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Advanced Drug Delivery Reviews 64 (2012) 756–762

Contents lists available at SciVerse ScienceDirect

Advanced Drug Delivery Reviews
journal homepage: www.elsevier.com/locate/addr

Genetic engineering with T cell receptors☆
Ling Zhang, Richard A. Morgan ⁎
National Institutes of Health, National Cancer Institute, Surgery Branch, Bethesda, MD 20892, USA

a r t i c l e

i n f o

Article history:
Received 9 August 2011
Accepted 21 November 2011
Available online 9 December 2011
Keywords:
T cell receptor
Gene therapy
Cancer immunotherapy

a b s t r a c t
In the past two decades, human gene transfer research has been translated from a laboratory technology to
clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients
with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens
by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made
to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein
modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss
the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients
with melanoma and other cancers.
Published by Elsevier B.V.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . .
TCR as a therapeutic tool . . . . . . . . . . .
2.1.
Improving transgenic T cell receptor gene
2.2.
TCR gene delivery systems . . . . . . .
3.
TCR gene transfer clinical studies . . . . . . .
4.
Strategies to improve TCR gene therapy . . . .
5.
Summary . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .

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1. Introduction
Adoptive cellular immunotherapy (ACT), the administration of autologous or allogenic tumor-reactive T cells into patients, is a promising
cancer therapy. It has been successful in achieving tumor regression in
transplant-related malignancies, leukemia, and melanoma. In melanoma, this process involves the identification of lymphocytes with high
tumor recognition that can be expanded in vitro, and administered
back to patients [1]. In pioneering clinical studies at the Surgery Branch,
US National Cancer Institute, it was shown that using selected tumor-

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Therapeutic Strategies for Controlling Metastasis and Recurrence of Cancers; Contribution
of drug Delivery Technologies”.
⁎ Corresponding author at: Surgery Branch, National Cancer Institute, Bldg 10 CRC
Room 3-5940, Bethesda, MD 20892, USA. Tel.: +1 301 594 9629; fax: +1 301 435 5167.
E-mail address: rmorgan@mail.nih.gov (R.A. Morgan).
0169-409X/$ – see front matter. Published by Elsevier B.V.
doi:10.1016/j.addr.2011.11.009

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reactive infiltrating lymphocytes (TIL) mediated a 50–70% response
rate in patients with metastatic melanoma when combined with lymphodepleting chemotherapy and IL-2 administration [2,3]. However,
the generation of such patient-specific tumor-reactive lymphocytes is
time-consuming and technically demanding, limiting the number of
cancer patients that receive treatment.
The success of ACT for the treatment of metastatic melanoma patients laid the foundation for the current interest in genetic engineering with T cell receptors to redirect effector T cell reactivity. Genes
encoding TCRs can be isolated from tumor-reactive T cell clones and
introduce into circulating lymphocytes for therapeutic application.
This approach has opened the possibility to treat patients with a variety
of cancer types other than melanoma. In addition to the generation of
anti-tumor effector cells, genetic engineering of T cell also offers a
strategy to introduce molecules that can augment T cell function or
overcome tumor evasion mechanisms, such as adding genes encoding
homeostatic or pro-inflammatory cytokines, chemokine receptors and
costimulatory factors, as well as elements to silence inhibitory molecules.

