TCRs with the highest avidity and best anti‐tumor recognition activity. TCRs derived from allorestricted, NY‐ESO‐1‐specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR‐transduced T cells for the treatment of NY‐ESO‐1‐expressing hematological malignancies and solid tumors. © 2009 UICC     

Human effector T cells derived from central memory cells rather than CD8T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy

Adoptive cell therapy provides an attractive treatment of cancer, and our expanding capacity to target tumor antigens is driven by genetically engineered human T lymphocytes that express genes encoding tumor-specific T cell receptors (TCRs). The intrinsic properties of cultured T cells used for therapy were reported to have tremendous influences on their persistence and antitumor efficacy in vivo. In this study, we isolated CD8⁺ central memory T cells from peripheral blood lymphocytes of healthy donors, and then transferred with the gene encoding TCR specific for tumor antigen using recombinant adenovirus vector Ad5F35-TRAV-TRBV. We found effector T cells derived from central memory T cells improved cell viability, maintained certain level of CD62L expression, and reacquired the CD62L⁺CD44^high phenotype of central memory T cells after effector T cells differentiation. We then compared the antitumor reactivity of central memory T cells and CD8⁺T cells after TCR gene transferred. The results indicated that tumor-specific TCR gene being transferred to central memory T cells effectively increased the specific killing of antigen positive tumor cells and the expression of cytolytic granule protein. Furthermore, TCR gene transferred central memory T cells were more effective than TCR gene transferred CD8⁺T cells in CTL activity and effector cytokine secretion. These results implicated that isolating central memory T cells rather than CD8⁺T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer.     

Generation of CD8+ T cells expressing two additional T-cell receptors (TETARs) for personalised melanoma therapy

Adoptive T-cell therapy of cancer often fails due to the tumor cells'' immune escape mechanisms, like antigen loss or down-regulation. To anticipate immune escape by loss of a single antigen, it would be advantageous to equip T cells with multiple specificities. To study the possible interference of 2 T-cell receptors (TCRs) in one cell, and to examine how to counteract competing effects, we generated TETARs, CD8⁺ T cells expressing two additional T-cell receptors by simultaneous transient transfection with 2 TCRs using RNA electroporation. The TETARs were equipped with one TCR specific for the common melanoma antigen gp100 and one TCR recognizing a patient-specific, individual mutation of CCT6A (chaperonin containing TCP1, subunit 6A) termed “CCT6A^m TCR.” These CD8⁺ T cells proved functional in cytokine secretion and lytic activity upon stimulation with each of their cognate antigens, although some reciprocal inhibition was observed. Murinisation of the CCT6A^m TCR increased and prolonged its expression and increased the lytic capacity of the dual-specific T cells. Taken together, we generated functional, dual-specific CD8⁺ T cells directed against a common melanoma-antigen and an individually mutated antigen for the use in personalised adoptive T-cell therapy of melanoma. The intended therapy would involve repetitive injections of the RNA-transfected cells to overcome the transiency of TCR expression. In case of autoimmunity-related side effects, a cessation of treatment would result in a disappearance of the introduced receptors, which increases the safety of this approach.     

Selection of highly responsive T cell receptors by an analysis combining the expression of multiple markers

The clinical success of T cell receptor (TCR) gene–transduced T (TCR-T) cell therapy is expected as one of the next-generation immunotherapies for cancer, in which the selection of TCRs with high functional avidity (high-functional TCRs) is important. One widely used approach to select high-functional TCRs is a comparison of the EC50 values of TCRs, which involves laborious experiments. Therefore, the establishment of a simpler method to select high-functional TCRs is desired. We herein attempted to establish a simple method to select high-functional TCRs based on the expression of T cell activation markers using the mouse T cell line BW5147.3 (BW). We examined relationships between the EC50 values of TCRs in interleukin-2 production and the expression levels of TCR activation markers on BW cells. In TCR-expressing BW cells stimulated with antigenic peptides, the CD69, CD137, and PD-1 expression was differentially induced by various doses of peptides. An analysis of TCRs derived from the tumor-infiltrating lymphocytes of murine melanoma and peripheral blood T cells of hepatocellular carcinoma patients treated with a peptide vaccination revealed that an analysis combining CD69, CD137, and PD-1 expression levels in BW cells stimulated with a single dose of an antigenic peptide selected high-functional TCRs with functional avidity assessed by EC50 values. Our method facilitates the section of high-functional TCRs among tumor-reacting TCRs, which will promote TCR-T cell therapy. The stimulation of BW cells expressing objective TCRs with a single dose of antigenic peptides and analysis combining the expression of CD69, CD137, and PD-1 allows us to select highly responsive TCRs.     

