TCR Gene Transfer Research Articles

TCR Gene Therapy of Spontaneous Prostate Carcinoma Requires In Vivo T Cell Activation

Analogous to the clinical use of recombinant high-affinity Abs, transfer of TCR genes may be used to create a T cell compartment specific for self-Ags to which the endogenous T cell repertoire is immune tolerant. In this study, we show in a spontaneous prostate carcinoma model that the combination of vaccination with adoptive transfer of small numbers of T cells that are genetically modified with a tumor-specific TCR results in a marked suppression of tumor development, even though both treatments are by themselves without effect. These results demonstrate the value of TCR gene transfer to target otherwise nonimmunogenic tumor-associated self-Ags provided that adoptive transfer occurs under conditions that allow in vivo expansion of the TCR-modified T cells.

Lentiviral TCR gene transfer for adoptive immunotherapy of cancer

Lentiviral TCR gene transfer for adoptive immunotherapy of cancer

Human TCR That Incorporate CD3ζ Induce Highly Preferred Pairing between TCRα and β Chains following Gene Transfer

TCR gene therapy is adversely affected by newly formed TCRalphabeta heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRalphabeta dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3zeta with specificities for three different Ags. Transfer of either TCRalpha:CD3zeta or beta:CD3zeta genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRalphabeta:zeta does not compromise surface expression and functions of an endogenous TCRalphabeta. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRalphabeta:CD3zeta is the first strategy that results in highly preferred pairing between CD3zeta-modified TCRalpha and beta chains as well as absence of TCR mispairing between TCR:CD3zeta and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3zeta chains is independent of endogenous CD3gamma, delta, and epsilon. Taken together, our data support the use of TCRalphabeta:CD3zeta to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.

Long-Term Functionality of TCR-Transduced T Cells In Vivo

To broaden the applicability of adoptive T cell therapy to cancer types for which tumor-specific T cells cannot routinely be isolated, an effort has been made to develop the transfer of tumor-specific TCR genes into autologous T cells as a novel immunotherapeutic approach. Although such TCR-modified T cells have been shown to react to Ag encounter and can be used to break tolerance to defined self-Ags, the persistence and capacity for renewed expansion of TCR-modified T cells has not been analyzed. To establish whether TCR-transduced T cells can provide recipients with long-term Ag-specific immune protection, we analyzed long-term function of TCR transduced T cells in mouse model systems. We demonstrate that polyclonal populations of T cells transduced with a class I restricted OVA-specific TCR are able to persist in vivo and respond upon re-encounter of cognate Ag as assessed by both proliferation and cytolytic capacity. These experiments indicate that TCR gene transfer can be used to generate long-term Ag-specific T cell responses and provide a useful model system to assess the factors that can promote high-level persistence of TCR-modified T cells.

Molecular Design of the Cαβ Interface Favors Specific Pairing of Introduced TCRαβ in Human T Cells

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRalpha- and TCRbeta-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRalpha- and TCRbeta-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Calpha and Cbeta domains and are related to each other by both a complementary steric interaction analogous to a "knob-into-hole" configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged "hole-into-knob" configuration. We show that this inversion in the CalphaCbeta interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

Cross-Reactive CD4+ T Cells against One Immunodominant Tumor-Derived Epitope in Melanoma Patients

TCRs exhibit a high degree of specificity but may also recognize multiple and distinct peptide-MHC complexes, illustrating the so-called cross-reactivity of TCR-peptide-MHC recognition. In this study, we report the first evidence of CD4(+) T cells recognizing the same tumor peptide-epitope from NY-ESO-1, in the context of multiple HLA-DR and HLA-DP molecules. These cross-reactive CD4(+) T cells recognized not only autologous but also allogenic dendritic cells previously loaded with the relevant protein (i.e., the normally processed and presented epitope). Using clonotypic real-time RT-PCR, we have detected low frequencies of CD4(+) T cells expressing one cross-reactive TCR from circulating CD4(+) T cells of patients with stage IV melanoma either spontaneously or after immunization but not in normal donors. The maintenance of cross-reactive tumor Ag-specific CD4(+) T cells in PBLs of cancer patients required the presence of tumor Ag/epitope in the context of the MHC molecule used to prime the Ag-specific CD4(+) T cells. Our findings have significant implications for the optimization of TCR gene transfer immunotherapies widely applicable to cancer patients.