the hybrid TCR exhibited enhanced expression leading to superior function of the engineered T cells including higher levels of cytokine release and cytolytic activity [15]. is the observed lack of peptide-specificity in some ultra-high affinity TCRs and. One of the potential problems in ectopic expression of tumor-specific TCR is the formation of mixed dimmers between endogenous and introduced TCR chains. It is known that the third complementarity determining region (CDR3) of the TCR is critical for antigen recognition and binding. (iii) the introduced α chain paired with endogenous β chain. and TNFα) upon encounter with antigen positive tumor cells.2.A. 1).19–21]. melanocyte differentiation antigens. However. have reported that small interfering RNA (siRNA) can be used to specifically down-regulate the endogenous TCR resulting in improved expression and reactivity of the transduced TCR [18]. Most significantly. it has been demonstrated that improved expression of TCR genes on the T cell surface can be achieved by codon optimization [23]. During the optimization process cis-acting AT or GC rich sequence stretches. Producing T cells with high functional avidity can be achieved by increasing the TCR affinity or increasing the expression of the TCR on the T cell surface. 2. a nonmolecular approach to avoid mis-pairing is to use γδ T cells for αβ TCR gene engineering. CEA [20] and MAGE-A3 [12]. Recently. the development of patient immune response did not affect the clinical outcome [25]. Single or dual amino acid substitutions in the CDR α or β chains have been shown to provide modest increase in TCR affinity that can enhance antigen-specific reactivity in T cells [12. the chimeric TCR chains preferentially associated with themselves and not the endogenous TCR chains. This approach bypasses the need to isolate tumor-specific T effector cells from individual cancer patients as the TCR-transduced T cells display tumor-specific recognition. progresses have been made to improve the efficiency of TCR gene transfer and expression with subsequent enhanced function of the TCR gene-engineered cells. Since then several reports demonstrated that transfer of a tumor antigen-specific TCR into T cells results in an antigen-specific T cell population [9–11]. Though there is no evidence of toxicity or autoimmunity in reported clinical trials. The most critical property for a TCR used for engineering T cells for clinical application is the requirement for high affinity specific recognition of the tumor antigen peptide-HLA complex. The identification of large numbers of tumor antigens has been essential to the development of TCR based immunotherapy [4]. As an alternative to protein engineering.1. tumor-specific antigens. TCR gene delivery systems The success of TCR gene transfer is built on the gene delivery system. An alternative biological strategy to generate high avidity TCRs is to immunize human-HLA-transgenic mice with human tumor antigen peptides. Antigen specificity is determined by the TCR heterodimer. co-expression of two α and two β chains could form four different TCRs: (i) the endogenous αβ heterodimer. The most widely studied antigens recognized by tumor infiltrating lymphocytes in human melanoma include. 2. Another approach to increase the specific pairing of TCR chains was to introduce additional cysteine residues within the constant region of the TCR α and β chains [16. R. It has demonstrated in vitro that TCR gene-engineered T cells secrete immunostimulatory cytokines (IFN-γ. Zhang. Morgan / Advanced Drug Delivery Reviews 64 (2012) 756–762 During the past decade. Murine CTL generated by this method is used to isolate murine TCR genes specific for HLA-restricted human peptides. Targeting T cells to tumor-associated CT antigens might selectively eliminate tumor cells and avoid or reduce toxicity to normal tissue. (ii) the introduced αβ heterodimer. At the level of mRNA. The clinical success of adoptive cell transfer therapy of TCR engineered T cells offers the possibility to treat patients who do not have natural tumor-reactive T cells. Some reports suggested that the removal of defined N-glycosylation motifs in the constant domains of TCR chains resulted in increased functional avidity