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Blood, 15 March 2007, Vol. 109, No. 6, pp. 2331-2338. Prepublished online as a Blood First Edition Paper on November 2, 2006; DOI 10.1182/blood-2006-05-023069.
GENE THERAPY Facilitating matched pairing and expression of TCR chains introduced into human T cells1 The Fred Hutchinson Cancer Research Center, Program in Immunology, Seattle, WA; 2 Department of Immunology, University of Washington School of Medicine, Seattle, WA; 3 Department of Hematology and Oncology, University of Mainz, Germany
Adoptive transfer of T lymphocytes is a promising treatment for a variety of malignancies but often not feasible due to difficulties generating T cells that are reactive with the targeted antigen from patients. To facilitate rapid generation of cells for therapy, T cells can be programmed with genes encoding the  and ß chains of an antigen-specific T-cell receptor (TCR). However, such exogenous and ß chains can potentially assemble as pairs not only with each other but also with endogenous TCR and ß chains, thereby generating ßTCR pairs of unknown specificity as well as reducing the number of exogenous matched ßTCR pairs at the cell surface. We demonstrate that introducing cysteines into the constant region of the and ß chains can promote preferential pairing with each other, increase total surface expression of the introduced TCR chains, and reduce mismatching with endogenous TCR chains. This approach should improve both the efficacy and safety of ongoing efforts to use TCR transfer as a strategy to generate tumor-reactive T cells.
The application of molecular technologies to identify proteins differentially expressed by transformed cells is providing large numbers of candidate antigens that can be potentially targeted to selectively eliminate tumor cells by cancer immunotherapy.1,2 Efforts to vaccinate patients to such antigens have yielded some provocative results, but only a small subset of patients have demonstrated therapeutic responses, likely reflecting the many in vivo obstacles to generating potent responses to these proteins, particularly in patients with an established malignancy.3 An alternative approach of isolating and expanding reactive T cells ex vivo followed by adoptive transfer into the patient circumvents many of these in vivo obstacles. Although this adoptive therapy approach has demonstrated significant clinical promise,4 generating the large numbers of T cells required for adoptive therapy of cancer patients, particularly within the time constraints posed by progressive tumors, is often not feasible. Molecular technologies have now provided a means to more broadly capture the therapeutic potential of this treatment strategy. Genes encoding the and ß chains of a T-cell receptor (TCR) can be isolated from a T cell reactive with the antigen of interest and restricted to a defined HLA allele, inserted into a shuttle expression vector, and then introduced into large numbers of T cells of individual patients sharing the restricting allele and the targeted protein.5 This approach is already being pursued clinically,6 and the goal is to establish a library of such defined TCR genes that could provide reagents for treating a diverse set of patients and diseases. Multiple virus- and tumor-reactive TCR genes have already been successfully isolated and re-expressed in T cells, including TCR genes with specificity for HLA*0201 (HLA-A2)–restricted epitopes from melanoma antigens7–9 and HLA-A2– and HLA*2402-restricted WT1-derived epitopes.10,11
The avidity of a T cell for its target reflects many factors, including the affinity of the TCR for its cognate antigen12 and the level of TCR expression.13–15 One difficulty with the TCR-transfer approach is that the TCR-transduced T cells are often of lower avidity than the parental T cell from which the TCR was derived due to failure to achieve wild-type levels of TCR expression, which likely contributed to the limited efficacy observed in the recently reported clinical trial pursuing this strategy.6 Thus, the TCR chains introduced into T cells need to be initially selected for appropriate affinity10,16 and inserted into vectors that can achieve and maintain high-level expression.17 However, even if these criteria are met, the introduced exogenous
To promote preferential pairing of introduced TCR chains with each other, several strategies might be pursued. Recently, we (Stanislawski et al21 and Voss et al22) and others (Cohen et al23) have been using murine constant TCR-chain domains to enhance expression of human TCR chains in human T cells. However, murine constant domains might be immunogeneic in patients, thereby limiting the survival of transgene-transduced T cells in patients.24 As the intracellular TCR-chain domains are relatively short and do not have a signaling function, these domains on each chain could potentially be replaced by or attached to zipper domains that promote interchain recognition and binding. This strategy has proven useful for producing soluble
We sought to determine if a disulfide bond previously shown to stabilize soluble TCR heterodimers that would require only single point mutations in the constant regions of the