Identification of Specific TRAV6 and TRAV23 Genes in Peripheral Blood T Cells from Patients with Diffuse Large B-Cell Lymphoma.

Antigen specific T cells were identified in peripheral blood from some malignancy, for example melanoma, as well as in B-ALL. The T cells proliferation in response to tumor antigen could be identified by analysis of T cell receptor repertoire, which can display the distribution of TRAV or TRBV subfamilies and the clonaltiy of different subfamily T cells, the clonal expanded T cells are thought to be relate to antigen specific T cells in non-T-cell malignancy. In order to identify the specific T cell clone which was expected to have a specific anti-DLBL (diffuse large B-cell lymphomas) effect and to use as immunotherapeutic cells to prevent minimal residual disease in patients with DLBL, TRAV subfamily genes from clonal expanded T cells were analyzed by using RT-PCR, genescan and DNA sequencing techniques. Peripheral blood mononuclear cells from 6 cases with DLBL were used in the study. By using RT-PCR with 29 specific TRAV subfamily primers, some of TRAV subfamilies could be detected in different samples, 4 monoclonal expanded TRAV subfamily T cells were identified by genescan analysis, in which PCR products direct sequencing was performed to identify the CDR3 sequence. Specific TRAV6 and TRAV23 sequences were obtained in 3 cases respectively, however the CDR3 sequence of TRAV6 or TRAV23 form different patients were not complete identical, suggesting that they recognized and bound specifically to different DLBL-associated antigens. And the CDR3 sequences of these 4 identified genes (two TRAV6 and two TRAV23) were not be reported in Genebank. The whole sequence of both TRAV6 and TRAV23 genes were obtained by RT-PCR amplification the global rearrangement TRAV6 or TRAV23 segments. There are being registered in NCBI Genebank. The sequences will be farther used in TCR gene transfer to T cells to establish the specific anti-DLBL T cell clone and to confirm its specific cytotoxicity.

Targeting the Wilms Tumor Antigen 1 by TCR Gene Transfer: TCR Variants Improve Tetramer Binding but Not the Function of Gene Modified Human T Cells

We have previously described the functional activity of a human TCR specific for an HLA-A2-presented peptide derived from the Wilms tumor Ag 1 (WT1). Recent studies showed that the expression and function of human TCR was improved by the introduction of an additional disulfide bond between the alpha- and beta-chains or by the exchange of the human constant region for murine sequences. In this study, we analyzed the functional activity of WT1-TCR variants expressed in Jurkat cells and in primary T cells. The introduction of cysteine residues or murine constant sequences into the WT1-TCR did not result in a global reduction of mispairing with wild-type TCR chains. Instead, the level of mispairing was affected by the variable region sequences of the wild-type TCR chains. The analysis of freshly transduced peripheral blood T cells showed that the transfer of modified TCR constructs generated a higher frequency of Ag-responsive T cells than the transfer of the wild-type TCR. After several rounds of peptide stimulation this difference was no longer observed, as all transduced T cell populations accumulated approximately 90% of Ag-responsive T cells. Although the Ag-responsive T cells expressing the modified TCR bound the HLA-A2/WT1 tetramer more efficiently than T cells expressing the wild-type TCR, this did not improve the avidity of transduced T cells nor did it result in a measurable enhancement in IFN-gamma production and cytotoxic activity. This indicated that the enhanced tetramer binding of modified WT1-TCR variants was not associated with improved WT1-specific T cell function.

Dual T Cell Receptor Expressing CD8+ T Cells with Tumor- and Self-Specificity Can Inhibit Tumor Growth without Causing Severe Autoimmunity

The engineering of Ag-specific T cells by expression of TCR genes is a convenient method for adoptive T cell immunotherapy. A potential problem is the TCR gene transfer into self-reactive T cells that survived tolerance mechanisms. We have developed an experimental system with T cells that express two TCRs with defined Ag-specificities, one recognizing a tumor-specific Ag (LCMV-gp(33)), the other recognizing a self-Ag in the pancreas (OVA). By using tumor cells expressing high and low amounts of Ag and mice expressing high and low levels of self-Ag in the pancreas (RIP-OVA-Hi and RIP-OVA-Lo), we show that 1) tumor rejection requires high amount of tumor Ag, 2) severe autoimmunity requires high amount of self-Ag, and 3) if Ag expression on tumor cells is sufficient and low in the pancreas, successful adoptive T cell therapy can be obtained in the absence of severe autoimmunity. These results are shown with T cells from dual TCR transgenic mice or T cells that were redirected by TCR gene transfer. Our data demonstrate that the approach of adoptively transferring TCR redirected T cells can be effective without severe side effects, even when high numbers of T cells with self-reactivity were transferred.