and enhanced recognition of tumor cells [22]. This approach has been shown feasible for peptides from the human gp100 [13].L. IL-2. Genes encoding the TCR α and β chains are molecularly cloned from highly reactive T cells that recognize and lyse target tumor cells and can be inserted into gene transfer vectors such as. The first TCR gene transfer to human peripheral blood lymphocytes conferring anti-tumor reactivity was reported in 1999 using a melanoma-antigen specific TCR [8]. In this approach rare codons used by the wild type human or murine derived TCR are replaced by those codons most frequently distributed in highly expressed human genes. The mixed pairing of TCR chains will not target tumor antigens and. Compared with human TCR. either αβ or γδ. but are also expressed in various tumor types [6. One potential concern regarding the generation of very high affinity TCRs. p53 [24]. Finally. Improving transgenic T cell receptor gene expression It is known that TCR function depends on the expression level of the α and β TCR chains.17]. several strategies have been developed to prevent mixed dimer formation while increasing TCR expression. which is composed of twochains. The aim of this review is to discuss the development of TCR gene transfer and update the clinical application of this cell-based drug delivery. however.14]. exhibit antigen-specific cytotoxicity. The first strategy to lessen mixed dimmer formation was the development of murine-human hybrid TCRs in which the constant region of the human TCR chains was replaced by their murine counterparts [15]. Okamoto et al. Several methods have been published recently to increase TCR affinity by modification of TCR genes.7]. Initial TCR gene transfer studies generated small numbers of effector T cells with poor function most likely caused by low-level expression of the introduced TCR chains. Alternatively. and are able to expand in response to the antigen stimulation [11. the function and persistence of γδ T cells in adoptive cell therapy are not well studied. TCR as a therapeutic tool The T cell antigen receptor is the protein expressed on the surface of T lymphocytes and mediates recognition of antigenic peptides presented by major histocompatability complex (MHC) proteins. circulating lymphocytes from cancer patients were engineering with TCRs against melanocyte differentiation antigens MART-1 and gp100 and the adoptive transfer of these cells into the lymphodepleted patients was shown to mediate cancer regression [13. Theoretically. The additional disulfide bond formed by the extra cysteine residues was demonstrated to enhance TCR expression and therefore improve the functional activity. many investigators have focused on cancer testis (CT) antigens as targets for therapeutic cancer vaccines and TCR based adoptive immunotherapy. retroviral or lentiviral vectors using recombinant DNA techniques. In early clinical studies. Identification of suitable target antigens for TCR gene therapy will be a high priority for the coming years. in theory. mutational inversions of the critical interacting amino acids in the TCR α and β chain constant regions favor the pairing of the introduced chains and also increase TCR reactivity [17]. It has been demonstrated that getting high efficiency and stable . Much progress has been made to improve the TCR gene transfer efficacy by optimizing the transduction protocol and by modifying TCR genes to improve specific chain pairing and expression (Fig. cryptic splicing and RNA instability motifs are also removed. and normal proteins highly overexpressed in tumors [5]. 2. and (iv) the endogenous α chain 757 paired with the introduced β chain. The genetic transfer of TCR α and β chains directed against specific tumor antigens can create antigen-specific T cells from any normal T cell.12]. It has been reported that some patients treated with murine TCR gene-engineered T cells developed antibodies directed to murine TCR variable regions. could result in T cells with novel reactivity that may be deleterious to the host. More than 110 CT antigens have been identified and these are normally expressed only in the human germ line. it is not clear what is the affinity threshold for optimal TCR function [26].