Peptides, antibodies, multimeric complexes, and cytokines
Peptides p53264-272 and WT1126-134 were synthesized by SynPep (Dublin, CA). Antihuman CD8-FITC/PerCP/PCY5, anti–human Vß21-FITC, anti–human Vß13-FITC, anti–human T-cell clones and cell lines The generation of WT1126-134-specific CD8+ T-cell clones RS28 and CE10 has been described.35 The TAP-deficient HLA-A2–positive cell line T2 and retroviral packaging lines 293T and Phoenix-Ampho were obtained from the ATCC (Manassas, VA). Fresh leukemia samples from patients with acute myeloid leukemia (AML1 and AML2) were a kind gift of Matthias Theobald (University of Mainz, Germany). Cloning of TCR genes and transduction, selection, and expansion of human T cells
The TCR chains expressed by CE10 and RS28 were amplified using a SMART RACE cDNA Amplification Kit (Clontech no. K1811-1; Palo Alto, CA) and sequenced. Full-length wild-type (wt) TCR
Cocultured packaging cells 293T and Phoenix-Ampho were transfected using Fugene transfection reagent (Takara, Gennevilliers, France) with TCR chain (pBullet), gag-pol (pHIT60), and env (pCOLT-GALV) vectors.21 Reverse transcriptase–PCR (RT-PCR), flow cytometry, and T-cell assays
Expression of introduced
Introducing cysteines in the constant domains of the TCR and ß chains (Cys TCR) promotes preferential assembly of appropriately matched TCR pairs
To determine if the creation of a disulfide interchain bond, previously used to stabilize soluble TCR chains,30 might promote preferential intracellular pairing of introduced TCR chains, the wild-type (wt) TCR v
To assess pairing of the introduced TCR chains at the cell surface, polyclonal CD8+ T cells transduced with wt and Cys chains were separately (Figure S1) or simultaneously (Figure 1A) stained with an anti-vß21 antibody and WT1126-134-specific multimers (expression of the v
The level of vß21 detected in transduced T cells directly correlated with the intensity of multimer staining (Figure 1A-D), implying that the goal of achieving higher levels of introduced TCR chains will in fact result in transduced T cells with higher avidity for targets. To determine if limitations on the levels of TCR that can be achieved on the cell surface existed, T cells transduced with mock, wt, or Cys-modified TCR chains were stained with an anti- ßTCR antibody. In 3 separate experiments, independent of the level of transduced chains attained, no differences in total ßTCR surface expression were found (Figure 1E left), suggesting that the total ßTCR surface expression is limited by factors other than just expression of more TCR chains, such as components of the TCR complex or adaptor proteins that affect assembly or export of TCR complexes.42,43 Thus, if TCR chains are being introduced into primary T cells, strategies that modify the chains to promote appropriate pairing and/or enhanced assembly and export have increased importance for expression of the desired TCR.
Differences in multimer binding by T cells transduced with wt and Cys-modified TCR chains expressing equivalent levels of v T cells with increased appropriately matched TCR pairs at the cell surface recognize target cells more efficiently
Increased expression of appropriately matched pairs of the introduced TCR chains should translate into more efficient target recognition if the receptors signal normally, since target avidity has been shown to correlate with level of TCR expression.14 Therefore, transduced T cells were sorted for comparable vß21 TCR surface expression, equivalent amounts of
The ability of gene-modified T cells to survive and proliferate in response to target recognition will likely contribute to therapeutic efficacy. To assess survival of T cells transduced with wt and Cys TCRs following antigen-specific stimulation, transduced T cells were selected for equivalent levels of vß21 expressions by flow cytometry and stimulated with WT1126-134-pulsed PBMCs, and activation-induced cell death (AICD) was assessed after stimulation by annexin V staining 4 days later. At 4 days, approximately 20% of cells from cultures of both wt and Cys-TCR–transduced T cells were annexin V+ (data not shown). Proliferation was also assessed 4 days after stimulation by both 3H-thymidine incorporation and CFSE dilution. After stimulation with the WT1126-134 peptide, a larger fraction of T cells transduced with Cys-modified TCR chains entered cycle and proliferated more extensively than T cells with wt TCR chains (Figure 2B-C), and enumeration revealed 2.1-fold more cells present at day 4. The failure of T cells expressing low levels of an introduced TCR to effectively eliminate cancer cells has been further suggested by the results of a recent clinical trial employing TCR gene-transfer technology.6 To test whether observed differences in avidity between wt- and Cys-TCR–transduced T cells could translate to improved recognition of leukemic blasts in T cells expressing only low levels of introduced TCR chains, T cells transduced with wt or Cys-modified TCR chains were selected for equivalent vß21 surface expression at an MFI of 7. Two different leukemic blast samples isolated from