NIH Public Access
`Author Manuscript
`Gene Ther. Author manuscript; available in PMC 2009 May 19.
`Published in final edited form as:
`Gene Ther. 2008 November ; 15(21): 1411–1423. doi:10.1038/gt.2008.90.
`
`Development of optimal bicistronic lentiviral vectors facilitates
`high-level TCR gene expression and robust tumor cell recognition
`
`S Yang, CJ Cohen, PD Peng, Y Zhao, L Cassard, Z Yu, Z Zheng, S Jones, NP Restifo, SA
`Rosenberg, and RA Morgan
`Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of
`Health, Bethesda, MD, USA
`
`Abstract
`In human gene therapy applications, lentiviral vectors may have advantages over γ-retroviral vectors
`in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and
`a potentially safer integration site profile. However, unlike γ-retroviral vectors it has been problematic
`to drive the expression of multiple genes efficiently and coordinately with approaches such as internal
`ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing
`two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1
`were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG)
`before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors
`and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor
`gene expression. Furthermore, we determined that the furin cleavage site was recognized in
`lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with
`an efficiency of 20–30%, which could not be increased by addition of multiple furin cleavage sites.
`The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to
`engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic
`activity. Such optimal vectors may have immediate applications in cancer gene therapy.
`
`Keywords
`T-cell receptor; adoptive immunotherapy; tumor immunity; lentivirus; 2A peptide
`
`Introduction
`The genetic engineering of human lymphocytes as a potential therapy for inherited, acquired
`or infectious disease requires efficient transfer and expression of the transgene. In the case of
`adoptive immunotherapy for cancer, naturally occurring antitumor T-cell receptors (TCRs)
`have been used to endow normal T cells with antitumor reactivity.1 In a recent clinical trial,
`we demonstrated the successful conferral of antitumor function through ex vivo γ-retroviral
`transduction of a MART-1-reactive TCR gene into the peripheral blood lymphocytes (PBLs)
`in patients with metastatic melanoma resulted in clinical tumor responses.2 Although
`successful in demonstrating the potential of this cancer gene therapy approach, there are several
`limitations to current γ-retroviral vector-based gene transfer systems.
`
`Correspondence: Dr RA Morgan, Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
`10 Center Drive, Building 10, CRC 3W-3864, Bethesda, MD 20892, USA. E-mail: E-mail: rmorgan@mail.nih.gov.
`Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)
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`In T-cells, the level of transgene expression is modulated by the activation state of the cell,3
`and γ-retroviral based vectors have been shown to be subject to gene silencing in certain cell
`types,4,5 and they have a preference for integration near transcription start sites,6 which may
`increase the potential for insertional mutagenesis. Furthermore, the current γ-retroviral vector
`based protocols require T cells to be fully activated for efficient transduction and this may be
`deleterious to their function.7,8 We previously reported that in a murine model, prolonged ex
`vivo stimulation yielded fully differentiated effector T cells which were capable of in vitro
`tumor killing and high-level INF-γ release but, exhibited inferior activity for in vivo tumor
`treatment compared to naive and less differentiated effectors.9 Finally, there is a growing need
`for gene delivery systems to carry multiple genes in one construct, and γ-retroviral vectors
`have a limited coding capacity (<7 kb).
`
`Lentiviral vectors with their larger coding capacity and ability to more easily express complex
`expression cassettes may be advantageous in this regard compared to γ-retroviral vectors.10,
`11 An example of a clinically important multi-gene construct is a tumor-associated antigen
`TCR, which is a heterodimer of TCR-α and -β chains.12 The TCRs for a variety of tumor-
`associated antigens (TAA) have been identified including the MART-1 and gp100 melanoma
`differentiation antigens, the NY-ESO-1 cancer-testis antigen and the p53 tumor suppressor.