T-cell receptor gene transfer by lentiviral vectors in adoptive cell therapy

Adoptive cell therapy can be envisioned as a promising strategy for tumour immunotherapy. However, existing protocols of adoptive cell therapy still require optimisation as many factors, such as specificity, avidity, level of differentiation and amount of transferred T lymphocytes, can influence their immunocompetence and in vivo functionality. In particular, the need to reduce the in vitro expansion phase and to obtain large numbers of tumour-reactive T cells, as a favourable condition for cancer regression, make TCR gene transfer a potentially ideal tool to overcome the limits of adoptive cell therapy strategies. Here, the authors review the state-of-the-art and recent advances in TCR transfer with particular emphasis on lentiviral vector systems. Initial data from preclinical models and recent clinical trials encourage optimisation of a safe, simplified and stable transfer system. In this regard, HIV-based vectors are emerging as good alternative candidates over the most widely used oncoretroviral vectors due to their peculiar molecular features that fit the ideal conditions for donor T cell in vitro manipulation.

CD8α coreceptor to improve TCR gene transfer to treat melanoma: down-regulation of tumor-specific production of IL-4, IL-5, and IL-10

CD8α coreceptor to improve TCR gene transfer to treat melanoma: down-regulation of tumor-specific production of IL-4, IL-5, and IL-10

CD8α Coreceptor to Improve TCR Gene Transfer to Treat Melanoma: Down-Regulation of Tumor-Specific Production of IL-4, IL-5, and IL-10

Therapeutic success of TCR gene transfer to treat tumors depends on the ability of redirected T cells to become activated upon tumor recognition in vivo. Help provided by tumor-specific Th1 cells is reported to relieve T cells from an anergized state and to induce tumor regression. We recently demonstrated the ability to generate melanoma-specific Th1 cells by genetic introduction of both a CD8-dependent TCR and the CD8alpha coreceptor into CD4+ T cells. In this study, we analyzed a TCR that binds Ag independently of CD8, a property generally preferred to induce tumor-specific T cell responses, and addressed the contribution of CD8alpha following introduction into TCR-transduced CD4+ T cells. To this end, primary human CD4+ T cells were gene transferred with a high-avidity TCR, and were shown not only to bind peptide/MHC class I, but also to effectively kill Ag-positive tumor cells in the absence of CD8alpha. The introduction of CD8alpha up-regulates the tumor-specific production of TNF-alpha and IL-2 to some extent, but significantly down-regulates production of IL-4, IL-5, and IL-10 in CD4+ T cells. The introduction of a mutated cysteine motif in CD8alpha, which prevents its binding to LCK and linker for activation of T cells, did not adversely affect expression and T cell cytotoxicity, but counteracted the CD8alpha-mediated down-regulation of IL-4 and IL-5, but not IL-10. In conclusion, CD8alpha down-regulates the production of major Th2-type cytokines, in part mediated by LCK and/or linker for activation of T cells, and may induce differentiation of tumor-specific Th1 cells, which makes this coreceptor an interesting candidate to improve the clinical potential of TCR gene transfer to treat cancer.

T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFNγ production

EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.

Optimizing TCR gene transfer

Optimizing TCR gene transfer

Prospects and Limitations of T Cell Receptor Gene Therapy

Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer, as it can circumvent the demanding process of in vitro generation of large numbers of specific immune cells and it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments and mouse model systems. Furthermore, we discuss the prospects of clinical application of this gene therapy approach, and the possible barriers on the route towards clinical use.

Fulminant experimental autoimmune encephalo-myelitis induced by retrovirally mediated TCR gene transfer

Although some efforts have been made to direct the antigen specificity of developing T cells by retroviral mediated expression of known TCR, it is not clear if the resultant T cells are fully functional. In this study retroviral gene transfer technology was used to introduce a cDNA encoding the TCR from a known encephalitogenic T cell into the bone marrow of mice. Activated T cells expressing this TCR, which is specific for the Ac1-11 peptide from myelin basic protein presented by I-A(u), cause rapid onset of experimental autoimmune encephalomyelitis (EAE). This enabled us to use the onset and progression of the disease as a direct measure of effector functions of T cells generated by this method. The data presented here show that recipients of bone marrow retrovirally transduced with this TCR rapidly develop full-blown EAE that results in paralysis. Therefore, retroviral TCR delivery into the bone marrow supports the development of T cells into fully functional effector cells.