HLA-A2+ AML patients demonstrated by RT-PCR to overexpress WT1 were used as targets in a 5-hour 51Cr release assay. The specificity of the observed lysis for the WT1126-135 antigen of leukemia cells was assessed by including as cold target inhibitors T2 cells pulsed with the WT1 or an irrelevant peptide but not labeled with 51Cr. T cells transduced with Cys-modified TCR chains lysed leukemic blasts in the presence of a 20:1 excess of T2 cells loaded with an irrelevant HLA-A–restricted peptide, whereas T cells transduced with the wt TCR chains lacked sufficient avidity to lyse these leukemic targets (Figure 2D). Addition of "cold" T2 target cells pulsed with the WT1 epitope inhibited lysis of the leukemia cells by T cells transduced with Cys-modified TCR chains, affirming the specificity of the lytic activity in these cells despite low levels of the TCR chains and demonstrating that the leukemic cells processed and presented the WT1126-134 epitope (Figure 2D). Transcription, translation, and expression of wt and Cys-modified TCR chains The Cys modifications of the TCR chains, which promoted preferential pairing, might also influence expression of the modified chains by modifying protein production, stability, or complex assembly. Therefore, T cells transduced with mock, wt, or Cys-modified TCR chains were stained for surface expression of vß21 TCR chain following drug selection with puromycin and neomycin. After each set of transductions, the parental T-cell clone was analyzed for comparison and always had the highest surface expression of the vß21 TCR chain (MFI 32). In 7 independent experiments, despite the use of identical expression vectors, promoters, and selection procedures for both the wt and Cys-modified TCR chains, surface expression of the vß21Cys TCR chain (MFI 24) was reproducibly significantly higher than expression of the vß21wt TCR chain (MFI 15; Figure 3A; Figures S1-2), suggesting that the Cys modification might be providing an additional benefit for expression beyond just preferential pairing.
It was possible that this increased surface expression might have merely reflected increased integration of retroviral vector copies in these experiments with subsequent augmented RNA levels. Therefore, RNA was isolated from T cells transduced with wt or Cys-modified TCR chains, and the vß21 chain message was quantitated by RT-PCR and normalized to ß-actin copies. No difference in v 21 or vß21 RNA expression was observed on T cells transduced with wt or Cys-modified TCR chains (Figure 3B), suggesting equivalent levels of transcription of the wt and modified genes.
Increased translation of transcripts from the point-mutated gene could also lead to augmented vß21 Cys expression. Therefore, intracellular vß21 TCR protein levels were assessed by flow cytometry. To permit analysis of intracellular and not surface TCR chains in single cells, cells were fixed with paraformaldehyde to mask antibody-binding sites and inhibit binding to the cell surface and then permeabilized. The efficiency of masking the binding site for the anti-vß21 antibody on surface molecules by paraformaldehyde was assessed by incubating the parental T-cell clone CE10 with the anti-vß21–FITC antibody in the presence or absence of paraformaldehyde, with or without saponin permeabilization. Paraformaldehyde completely blocked anti-vß21–FITC surface binding, but intracellular vß21 remained detectable after permeabilization with saponin (Figure 3C-D). Intracellular vß21 protein levels in the parental clone were highest (MFI 21), but, in contrast to the differences in surface expression of wt or Cys-modified vß21, intracellular vß21 in T cells transduced with wt or Cys-modified TCR chains were almost identical (both MFI 18; Figure 3D) and nearly equivalent to the parental clone. Thus, the higher levels of Cys-modified TCR chains detected on the surface did not appear to reflect enhanced transcription or translation, suggesting that exogenous TCR
Increased surface expression of Cys-modified TCR chains is not unique to a single TCR
To determine if the increased surface expression of Cys-modified TCR chains only reflected a unique property of chains from clone CE10, the same Cys modifications were introduced into TCR chains isolated from clone RS28, which is also specific for WT1126-134 but employs different
To determine if the effect of Cys modifications on expression would occur even if the TCR chains were not selected based on natural pairing, T cells were transduced with either the wt or Cys-modified chain of clone CE10 (v21) and the wt or Cys-modified ß chain of clone RS28 (vß13). T cells expressing such unrelated TCR chains predictably did not acquire specificity for WT1126-134 (data not shown). However, as observed for T cells transduced with the and ß chains from clone CE10 (Figure 3A) or RS28 (Figure 4A), transduction with the combination of the chain from clone CE10 (v21) and the ß chain from clone RS28 (vß13) led to more efficient expression if both chains were Cys modified (Figure 4B), demonstrating the effect of the Cys mutations on expression was independent of the specificity of paired variable TCR domains.