`13–18 An important aspect of TCR expression vector design is that, the TCR-α- and -β chains
`must be coordinately expressed for proper biologically activity.12
`
`The most common strategy for the expression of multiple genes has been based on internal
`ribosome entry site (IRES) elements.19 However, bicistronic lentiviral vectors containing
`IRES elements have consistently demonstrated a biased expression of two transgenes with the
`second gene being under expressed.20–22 We confirmed these reports (data not shown) and
`thus assembled a series of dual promoter-containing lentiviral vectors to express the α- and β-
`TCR chains, but consistently failed to achieve a high percentage of TCR expression in PBL (S
`Jones, data not shown). Naldini and co-workers recently reported that lentiviral vectors
`coordinately expressing two genes could be assembled using a synthetic bidirectional
`promoter,21 but this synthetic promoter exhibited poor activity in PBL (approximately 5–10%
`of transduced cells expressed both genes). An alternative to these approaches is the use of
`ribosomal skipping via 2A peptides.23,24 Several viruses use 2A peptides to mediate protein
`cleavage, including foot-and-mouth disease virus (F2A), equine Rhinitis A virus, porcine
`teschovirus-1 (P2A) and Thosea asigna virus (T2A). The 2A peptide consensus motif
`(DVEXNPGP) is extremely rare and is associated with cleavage-like activity between 2A and
`2B genes through a ribosomal skip mechanism; the 2A peptide impairs normal peptide bond
`formation between 2A and 2B without affecting the translation of 2B. 2A peptides have been
`shown in γ-retroviral vectors to initiate the production of up to four proteins both in vitro and
`in vivo.24,25 When exogenous genes are linked using this approach, about 18 amino acids are
`left on the C terminus of the first gene and a single proline residue remains on the N terminus
`of the second gene. Although linking of the TCR-α and -β chains by 2A peptide has been
`reported,25 the effect of the residual amino acids on the function of expressed TCR has not
`been fully explored. In this study, we inserted a furin protease recognition site ahead of the 2A
`peptide to obtain a more native TCR chain by post-translational processing. Furin is a
`ubiquitous subtilisin-like proprotein convertase, whose natural substrates include certain serum
`proteins and growth factor receptors, such as the insulin-like growth factor receptor. The
`consensus sequence for furin cleavage is RXXR but the potential for actual cleavage is
`dependent on substrate tertiary structure and the amino acids immediately surrounding the
`recognition site.26
`
`Herein, we developed a novel bicistronic lentiviral vector that combines a furin cleavage site,
`and an aminoacid spacer followed by a 2A ribosomal skip peptide. When the spacer sequence
`was augmented by the addition of a synthetic V5 peptide tag sequence, this specifically boosted
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`protein processing and resulted in a lentiviral vector capable of mediating high-level TCR
`expression in transduced lymphocytes.
`
`Results
`2A peptides along with furin and spacer facilitate transgene expression
`In preliminary experimentation, we investigated several strategies such as dual promoters or
`IRES, to drive a two-gene TCR expression cassette, but none of these lentiviral vectors yielded
`significant levels of TCR expression (data not shown). Encouraged by the successful use of
`2A peptides in γ-retroviral vectors to express multiple genes in one construct,24,25,27 we
`attempted to develop lentiviral vectors expressing antitumor antigen-specific TCRs using
`different 2A peptides. In Figure 1a, the amino acids from different 2A peptides are illustrated
`and the conservative domains highlighted. We utilized T2A, F2A and P2A to link the α- and
`β-chains of a murine TCR that specifically recognized the human melanoma differentiation
`antigen gp100 (154–162) (Figure 1b). It has been reported that in γ-retroviral constructs,
`expression of multiple genes linked with 2A peptides can be facilitated by a spacer sequence
`(GSG) ahead of the 2A peptides.25,27 We designed several constructs combining a spacer
`(SGSG or GSG) and furin (R-A-K-R)28 cleavage site with different 2A peptides in bicistronic
`lentiviral vectors (Figure 1b).