In vivo antitumor function of tumor antigen‐specific CTLs generated in the presence of OX40 co‐stimulation in vitro

Adoptive cell transfer (ACT) is an emerging and promising cancer immunotherapy that has been improved through various approaches. Here, we described the distinctive characteristics and functions of tumor Ag‐specific effector CD8⁺ T‐cells, co‐cultured with a tumor‐specific peptide and a stimulatory anti‐OX40 antibody, before being used for ACT therapy in tumor‐bearing mouse recipients. Splenic T‐cells were obtained from wild‐type FVB/N mice that had been injected with a HER2/neu (neu)‐expressing tumor and a neu‐vaccine. The cells were then incubated for 7 days in vitro with a major histocompatibility complex (MHC) class I peptide derived from neu, in the presence or absence of an agonistic anti‐OX40 monoclonal antibody, before CD8⁺ T cells were isolated for use in ACT therapy. The proliferative ability of OX40‐driven tumor Ag‐specific effector CD8⁺ T‐cells in vitro was less than that of non‐OX40‐driven tumor Ag‐specific effector CD8⁺ T‐cells, but they expressed significantly more early T‐cell differentiation markers, such as CD27, CD62L and CCR7, and significantly higher levels of Bcl‐2, an anti‐apoptotic protein. These OX40‐driven tumor Ag‐specific effector CD8⁺ T‐cells, when transferred into tumor‐bearing recipients, demonstrated potent proliferation capability and successfully eradicated the established tumor. In addition, these cells exhibited long‐term antitumor function, and appeared to be established as memory T‐cells. Our findings suggest a possible in vitro approach for improving the efficacy of ACT, which is simple, requires only a small amount of modulator, and can potentially avoid several toxicities associated with co‐stimulation in vivo.     

Development of a T‐cell receptor multimer with high avidity for detecting a naturally presented tumor‐associated antigen on osteosarcoma cells

For efficacy of peptide vaccination immunotherapy for patients with cancer, endogenous expression of the target peptide/human leukocyte antigen (HLA) on cancer cells is required. However, it is difficult to evaluate the expression status of a peptide/HLA complex because of the lack of a soluble T‐cell receptor (TCR) that reacts with tumor‐associated antigen (TAA) with high avidity. In the present study, we developed two soluble TCR‐multimers that were each directed to TAA, survivin‐2B (SVN‐2B) and PBF in the context of HLA‐A24 (SVN‐2B TCR‐multimer and PBF TCR‐multimer, respectively), from CTL clones that were established from peptide‐vaccinated patients. Both TCR multimers could recognize cognate peptide‐pulsed antigen‐presenting cells, C1R‐A24 cells, in a CD8‐independent method. Moreover, the PBF TCR‐multimer successfully recognized a PBF peptide naturally presented on HLA‐A24⁺PBF⁺ osteosarcoma cells. Taken together, the results indicated that a TCR‐multimer might be useful for detection of a TAA‐derived peptide presented by HLA in patients receiving immunotherapy.     

NY‐ESO‐1 antigen‐reactive T cell receptors exhibit diverse therapeutic capability

The cancer‐testis antigen NY‐ESO‐1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen‐specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross‐reactive to other antigens. We characterized three NY‐ESO‐1 antigen‐reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC‐multimers loaded with NY‐ESO‐1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA‐A2 independent of the bound peptide beside its much stronger recognition of NY‐ESO‐1 bound to HLA‐A2. A third TCR displayed an intermediate but peptide‐specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene‐modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.     

Identification of HLA‐A2‐ and A24‐restricted T‐cell epitopes derived from SOX6 expressed in glioma stem cells for immunotherapy

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma‐bearing mice. In this study, we attempted to identify SOX6‐derived peptides as specific targets for effective and safe T‐cell‐mediated immunotherapy targeting SOX6‐positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)‐A*2402 (A24)‐restricted peptides, RFENLGPQL (SOX6₅₀₄) and PYYEEQARL (SOX6₆₂₈) or the HLA‐A*0201 (A2)‐restricted peptide, ALFGDQDTV (SOX6₄₄₇) was capable of inducing SOX6 peptide‐specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I‐restricted and an antigen‐dependent manner. Furthermore, peptide vaccines of SOX6₆₂₈, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H‐2^d, induced CTLs specific for SOX6₆₂₈ in H‐2^d mice. Normal autologous cells from mice, in which SOX6‐specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA‐A24 or ‐A2 positive glioma patients and should be considered as a promising strategy for safe and effective T‐cell‐based immunotherapy of patients with gliomas.     