Shown are the varieties of different strategies that can be used to potentially improve TCR activity (see text for details). and one experienced a measurable response. R. future TCR gene therapies should choose antigens that are . three patients with metastatic colorectal cancer were treated. Severe transient colitis was also observed in the patients presumptively due to targeting CEA. Initial bicistronic vectors were generated with dual promoters or the chains were linked with IRES sequences [9]. Fifteen patients were treated with MART-1 TCR gene-engineered T cells plus IL-2 after nonmyeloablative chemotherapy and we observed durable tumor regression in 2 out of 15 patients [14]. TCR gene transfer clinical studies The first human clinical trial of melanoma TCR gene therapy was reported in 2006 from our group [14]. The gene inserted downstream of the IRES is expressed poorly compared to the gene positioned upstream. More recent vectors are constructed using picornavirus ribosomal skip peptides to link TCR α and β chain expression and this affords optimal stoichiometric expression of the TCR chains [13. leading to suboptimal expression of the TCR chains and poor T cell reactivity. Morgan / Advanced Drug Delivery Reviews 64 (2012) 756–762 Fig. This trial revealed that T cells expressing highly reactive TCRs might target cognate-antigen expression on the normal cells and cause on-target/off-tumor toxicity [13]. To date. such as Sleeping Beauty and PiggyBac. RNA-based therapy would eliminate the safety concern of gene transfer caused by genome integration [37].758 L. CEA is a 180-kDa tumorassociated glycoprotein over-expressed in many epithelial cancers. Mutagenesis of amino acids in the complementarity determining region 3 (CDR3) and knocking down endogenous TCR gene expression using short interfering RNAs (siRNA) can also increase the overall reactivity of the introduced TCR. It is also reported that lentiviral vectors offer a larger gene insertion capacity and may be less prone to insertional mutagenesis due to their random integration unlike gamma-retroviral vectors that have preferential integration near the transcription start sites of genes [32]. which may be beneficial as less-differentiated or naïve T cells have been reported to be superior for adoptive immunotherapy in animal models [30. This study provided the first proof-of-principle for this novel genetic immunotherapy involving TCR modified T cells. Alternatives to gamma-retroviral vectors are lentiviral vectors' expression systems. As reported by Parkhurst et al. most of the reported TCR gene transfer studies are based on viral vector-based expression system. Zhang. and MSGV1based vector systems [27–29].31]. 1. Design of the optimal TCR expression cassette is critical for efficient expression in viral vectors.. as well as adding extra disulfide bonds. In the early studies. The observation of on-target/off-tumor toxicities suggests that ideally. objective cancer regression was observed in 6 of 20 (30%) and 3 of 16 (19%) patients respectively. In an attempt to improve the efficacy of TCR based therapy. This is another example of how targeting self-antigens with a high affinity TCR can mediate cancer regression. gene transfer in T cell using traditional chemical methods is difficult. TCR α and β chains were assembled in different configurations in retroviral vectors either alone or together. The first clinical trial utilizing lymphocytes transduced with a TCR targeting carcinoembryonic antigen (CEA) to treat metastatic colorectal cancer was reported by Parkhurst et al.30]. Transposonbased non-viral gene delivery systems. Strategies to improve TCR activity. In a second reported TCR gene therapy trial targeting either MART-1 or gp100. In this trial HLA-A2 positive metastatic melanoma patients were treated with retrovirally transduced autologous PBL expressing a TCR against the MART-1:27–35 epitope. patients also exhibited destruction of normal melanocytes in the skin. The approach to redirect effector cells using genetically modified T cells with tumor-specific T cell receptors (TCR) has opened the possibility to treat patients with a variety of cancer types other than melanoma. and ear and required local steroid administration. recently [38]. but can also result in recognition and destruction of normal tissue(s). including MFG/SFG-. Although the short half-life of RNA expression posttransfer may limit its clinical application. efforts were made to isolate higher avidity TCRs [13]. which is also expressed in normal intestinal epithelial cells [38]. These vectors have been used in several TCR clinical studies in which they were shown to have good transduction efficiency and no vector-associated toxicity has been reported to date. Lentiviral vectors can transduce non-dividing or minimal stimulated T cells. most notably in colorectal adenocarcinoma. also have random integration profiles with acceptable gene transfer efficiency and are under development for potential clinical application [33–35]. These methods include creating hybrid molecules containing the constant region (CR) from murine TCR chains. all patients experienced a decrease in serum CEA levels (74–99%). and altering amino acids or glycosylation sites within the TCR chains. which may be a limitation to this treatment. Gamma-retroviral vectors have been used as gene transfer reagents in human application for more than 20 years. eye.A. It has been reported that electroporation/ nucleofection yielded good gene transfer with RNA-based expression systems [36]. However. 3. MP71/SF91-.