`
`To compare the ability of different 2A peptide linkers to express TCRs, lentiviral vectors were
`used to transduce human T-cell lines Jurkat-T3 and SupT1. All the constructs, irrelevant to the
`origin of 2A peptides (T2A, F2A, P2A), functioned to produce the TCR in these two cell lines
`as demonstrated by fluorescence-activated cell sorting (FACS) analysis following gp100
`tetramer staining (Figure 1c). In contrast to a previous report,29 the addition of furin to the 2A
`peptide inhibited TCR expression, whereas furin plus the addition of spacer sequences (GSG
`or SGSG) enabled highly efficient gene expression (up to 95%). While active in T-cell lines,
`these constructs produced significantly less TCR in primary PBL (<34% tetramer+;
`Supplementary Figure 1).
`
`Addition of peptide tags increases TCR expression
`In an attempt to optimize TCR expression in primary human T cells, we determined the
`maturation events of the expressed precursors proteins. The lack of available α- and β-chain
`antibodies that functioned in western blot analysis necessitated the addition of peptide tags.
`The V5 synthetic peptide tag was added between the furin cleavage site and the amino-acid
`spacer-F2A sequence (at the C terminus of α-chain) and a hemagglutinin (HA) tag was added
`to the C terminus of the β-chain (Figure 2a). As depicted in Figure 2a, when 2A peptides initiate
`a ribosomal skip during translation, two molecules can result: the TCR-α chain with an F2A
`residual peptide (including the V5 tag), and the TCR-β chain with an HA tag. If the furin site
`is additionally recognized, this leads to the production of the TCR-α chain without the V5 tag.
`Western blotting with anti-HA antibody demonstrated approximately 90% efficiency of
`processing of the β-chain in the T-cell lines (Figure 2b, quantitation not shown). When the
`anti-V5 antibody was used for western blot analysis, a relatively lower percentage of the α-
`chain was detected, which was likely caused by the loss of the V5 tag following furin cleavage
`(Figure 2a). On the basis of the difference in processing detected by the two antibodies, we
`estimated that cleavage at the furin site occurred at a post-translational level of 28% in SupT1
`and 19% in Juarkt-T3 cells (Figure 2b, compare HA and V5 blots, quantitation not shown).
`Interestingly, this ‘tag-modified’ vector also showed enhanced tetramer staining in the two
`transduced T-cell lines (data not shown).
`
`We next tested whether the superior performance of the tag-modified vector also applied to
`primary human PBL. PBLs were transduced with peptide tag vectors with or without a furin
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`cleavage site (Figure 2c). By western blot analysis we observed a similar pattern of TCR chain
`maturation (Figure 2b, right panel) as described in T-cell lines, where furin contributed to 28%
`cleavage at the post-translational level in primary PBL (quantitation not shown). The
`unexpectedly high protein processing of the F2A fusion protein with the V5 and HA peptide
`tags led us to further investigate the biological properties of this tag-modified vector. When
`primary PBLs were transduced with the tag-modified vectors, we observed a significant
`enhancement in TCR expression measured by tetramer staining using the vector with a furin
`site and peptide tags (Figure 2c). The lower tetramer staining observed without the furin site
`suggested that post-translational cleavage at the furin site may be important in the maturation
`of the TCR heterodimer in transduced human PBL.
`
`Furin plus V5 is critical for optimal TCR expression in PBL
`We next systematically tested the biological activities of several 2A linker modifications
`following transduction of PBL from melanoma patients. Analysis of PBL transduced with the
`vectors containing the furin site and peptide tags demonstrated a significant and reproducible
`improvement in TCR production (Figure 3a) as measured by both an increased transduction
`rate (66% versus 38%) and enhanced expression per cell mean fluorescence intensity ((MFI)
`211 versus 84). To test the antigen reactivity of the expressed TCR, transduced cells were co-
`cultured with peptide-pulsed cells and specific dose-dependent induction of interferon (IFN)-
`γ was demonstrated (Figure 3a, right panel). The level of IFN-γ induction was correlated with
`the transduction rate and MFI, that is, the TCR with the peptide tags was more active than TCR
`without the peptide tags. To further define the role of the tag sequences, the V5 and HA tags
`were removed and the various combinations tested in one experiment (Figure 3b). As depicted
`in Figure 3c, the combination of a furin cleavage site followed by the V5 peptide tag sequence
`showed the highest amount of TCR expression in transduced PBL (image 5). Removal of the
`HA tag did not affect the performance of the vector (compare images 5 versus 6). Removal of
`the furin site significantly decreased TCR expression from the vector (compare images 5 and
`7). The tag-modified vectors also demonstrated superior biological activity when transduced
`PBLs were co-cultured with peptide-pulsed T2 cells (Figure 3d). To address whether the few
`residual amino acids from 2A peptide affect the functionality of expressed TCR, we monitored
`TCR activity using a γ-retroviral construct where TCR-α and -β chains were linked by an IRES
`(this arrangement produced α- and β-chains without residual amino acids). When compared to
`PBL expressing similar amounts of TCR by tetramer staining (images 4 versus 8) the level of
`IFN-γ induction was similar in both vectors (Figure 3d), indicating that the extra amino acids
`added to the TCR-α and -β chains using the 2A linker approach did not significantly affect the
`functionality of this TCR.