Dendritic Cells Pulsed with Total Tumor RNA for Activation NK-like T Cells Against Glioblastoma Multiforme

Summary Dendritic cells (DCs) are potent antigen presenting cells and play critical role in T cell-mediated immunity. DCs have been shown to induce strong anti-tumor responses both in vitro and in vivo. Their efficacies in tumor therapy are being investigated in clinical trials. Previous evidence has shown that these DCs enhance the cytotoxicity of NK cells. We generated NK-like T cells (CD3⁺CD56⁺), a novel type of effector cells differentiated from normal lymphocyte, which is now being used for adoptive immunotherapy in clinical trials. This study aimed to elucidate the effects of NK-like T cells after co-culturing with DCs against tumor cells. The result revealed that tumor-derived RNA-pulsed DCs can enhance the immune responses of NK-like T cells against glioblastoma multiforme cell line but these effector cells did not appear to have the cytotoxic effect against normal cells (human umbilical vein endothelial cells (HUVEC) and fibroblasts) in vitro. This study may be beneficial for the development of new immunologic effector cells for using in adoptive immunotherapy for glioblastoma multiforme in the future.     

Chimeric antigen receptor‐engineered cytokine‐induced killer cells overcome treatment resistance of pre‐B‐cell acute lymphoblastic leukemia and enhance survival

Pre‐emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine‐induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft‐versus‐host‐disease. CIK cells are a heterogeneous effector cell population including T cells (CD3⁺ CD56^?), natural killer (NK) cells (CD3^?CD56⁺) and natural killer T (T‐NK) cells (CD3⁺ CD56⁺) that exhibit non‐major histocompatibility complex (MHC)‐restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)‐γ, anti‐CD3 antibody, interleukin‐2 (IL‐2) and interleukin‐15 (IL‐15). To facilitate selective target‐cell recognition and enhance specific cytotoxicity against B‐cell acute lymphoblastic leukemia (B‐ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28‐CD3ζ domain for signaling and a CD19‐specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19‐targeted CIK/63.28.z cells against otherwise CIK‐resistant cancer cell lines and primary B‐ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre‐B‐ALL. Our results demonstrate potent antileukemic activity of CAR‐engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre‐B‐ALL.     

Generation and application of human induced‐stem cell memory T cells for adoptive immunotherapy

Adoptive T‐cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen‐specific stem cell memory T (T_SCM) cells is expected to overcome this shortcoming as T_SCM cells are close to naïve T cells, but are also highly proliferative, long‐lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into T_SCM‐like cells (iT_SCM) by coculturing with OP9 cells expressing Notch ligand, Delta‐like 1 (OP9‐hDLL1). Here we show the methodological parameters of human CD8⁺ iT_SCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti‐CD3/CD28 antibodies or by antigen‐presenting cells, human iT_SCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9‐hDLL1 cells, interleukin (IL)‐7 and IL‐15 (but not IL‐2 or IL‐21) could efficiently generate iT_SCM cells. Epstein–Barr virus‐specific iT_SCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein–Barr virus transformed‐tumor model mice. Thus, adoptive T‐cell therapy with iT_SCM offers a promising therapeutic strategy for cancer immunotherapy.     

Alphabeta T-cell receptor engineered gammadelta T cells mediate effective antileukemic reactivity

Retroviral transfer of T-cell receptors (TCR) to peripheral blood-derived T cells generates large numbers of T cells with the same antigen specificity, potentially useful for adoptive immunotherapy. One drawback of this procedure is the formation of mixed TCR dimers with unknown specificities due to pairing of endogenous and introduced TCR chains. We investigated whether gammadelta T cells can be an alternative effector population for TCR gene transfer because the gammadeltaTCR is not able to form dimers with the alphabetaTCR. Peripheral blood-derived gammadelta T cells were transduced with human leukocyte antigen (HLA) class I- or HLA class II-restricted minor histocompatibility antigen (mHag) or virus-specific TCRs. Because most gammadelta T cells do not express CD4 and CD8, we subsequently transferred these coreceptors. The TCR-transduced gammadelta T cells exerted high levels of antigen-specific cytotoxicity and produced IFN-gamma and IL-4, particularly in the presence of the relevant coreceptor. gammadelta T cells transferred with a TCR specific for the hematopoiesis-specific mHag HA-2 in combination with CD8 displayed high antileukemic reactivity against HA-2-expressing leukemic cells. These data show that transfer of alphabetaTCRs to gammadelta T cells generated potent effector cells for immunotherapy of leukemia, without the expression of potentially hazardous mixed TCR dimers.     