MDSC. myeloid-derived suppressor cells. Several reasons could contribute to this including the fact that antigen targets may not be the same as TIL. LTV. and lung [6. . antibodies to CTLA-4 or PD-1 could be co-administered with TCR gene-engineered T cells. whose cells do not express HLA molecules. Zhang.12 expressing metastatic cancer Melanoma Myeloma gp100 DR4‐TRAIL p53 Y Y Y RTV RTV RTV NCT00509288 NCT00910650 NCT00509496 NCT00923390 NCT00393029 HIV‐Gag CEA N Y LTV RTV NCT00991224 NCT00923806 NYE‐ESO‐1 Y RTV NCT00670748 MAGE‐A3/12 Y RTV NCT01273181 NY‐ESO‐1/MAGE‐A3 MAGE‐A3/NY‐ESO Y N LTV LTV NCT01350401 NCT01352286 Abbreviation: RTV. T cells could be further modified to resist apoptosis. liver. or to be resistant to inhibitory factors at the tumor environment and to deliver inflammatory cytokines to tumor site. Disease TCR‐target Lymphodepletion Gene transfer ClinicalTrial.gov identifier Melanoma MART‐1 Y RTV Melanoma Renal cancer p53 expressing metastatic cancer HIV infection CEA expressing metastatic cancer NY‐ESO‐1 expressing metastatic cancer MAGE‐A3. These encouraging results point to the feasibility of transferring T cells engineered with TCR to treat solid tumors. the durable objective response rate of this approach is generally lower than the rate seen in TIL therapy. Fig. To potentially lessen T cell anergy. are currently under clinical investigation (Table 1). In contrast to the vigorous ontarget/off-tumor toxicity seen using TCRs targeting melanoma differentiation antigens and in the CEA TCR trial. Morgan / Advanced Drug Delivery Reviews 64 (2012) 756–762 759 Table 1 TCR gene therapy trials registered in the United States. Their expression is restricted in normal adult tissues to the testes. These objective regressions with the concomitant lack of toxicity exemplify the use of CT antigens as targets in adoptive cell therapy to treat established solid tumors without damage to normal tissues. In this trial reported by Robbins et al. Strategies for improving ACT to treat human cancer.. retrovirus.L. The first clinical trial using adoptive transfer of autologous lymphocytes genetically engineered to express a TCR against CT antigen-NY-ESO-1 has recently been conducted at Surgery Branch. none of the patients who received NY-ESO-1 TCR gene-modified T cells experienced toxicity related to the infused cells. DN-TGFβR indicates dominant-negative TGFβ receptor. Shown are the potential ways to facilitate engraftment. to preferentially migrate to tumors. CCR.7]. lentivirus. Other CT antigens. T regulatory cell. there was a measurable response rate in synovial cell sarcoma patients of 66% (4/6) and in melanoma patients of 45% (5/11) with two melanoma patients being ongoing complete responders [39]. 2. R.A. Data from: ClinicalTrial. and activity of adoptively transferred TCR gene-modified cells (see text for details).gov 08/04/2011. and are thus not susceptible to recognition by a TCR. In addition TCR gene engineering. such as MAGE-A3 [12]. and carcinomas of the bladder. However. Treg. NCI. Phenotype of the PBL for used TCR engineering may also be different from that of the TIL in terms of the expression of homing and other molecules necessary for the efficient tumor trafficking and/or effector function. persistence. only expressed by the tumor or nonessential organs. chemokine receptor. Cancer testis (CT) antigens are expressed in a wide variety of epithelial cancers including melanoma.