`
`We next screened a series of linker peptides inserted between furin and F2A sequences to
`determine if the specific amino acids in the V5 peptide influenced the enhanced biological
`activity of this construct (Figure 3e). In two constructs we dissected the V5 into two parts and
`accordingly made the two constructs as depicted in Figure 3e (second and third images). Next,
`we replaced the V5 peptide with a synthetic hinge region commonly used in chimeric TCR
`constructs30 ((G4S)2, construct 4) or naturally occurring hinge regions from primate
`immunoglobulins31 (constructs 5–8). The original vector with the furin site plus the complete
`V5 peptide (construct 9) consistently displayed the highest level of TCR expression, whereas
`other vectors showed a performance similar to the vector where furin was linked directly to
`F2A (construct 10).
`
`To verify the applicability of this novel molecular strategy to other TCR, we constructed a
`human anti-MART-1 TCR linked by furin-V5-SGSGF2A in the same lentiviral vector
`backbone (Figure 4a). This TCR, DMF5 was previously shown to be highly reactive against
`MART-1 expressing melanoma tumor cell lines.32 Similar to the mouse anti-gp100 TCR
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`vector, superior performance was observed in two patient PBL transduced with a vector where
`the TCR chains were linked with a furin site plus the V5 peptide (Figure 4b). We observed
`enhancement not only in the number of tetramer-positive cells (60–69 and 61–75%) but also
`in expression measured by MFI (74–88 and 63–78). When transduced PBLs were co-cultured
`with antigen expressing human lymphocyte antigen (HLA)-A2-matched tumor line 624, we
`observed a significant increase in IFN-γ production in one of the donors tested, while a modest
`increase in cytokine production was observed with the other donor (Figure 4c). As the
`magnitude of increased biological activity was less pronounced than noted for the gp100 TCR,
`thus it is possible that the improved function of V5 peptide linked TCR constructs may vary
`depending on the genes involved.
`
`One furin site is sufficient for TCR maturation
`In a last series of vectors, we tested alternatives to the single furin cleavage site. Some naturally
`occurring growth factors like insulin-like growth factor I are synthesized as a proprotein that
`is converted to the mature form by cleavage within a unique pentabasic-processing motif
`(KXXKXXRXXRXXR) containing four furin recognition sites. We tested if these tetra-furin
`sites when engineered into the vector could further improve the performance of the vector. In
`Figure 5, we assembled two constructs where tetra-furin sites were inserted either with the V5
`tag or directly fused to the F2A peptide. These vectors were used to transduce PBL, and TCR
`expression assayed by tetramer staining. FACS data demonstrated that the vectors with tetra-
`furin sites did not improve TCR expression compared to the vector with a single furin site
`(compare images 3–5 and images 2–4). When tetra-furin was combined with the V5 peptide,
`we again observed an improvement in TCR expression (compare images 4 and 5), indicating
`that the V5 peptide tag facilitated the cleavage by endoproteolysis at the tetra-furin site.