Primary Human Lymphocytes Transduced with NY-ESO-1 Antigen-Specific TCR Genes Recognize and Kill Diverse Human Tumor Cell Lines

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.

Generation of Tumor-Specific CD8+ Cytotoxic T Lymphocytes and CD4+ Th1 Helper T Cells by Transfer of T-Cell Receptor Genes Isolated from a WT1-Specific CD8+ Cytotoxic T-Lymphocyte Clone.

Background: Cytotoxic T lymphocytes (CTLs) and T-helper type 1 (Th1) cells undoubtedly play a crucial role in the eradication of tumors in vivo. However, the production of Th1 cytokines such as IL-2 and IFN-γ is markedly suppressed in the majority of tumor-bearing hosts. Such defects in Th1-mediated immunity in cancer patients have made it difficult to induce tumor-specific CTLs that promote tumor rejection. Adoptive transfer of tumor-specific CTLs and Th1 cells can overcome the difficulty to induce tumor-specific immune response in cancer patients; however, the generation and expansion of tumor-specific CTLs and Th1 cells in vitro are not easy. In the present study, to overcome this problem, we isolated TCR-α and -β chain genes from a WT1-specific CD8+ CTL clone, which had been shown to exert strong cytotoxicity against hematopoietic malignancies and solid tumors in an HLA-A24-restricted manner, and transduced them into nonspecifically activated human CD8+ and CD4+ T cells. Consequently, both CD8+ and CD4+ T cells appeared to acquire WT1-specific function in an HLA-A24-restricted manner.Methods: A WT1 peptide (CMTWNQMNL)-specific CD8+ CTL clone, TAK-1, was established as reported previously (Blood 95:286, 2000). TAK-1 exerts cytotoxicity against variety of tumor cells including leukemia, myeloma, and lung cancer cells but not against normal cells in an HLA-A24-restricted manner. cDNAs encoding TCR-α and -β chain genes were amplified from cDNA of TAK-1 by RT-PCR. TCR-α and -β chain cDNAs were inserted into the plasmid vector. Preparation of lentiviral vectors for transduction of TCR-α and -β chain cDNAs was performed as described previously (Cancer Res 64:1490, 2004). Peripheral blood CD4+ and CD8+ T cells isolated from healthy individuals were cultured with anti-CD3 mAb and retronectin and then infected twice with lentivirus vectors. The infected cells were expanded by culture in the presence of IL-2, IL-12, IFN-γ and anti-IL-4 mAb. Cytotoxicity of CTLs against WT1-peptide-loaded cells and various human tumor cells was examined by a standard 51Cr-release assay. Recognition of tumor cells by Th1 cells was examined by measuring IFN-γ production by ELISA.Results: CD4+ T-cell line (CD4-TCR) and CD8+ T cell line (CD8-TCR) expressing TCR-α and -β chains of TAK-1 were established. Both CD4-TCR and CD8-TCR cells exerted cytotoxicity against WT1 peptide-loaded HLA-A24-positive but not -negative cells. CD8-TCR cells appeared to be cytotoxic against human tumor cells including leukemia, myeloma, and lung cancer cells in an HLA-A24-restricted manner, but did not show any cytotoxicity against HLA-A24-positive normal cells. CD4-TCR cells produced IFN-γ in response to stimulation with HLA-A24-positive but not -negative leukemia cells.Conclusion: The present data demonstrate the functional reconstitution of CD4+ as well as CD8+ T cells by transfer of the αβ TCR complex of a WT1-specific CD8+ CTL clone. Since WT1 is a universal tumor-associated antigen, transfer of TCR genes of WT1-specific CTLs into CD4+ and CD8+ T cells would be useful for Th1-based immunotherapy of various malignancies.

T Cell Receptor Gene Transfer to Produce MHC Class I-Restricted CD4 Helper T Cells: Implications for Immunotherapy of Leukaemia.

