No abstract

Adoptive transfer of T cell receptor (TCR)-transduced T cells may be an attractive strategy to target both hematological malignancies and solid tumors. By introducing a TCR, large numbers of T cells with defined antigen (Ag) specificity can be obtained. However, by introduction of a TCR, mixed TCR dimers can be formed. Besides the decrease in TCR expression of the introduced and endogenous TCR, these mixed TCR dimers could harbor potentially harmful specificities. In this study, we demonstrate that introduction of TCRs resulted in formation of neoreactive mixed TCR dimers, composed of the introduced TCR chains pairing with either the endogenous TCR α or β chain. Neoreactivities observed were HLA class I or class II restricted. Most neoreactive mixed TCR dimers were allo-HLA reactive; however, neoreactive mixed TCR dimers with autoreactive activity were also observed. We demonstrate that inclusion of an extra disulfide bond between the constant domains of the introduced TCR markedly reduced neoreactivity, whereas enhanced effectiveness of the introduced TCR was observed. In conclusion, TCR transfer results in the formation of neoreactive mixed TCR dimers with the potential to generate off-target effects, underlining the importance of searching for techniques to facilitate preferential pairing.

Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)-specific T cells by transfer of minor histocompatibility antigen HA-2-specific T-cell receptors (TCRs). HA-2-TCR-transferred CMV-specific T cells were potent effectors against HA-2-expressing leukemic cells, as well as CMV-expressing cells.Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cellsurface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties. (Blood. 2007;109: [235][236][237][238][239][240][241][242][243]

JHFF and MG are inventors on a pending patent for the nonoptimized and optimized TRAV and TRBV sequences of TCRs for mutated NPM1 as well as cells containing these TCR sequences (no. 2019156).

T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR–transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2− individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti–HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2–expressing patients transplanted with HLA-A2–matched or –mismatched donors.

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. Clin Cancer Res; 17(17); 5615–25. ©2011 AACR.

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 ;D T cells can be an alternative effector population for TCR gene transfer because the ;DTCR is not able to form dimers with the ABTCR. Peripheral bloodderived ;D 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 ;D T cells do not express CD4 and CD8, we subsequently transferred these coreceptors. The TCR-transduced ;D T cells exerted high levels of antigen-specific cytotoxicity and produced IFN-; and IL-4, particularly in the presence of the relevant coreceptor. ;D 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 ABTCRs to ;D T cells generated potent effector cells for immunotherapy of leukemia, without the expression of potentially hazardous mixed TCR dimers. (Cancer Res 2006; 66(6): 3331-7) IntroductionCellular immunotherapy is a promising strategy for the treatment of cancer (1). However, adoptive transfer of sufficient numbers of antigen-specific T cells requires complex isolation methods and laborious and time-consuming tissue culture procedures. An alternative method to obtain large numbers of T cells with a defined antigen specificity is the retroviral transfer of a T-cell receptor (TCR). Because T-cell specificity is exclusively determined by the TCR, T-cell specificity can be functionally transferred to other T lymphocytes by retroviral TCR gene transfer. We and others have shown that transfer of human leukocyte antigen (HLA) class I-and HLA class II-restricted TCRs to CD8 + and CD4 + T cells, respectively, generated T cells with converted antigen-specific cytolytic activity and cytokine production (2-10).The potential in vivo efficacy of TCR-transferred T cells was shown in mouse models (8,9). The TCR-transferred T cells were activated in vivo, homed to effector sites, and contributed to tumor clearance.A potential disadvantage of TCR gene transfer to other ah T cells is the formation of mixed TCR dimers. Chains of the introduced TCR can pair with the endogenous TCR chains naturally expressed by the TCR-transferred T cells. The specificity of these mixed TCR dimers is unknown and, therefore, autoreactivity cannot be excluded. To limit the number of T cells with different TCR chains and thus the chance to generate autoreactive T cells, T cells with defined antigen specificity and, therefore, with a limited TCR repertoire can be selected as host cells for TCR gene transfer. We previously showed the reprogramming of cytomegalovirus (CMV)-specifi...

Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.