`
`Both TCR-engineered CD4 and CD8 cells recognize and kill tumor cells
`As a final test of the optimized vector design, we transduced melanoma patient PBL with the
`anti-gp100 TCR vectors and analyzed these engineered cells for anti-melanoma activity. The
`ability to expand following encounter with tumor antigen in vivo may be essential to successful
`TCR gene therapy. To test the proliferative capacity the V5 peptide-containing vectors, we
`transduced PBL from a melanoma patient with the V5 tag-containing F2A vectors and
`determined antigen-specific cell proliferation (Figure 6a, left panel). When the TCR-
`engineered PBLs were co-cultured with melanoma tumor cell lines, the gp100 TCR-engineered
`PBL underwent proliferation with HLA-A2+-matched gp100+ melanoma cell line 624, but not
`with control tumor lines (Figure 6a). The specific TCR used in these studies, gp100 (154–162),
`has been determined to be CD8-independent (L Cassard, manuscript in preparation). To test
`TCR activities in potential helper versus killer T cells, we separated CD4 and CD8T cell subsets
`from PBL by negative selection, and the purified lymphocytes were transduced with the V5
`peptide tag-containing TCR lentiviral vector. The expression of TCR and purity of CD4 or
`CD8 was analyzed by FACS analysis (Figure 6b). The CD4 cells were 89% tetramer-positive,
`whereas the CD8T cells were 56% tetramer-positive (the difference in transduction efficiency
`may relate to the growth of the cells during the time of transduction, as the growth rate of CD4
`was greater than CD8 cells, data not shown). The biological activity of the TCR-engineered
`CD4 and CD8T cells were tested by co-culture with melanoma tumor lines. Both T-cell subsets
`exhibited significant release of IFN-γ to HLA A2-matched tumor cells (Figure 6c). Lastly,
`tumor cell lysis by the TCR-engineered CD4/CD8 lymphocytes was determined by 51Cr release
`assay following co-culture with two HLA A2+ and two HLA A2− melanoma lines (Figure 6d).
`Both CD4 and CD8 TCR-transduced T cells demonstrated specific lytic activity and as
`expected, CD8T cells lysed tumors to a greater extent than CD4T cells.
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`Discussion
`Lentiviral vectors were developed in the mid-1990s by adopting strategies defined over the
`past decade to optimize γ-retroviral vectors for safety and effectiveness.33,34 Lentiviral
`vectors can integrate in nondividing cells, making them more effective than γ-retroviral vectors
`for gene transfer to postmitotic or slowly dividing cells, which may include hematopoietic stem
`cells and naive T cells.35 The lentiviral vector used in this report contain several elements
`previously shown to enhance vector function, including a central polypurine tract (cPPT) for
`improved replication and nuclear import,36 a promoter from the murine stem cell virus
`(MSCV), which has been shown to lessen vector silencing in some cell types,37,38 a
`woodchuck hepatitis virus post-transcriptional responsive element (WPRE) for improved
`transcriptional termination,39 and the backbone was a deleted 3′-LTR self-inactivating (SIN)
`vector design that may have improved safety, sustained gene expression and antisilencing
`properties.5,40–42
`The ability of lentiviral vector to transduce minimally stimulated T cells43 may have significant
`advantages. In vitro analysis of human T cells transduced with γ-retroviral vectors following
`T-cell activation indicated that both TCR Vβ usage and global gene expression patterns were
`altered44,45 and such changes may affect the in vivo survival and function of gene-modified
`T cells. The use of a transgenic mouse model of adoptive cell transfer (ACT)46 also indicated
`that ex vivo stimulated and fully differentiated effector T cells, where inferior for in vivo tumor
`treatment compared to naive or less differentiated T cells (this was despite the fact that they
`demonstrated superior in vitro tumor killing and high-level IFN-γ release).9 We recently
`confirmed and extended these observations in this murine model of ACT by demonstrating
`that TCR gene transfer into stimulated murine splenocytes was less efficacious than T cells
`containing the same TCR isolated from transgenic mice.47 These reports suggested that the
`ability to use lentiviral vectors to genetically modify T lymphocytes at a less differentiated
`stage may have important applications in cancer immunotherapy.