CD4 helper T cells play a critical role in the anti-tumour immune response. Cytokines secreted by CD4 T cells can have a direct effect on tumour cells and provide help for CTL priming and effector function. In this study we tested if it was possible to generate MHC class I-restricted helper T cells by retroviral TCR gene transfer into CD4 lymphocytes. Methods: We used a TCR (utilising V11) that recognises the influenza virus A nucleoprotein (NP366–379) peptide in the context of murine Db MHC class I. Murine splenocytes were isolated from C57BL/6 mice (H2b) and activated with conconavalin A and IL-7, and after 48 hours transduced with the pMX-TCR-IRES-TCR retroviral vector. The transduced splenocytes were then cultured in the presence of IL2 for a further 48 hours before staining with anti-murine CD4, CD8 and V11 antibodies and sorting into CD4+ V11+ and CD8+ V11+ populations. Sorted cells were expanded for a further 48–72 hours prior to functional assays. Functional Assays: Purified TCR-transduced (TCR-Td) CD8+ cells and purified TCR-Td CD4+ cells were tested for IFN secretion in response to dendritic cells (DCs) pulsed with NP peptide, an irrelevant peptide (pMDM100) or no peptide. Further experiments examined IFN secretion in response to peptide-loaded tumour cells (EL4 murine lymphoma cells) or transfected tumour cells expressing NP endogenously. Secretion of IFN was measured by ELISA. Results: (1) Antigen-specific IFN secretion was observed by both CD8+ (100% purity) and CD4+ cells (99.93% purity) expressing the class I-restricted TCR when incubated with peptide-loaded DCs. When tested with no peptide or irrelevant peptide, no IFN secretion was observed. The CD8+ cells were more sensitive, recognizing lower concentrations of peptide (10pM) than CD4+ cells (100pM). With peptide-coated EL4 tumour cells as stimulator cells, CD8+ cells showed a peptide-specific response. In contrast, the TCR-Td CD4+ cells were only able to elicit a weak peptide-specific response. Similarly, TCR-Td CD8+ cells were able to recognise NP transfected EL4 tumour cells (EL4NP68), whereas the CD4+ cells were unable to. However, the addition of syngeneic DCs restored the CD4+ cell response to NP transfected EL4 tumour cells to one equivalent to that seen with the TCR-Td CD8+ populations (Table 1). Summary: We have demonstrated that it is feasible to generate MHC class I-restricted CD4+ helper T cells, that are specific for peptide epitopes presented in the context of MHC class I. The CD4+ T cells can recognise antigen-expressing tumour cells in the presence of professional APC, such as DCs. The mechanism by which APC restore tumour recognition may involve trans-costimulation or cross presentation. The data suggest that class I-restricted CD4+ T cells may be able to contribute to enhanced anti-tumour immunity.αββββγγγγγβγIFN Secretion (ng/ml) After Stimulation with DCs or Tumour CellsT Cell (Responder Cell)Stimulator Cell/sNo PeptideNP (100nM)pMDM100 (100nM)TCR-Td CD8+DCs0.1163.20.7TCR-Td CD8+EL40.119.90.2TCR-Td CD8+EL4NP6816.6NDNDTCR-Td CD8+EL4NP68 + DCs31.2NDNDTCR-Td CD4+DCs0.1163.90.2TCR-Td CD4+EL40.10.80.0TCR-Td CD4+EL4NP680.2NDNDTCR-Td CD4+EL4NP68 + DCs25.3NDNDAbbreviations: ND not done; DC, EL4 and EL4NP68 as indicated in text.

WT1-targeted immunotherapy of leukaemia

Since malignant cells are derived from normal cells, many tumour-associated antigens are also expressed in normal tissues. For examples, WT1 is expressed at elevated levels in most leukaemias, but it is also expressed at reduced levels in normal CD34+ haematopoietic stem cells and in progenitor cells of other tissues. Antigen expression in normal tissues is likely to trigger immunological tolerance and thus blunt T cell responses. This could explain the observation that WT1 vaccination in mice frequently fails to stimulate high avidity cytotoxic T cell responses. In order to circumvent tolerance, we have isolated from HLA-A2-negative donors high avidity CTL specific for HLA-A2-presented peptide epitopes of WT1. These allorestricted CTL efficiently kill HLA-A2-positive leukaemia cells but not normal CD34+ haematopoietic stem cells. However, adoptive cellular therapy with allorestricted CTL could only be performed in leukaemia patients rendered tolerant to the infused CTL by prior allogeneic stem cell transplantation. In order to circumvent this limitation, we propose to exploit the TCR of allorestricted CTL as therapeutic tool. TCR gene transfer can be used to take advantage of the specificity of allorestricted CTL and transfer it to patient CTL, while avoiding the transfer of immunogenic alloantigens from the donor CTL to the patient.