Clonal expansion of bone marrow CD8+ T cells in acute myeloid leukemia patients at new diagnosis and after chemotherapy

CD8⁺ T cells are crucial adaptive immune effectors and express receptors (T cell receptors, TCRs) that specifically recognize and eradicate tumor cells. The diversity of the TCR repertoire is generated by specialized genetic diversification mechanisms, which lead to an extremely variable TCR repertoire that is capable of recognizing a wide range of antigens. However, the variations in CD8⁺ TCR diversity and their clinical implications in acute myeloid leukemia (AML) patients remain unknown. CD8⁺ T cells were enriched from 10 healthy donors and 31 AML patients at diagnosis and after chemotherapy, and TCRβ deep sequencing was performed to analyze CD8⁺ T cell clonal expansion and TCR repertoire diversity. Diminished TCR repertoire diversity and increased T cell clone expansion were noted in the bone marrow of AML patients. In relapsed patients, T cells were found to be more clonally expanded after chemotherapy than at new diagnosis. Moreover, significantly more expanded TCRβ clonotypes were noted in CD8⁺ PD-1⁺ T cells than in CD8⁺ PD-1^- T cells regardless of the time of examination. Our systematic T cell repertoire analysis may help better characterize CD8⁺ T cells before and after chemotherapy in AML, which may provide insights into therapeutic strategies for hematological malignancies.     

Blood monocytes sample MelanA/MART1 antigen for long‐lasting cross‐presentation to CD8+ T cells after differentiation into dendritic cells

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross‐presenting antigens to CD8⁺ T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen‐specific CD8⁺ T cell clones after 5 days of differentiation into monocytes‐derived dendritic cells (MoDCs). Tagged‐long peptides can be visualized in electron‐dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14⁺ monocytes from two patients effectively activate a MelanA‐specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor‐specific immune response and should be taken into account for cancer immunotherapy.     

In situ thermal ablation augments antitumor efficacy of adoptive T cell therapy

Abstract

Purpose: The aim of this study is to investigate whether radiofrequency ablation (RFA) improves the efficacy of adoptive T cell immunotherapy in preclinical mouse cancer models.

Method: Mice implanted subcutaneously (sc) with syngeneic colon adenocarcinoma or melanoma were treated with sub-curative in situ RFA (90?°C, 1?min). Trafficking of T cells to lymph nodes (LN) or tumors was quantified by homing assays and intravital microscopy (IVM) after sham procedure or RFA. Expression of trafficking molecules (CCL21 and intercellular adhesion molecule-1 [ICAM-1]) on high endothelial venules (HEV) in LN and tumor vessels was evaluated by immunofluorescence microscopy. Tumor-bearing mice were pretreated with RFA to investigate the therapeutic benefit when combined with adoptive transfer of in vitro-activated tumor-specific CD8⁺ T cells.

Results: RFA increased trafficking of naïve CD8⁺ T cells to tumor-draining LN (TdLN). A corresponding increase in expression of ICAM-1 and CCL21 was detected on HEV in TdLN but not in contralateral (c)LN. IVM revealed that RFA substantially enhanced secondary firm arrest of lymphocytes selectively in HEV in TdLN. Furthermore, strong induction of ICAM-1 in tumor vessels was associated with significantly augmented trafficking of adoptively transferred in vitro-activated CD8⁺ T cells to tumors after RFA. Finally, preconditioning tumors with RFA augmented CD8⁺ T cell-mediated apoptosis of tumor targets and delayed growth of established tumors when combined with adoptive T cell transfer immunotherapy.

Conclusions: These studies suggest that in addition to its role as a palliative therapeutic modality, RFA may have clinical potential as an immune-adjuvant therapy by augmenting the efficacy of adoptive T cell therapy.     

Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability

Little is known about the biology of murine T-cell receptors (TCR) expressed in human cells. We recently observed that a murine anti-human p53 TCR is highly functional when expressed in human lymphocytes. Herein, we compare human and mouse TCR function and expression to delineate the molecular basis for the apparent superior biological activity of murine receptors in human T lymphocytes. To this end, we created hybrid TCRs where we swapped the original constant regions with either human or mouse ones, respectively. We showed that murine or "murinized" receptors were overexpressed on the surface of human lymphocytes compared with their human/humanized counterparts and were able to mediate higher levels of cytokine secretion when cocultured with peptide-pulsed antigen-presenting cells. Preferential pairing of murine constant regions and improved CD3 stability seemed to be responsible for these observations. These enhanced biological properties translated into significantly greater antitumor response mediated by TCR with mouse constant regions. Furthermore, we were able to circumvent the natural low avidity of class I MHC TCR in CD4(+) cells by introducing the murinized TCR into CD4(+) lymphocytes, giving them the ability to recognize melanoma tumors. These findings have implications for human TCR gene transfer therapy and may provide new insights into the biology of the TCR/CD3 complex.