`
`The individual α- and β-chains of the TCR heterodimer must be expressed at equal levels for
`full biologically activity.12 In the case of TCR gene transfer into mature T cells, there is an
`additional complication that the introduced genes must compete with the endogenous TCR
`chains for TCR complex assembly. If mixed heterodimers were formed, they would not
`recognize their appropriate target antigens, and while mixed heterodimer formation can be
`lessened by protein modifications,48,49 the individual α- and β-chains must still be optimally
`expressed. A common strategy for multiple gene expression has relied on IRES element, but
`the use of these elements in bicistronic lentiviral vectors was demonstrated to lead to biased
`expression of the transgenes.20–22,50 Although it has been reported that lentiviral vectors
`containing two independent internal promoters transferred high-level expression of multiple
`transgenes,20 we consistently failed to achieve a high percentage of TCR expression using this
`approach in PBL (data not shown). Naldini and co-workers recently developed lentiviral
`vectors coordinately expressing dual-genes driven by synthetic bidirectional promoters.21
`While they observed coordinated gene expression in various cells and tissues, these synthetic
`promoters exhibited lower activities in activated (10%) and naive PBL (5%).
`
`Encouraged by the successful use of 2A peptides in γ-retroviral vectors to express multiple
`genes in one construct,24,25,27,51 we attempted to develop lentiviral vectors expressing
`antitumor antigen TCR using different 2A peptides for adoptive immunotherapy. The main
`advantage of using 2A peptides in the construction of bicistronic vectors is the potential for
`co-expression of both genes at equal levels. Our western blot analysis indicated that cleavage
`of the polyprotein occurred approximately 90% of the time (Figure 2), thus assuring near
`stoichiometric synthesis of the TCR-α- and -β chains. A potential disadvantage in the use of
`2A peptides is the residual amino acids left on the C terminus of the first protein with the
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`potential for inappropriate targeting of proteins to different subcellular organelles.52 The
`residual 2A peptide left on the α-chain did not appear to significantly affect TCR function in
`comparison to an α-chain without these residues (Figure 3), but this observation is likely gene
`specific as we have observed the loss of biological activity of cytokine genes produced using
`2A peptides (data not shown).
`
`The concern for optimizing TCR gene expression led us to compare three 2A peptides-T2A,
`F2A and P2A,25,29 and consistent with published observations,21,50 2A peptides alone failed
`to achieve high level of TCR expression (Figure 1). Interestingly, when these 2A peptides were
`combined with spacer sequences (SGSG or GSG) and a furin cleavage site, there was
`significant increase in TCR expression (>95%) in T-cell lines, but expression in transduced
`PBL was significantly lower (generally 20%; Supplementary data). This result suggested a
`failure of 2A peptide-mediated ribosomal skipping in PBL, and led us to perform protein-
`processing studies using TCRs modified with peptide tags compatible with western blot
`analysis. This analysis led to the surprising observation that a vector in which the furin cleavage
`site was followed by a V5 peptide tag significantly improved TCR expression compared to
`TCR vectors without the tag. Although the precise mechanism that accounts for enhanced
`processing is unknown, we speculate that the specific V5 peptide sequence may permit
`enhanced ribosomal skipping in this context. Attempts to substitute other sequences for the V5
`peptide were unsuccessful (Figure 3e), suggesting that the specific amino-acid sequence of the
`V5 peptide may be involved in the enhanced activity. This effect was observed with both
`murine and human TCRs.
`
`By western blotting (Figure 2), we saw a robust processing of the TCR 2A fusion protein into
`the individual α- and β-chains (more than 90% of the initial polyprotein as processed). By
`analyzing TCR-α chain fused with V5 tag we observed that post-translational cleavage at furin
`site occurred approximately 30% of the time. Cleavage could not be enhanced using a naturally
`occurring tetra-furin sequence and this sequence may have inhibited the ribosomal skip, as
`TCR expression was lower in tetra-furin sequence-containing vectors (Figure 5). Taken
`together, the combination of a single furin cleavage site followed by a V5 peptide tag plus a
`2A ribosomal skip peptide represents a unique molecular strategy, which may overcome
`technical difficulties using 2A peptides in lentiviral vectors to express multiple genes in one
`construct.