E. VanWaes. Immunol.F. Z. Sachs. [14] R. Rev. Leitman. Cancer immunotherapy: a treatment for the masses. Immunol. A TCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in several types of cancer.J. D.R. Morgan / Advanced Drug Delivery Reviews 64 (2012) 756–762 Alternatively the immune response by the TIL against the tumor may be of polyclonal nature. L. Based on these results. Hubicki. gametogenesis and cancer. M. Laurencot. Morgan. [7] A. J. P.L. J. Royal. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. D. Schrump.E. P. P. Zhang.760 L.A. 26 (2008) 5233–5239. Chen. Blattman. Chinnasamy.P.A. Rosenberg.F. Royal. The human T cell response to melanoma antigens. The anti-tumor activity of recombinant IL-12 was tested in a variety of murine tumor models where it caused tumor regression and prolonged the survival of tumor-bearing animals [67–70]. S. Duray. J. Sherry. Rosenberg. L. C. M. Z.P. Kim.E. efforts have been made to improve treatment efficacy of adoptive cell therapy. S.F. Davis. R. Kerkar et al. Dudley.A. J.S. M. R. Z. Nat. Wunderlich. Cancer/testis antigens.E.L. M. Feldman. Rosenberg. its clinical application has been hindered by the toxicity associated with its system administration. Sherry. J. J. S. Hong.A. such as using artificial APC. T cells genetically modified with a tumorspecific TCR and NFAT-IL12 demonstrated powerful therapeutic efficacy in mouse B16 tumor model [72]. M.J. [13] L. Morton. R.E.A. Essers. S. J. Wunderlich. High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. T cells can undergo apoptosis after infusion due to the lack of growth factors or costimulation.P. R.J. Summary Recent TCR based clinical trials in melanoma patients have highlighted the promise as well as the challenges facing this therapy. The identification of best subset of T cell to engineer and development of novel in vitro expansion protocols. Clin. Restifo.59].L. Wunderlich. J. Sherry. U. Caballero. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. R. Hughes. M. Restifo. strategies like the expression of inhibitory RNAs designed to suppress the expression of PD-1 or CTLA-4 may potentially improve the functionality of TCR engineered lymphocytes [55. Willemsen. Royal. Zheng. Cassard. de Vries. Rosenberg. P. N. Nat.A. TGF-β [42–44]. N.L. J. Blood 114 (2009) 535–546.E. Rosenberg. Brewer. Kammula. R.L. Adv. Morgan. S. J. Rosenberg.M. R. Morgan. Morgan.F. Various factors will contribute towards the clinical efficacy of TCR gene therapy including.S. Wargo. P. In an attempt to blunt the inhibitory molecules expressed on the tumor-active T cell. Downey. C. Based on preliminary clinical results.A. A. Hughes. J. Custer. Khong.M. Simpson. To further limit potential toxicity related to constitutive express of IL12.E.C. Robbins.A. Y.66]. Nathan. R. Sherry. Cerottini. S. P. Restifo.J. a phase I/II clinical study using the administration of NFAT-IL12 vector-transduced TIL to patients with metastatic melanoma is in progress. Bishop. [6] A. [10] N. D. Suri. Robbins. J.P. M. Strategies to improve TCR gene therapy Though highly effective T cells can be generated against the desired tumor antigens ex vivo. 6 (2006) 379–389. M. R. However. Theoret. Parkhurst. Hwu. recently demonstrated that co-expression of IL-12 in TCR gene-engineered lymphocytes dramatically augmented tumor treatment efficacy of adoptive transfer in the B16 murine melanoma model without need the administration of IL-2 and vaccine [71].R. J. Topalian. Lee. Romero. Debets. 2). and mediating the differentiation of naïve CD4+ T cells to Th1 cells [65.T. Y. Zheng. El-Gamil. 171 (2003) 3287–3295. U. [3] M. 174 (2005) 4415–4423. T cells were modified to express a dominantnegative or soluble form of the TGF-β receptor. Since the first report of the use of TCR engineered lymphocytes in human in 2006. lymphodepletion of the host increases the effectiveness of cell transfer therapy by eliminating cellular elements like regulatory T cells and myeloid derived suppressor cells and also by removing the lymphocyte pool in the body that might compete for available growth factors or cytokines [54]. R. Morton.J. J. Lankiewicz.F. Robbins. Citrin. Wunderlich.T. Expert. C. J. Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability. M. J.A. S. Restifo. Raffeld. Old. Huang. L. Hughes. Immunol. P. J.G. Greenberg. Rosenberg. J. The mechanism of the local suppression of effector T cell function at the tumor is not fully understood. R. T. Robbins.S. the maintenance of TCR gene expression over time. Yu.F. [9] R. Cohen. Rogers-Freezer. It has been shown in some cases that normal tumor growth proceeds despite high levels of circulating anti-tumor CD8 cells [40]. Rev.C. J. R. it is likely that this therapy will continue to improve and potentially be of benefit to patients with a variety of cancers.F. R. O. B. To block TGF-b signaling.C. Speiser. Immunol. Riley.R. A.N. B. [8] T. Figdor. A. Mavroukakis.A.A. P. As reported recently. M.P. potentially beneficial for the treatment of infectious and malignant diseases by enhancing the cytotoxic activity of NK cells and cytotoxic T lymphocytes (CTL) [63. Tumors display multiple immune suppressive mechanisms. Survival of tumor-specific T cells may be enhanced by engineering them to co-express anti-apoptotic genes like Bcl-2 [58. Adema. K. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.A.R.56]. 5. Y.C. Cancer 5 (2005) 615–625. Kammula. Robinson. Cancer regression in patients after transfer of genetically engineered lymphocytes. S. Z.A. Y. Immunol. Cancer Res. Strategies to address some of these issues are already incorporated in the current clinical trials involving TCR modified T cells. Restifo. Hwu. Science 305 (2004) 200–205. Yang.P. N. Jungbluth. M. Restifo. D. H. R. Thomasian. S. A. Morgan. Rogers-Freezer.A. Yang. Rosenberg.F.S. Restifo.M.M. Alternative strategies to improve the adoptive cell therapy could be to co-administer TCR gene-engineered T cells with antibodies that block T cell negative regulatory activity. Dudley.Y. Zheng. Hughes.J. 163 (1999) 507–513.D. Zhao.A. M. Schwartzentruber. Clay. [11] Y. Lessons learned from the clinical response of patients treated by TCR gene therapy demonstrated the importance of choice the tumor antigen in order to limit off-tumor/on-target toxicity. Science 314 (2006) 126–129. J. J. 92 (2006) 187–224. Morgan.R. Li. 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