`
`This report also points out the importance of testing transgene expression in the ultimate target
`cell. On the basis of the data from human T-cell lines (where >95% of the simple 2A constructs
`were processed and expressed), we might have concluded TCR vector design to be optimized.
`The fact that TCR expression in PBL was generally less than one quarter of the activity in T-
`cell lines lead to the continued refinement in vector design such that all current TCR vectors
`now produce robust expression in transduced primary human lymphocytes. Recently, Levine
`et al.53 reported on a phase 1 clinical trial using lentiviral vectors that demonstrated efficient
`and safe gene delivery to patients’ T cells with good persistence in vivo. The novel bicistronic
`lentiviral vectors harboring specific antitumor TCR described herein provide the opportunity
`not only for sustained transgene expression but also new methodologies to modify T
`lymphocytes at a less differentiated stage, and may have important applications in cancer
`immunotherapy.
`
`Materials and methods
`Cell culture
`
`PBLs used in this study were from metastatic melanoma patients seeking treatments at Surgery
`Branch, National Cancer Institute. Briefly, PBLs were collected by leukapheresis, and
`lymphocytes were separated by Ficoll/Hypaque cushion centrifugation, washed and
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`resuspended at a concentration of 1 × 106 per ml in AIM-V medium (Invitrogen, Carlsbad,
`CA, USA) supplemented with 300 IU ml−1 interleukin IL-2 and 5% heat-inactivated human
`AB serum (Valley Biomedical, Winchester, VA, USA). CD4+- and CD8+-enriched
`populations were separated using a magnetic beads-based method for both negative and
`positive selection of these subsets (Invitrogen). The other cell lines used in this study, including
`a human lymphoid cell line SupT1, (ATCC, CRL-1942), a T-cell acute leukemia, Jurkat
`(ATCC, TIB-152) and a lymphoblastoid cell line deficient in TAP function, T2 (Starter, 1985)
`were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with heat-
`inactivated 10% fetal calf serum (FCS), 100 U ml−1 penicillin/streptomycin, 2 mM L-glutamine
`and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution.
`Melanoma cells included MART-1-positive HLA-A2+ 624, 526 and two MART-1-positive
`HLA-A2− cells 888 and 938. Other lines included breast cancer line MDA231 (HLA-A2+
`MART-1 negative), 293T and 293FT (Invitrogen) that were cultured in Dulbecco’s modified
`Eagle’s medium (DMEM) supplemented with 10% FCS, 100 U ml−1 penicillin/streptomycin,
`2 mM L-glutamine, 20 μM 2-mercaptoenthanol and 25 mM HEPES buffer solution. All cell
`lines were cultured at 37 °C in a 5% CO2-humidified incubator.
`
`Peptide synthesis
`Peptides in this study were synthesized using a solid-phase method on a peptide synthesizer
`(Gilson, Middleton, WI USA) at the Surgery Branch (NCI). The quality of peptide was
`evaluated by the mass spectrometry (Biosynthesis, Lewisville, TX, USA). The peptides used
`in this study are as follows: gp100 (154–162) (KTWGQYWQV) and control influenza peptide
`(GILGFVFTL).
`
`Vector construction
`The lentiviral constructs utilized are from pLenti6/V5-D-TOPO vector (Invitrogen) and
`pRRLSIN.cPPT. MSCV/GFP.WPRE harboring a green fluorescent protein (GFP) gene driven
`by MSCV U3 promoter (S Jones, unpublished data). Mouse TCR (sp0.01 A-α and -β,
`DQ452619 and DQ452620) targeting gp100 (154–162) melanoma differentiation antigen was
`screened and established in Surgery Branch (L Cassard, unpublished data). MSGV1-154-AIB
`(γ-retroviral construct with gp100 (154) TCR gene linked with IRES) and MSGV-154-A2AB
`(γ-ret