To determine the effect of having the Cys modification on only 1 chain, T cells were transduced with combinations of v
The reduced expression of a Cys-modified ß chain when expressed in combination with an introduced wt
The introduction of an additional set of and ß TCR chains into a T cell creates a situation in which the cell can express 4 different TCR pairs: the natural endogenous and introduced exogenous TCRs and 2 additional TCRs formed by mismatched pairing of the introduced and ß TCR chains with the endogenous ß and TCR chains, respectively. Such mismatched pairing poses potentially substantive problems for the use of TCR transfer as a clinical strategy, since the specificity of the mismatched pairs will be unknown and potentially autoreactive. In the absence of any preferential pairing of the 2 introduced chains with each other or between the 2 endogenous TCR chains, only 50% of introduced TCR chains would be predicted to be paired with each other. Although recent reports have demonstrated that preferential pairing can occur between selected and ß TCR chains,45 such events appear relatively rare and not sufficiently predictable to readily permit selection of TCR chains for use in TCR transfer that have unique naturally high affinities for each other.46 Using antigen-specific down-modulation of the introduced ß TCR chain derived from the WT1-specific clone CE10 to reflect incorporation into matched versus mismatched pairs, our data demonstrated that, depending on the level of expression of introduced TCR chains, approximately 40% to 60% of wt ß chains were not down-modulated following antigen-specific stimulation in T cells expressing lower levels of introduced TCR chains. Thus, the introduced wt TCR chains did not exhibit a significant preference to assemble with each other rather than the endogenous TCR chains. However, engineering point mutations in each of the introduced chains that can promote formation of a disulfide bond between the constant ectodomains significantly altered these apparently largely random interactions between and ß chains, with approximately 95% of the TCR complexes containing the introduced Cys-modified ß chain down-modulated by specific stimulation with antigen independent of the level of chain expression achieved, suggesting that the exogenous Cys-modified TCR chains were overwhelmingly pairing with each other. This might reflect the cysteine bonds promoting preferential interactions between the chains and/or increasing the conformational stability of the proteins after interacting,47 since these particular cysteine modifications have been shown to stabilize a number of soluble TCR ß chains.30–32 This preferential pairing should improve the safety of TCR transfer, since, with this approximately 1-log reduction in expression of mismatched pairs, the avidity of T cells expressing these low levels of mismatched TCR chains even if potentially autoreactive should be greatly reduced for the relevant target.14
The insertion of these cysteines in the TCR chains appeared to have another unanticipated and beneficial activity. In particular, not only was the percent of matched TCR chains at the cell surface increased but the total amount of introduced TCR chains at the cell surface was also increased. This outcome might again reflect formation of more intrinsically stable conformations between the modified chains, providing these pairs of TCR chains with a competitive advantage for assembly and incorporation into more stabilized complete TCR-CD3 complexes that then get exported to the cell surface. Alternatively, it might reflect the cysteine modifications leading to a conformation more favorable for interacting with limiting components of the TCR-CD3 complex. The observed increase in expression of introduced chains occurred in the context of evidence that the total amount of TCR-CD3 complexes on the cell surface, composed of the sum of receptors with endogenous and exogenous chains, did not differ from nontransduced T cells, consistent with evidence that the total amount of TCR-CD3 complexes expressed on the T-cell surface on T cells is regulated and limited. Such a limit has also been observed after transfer of The observed enhanced formation of matched pairs of cysteine-modified introduced TCR chains clearly had the desired outcome of reducing formation of unwanted mismatched pairs of unknown specificity. However, the efficiency with which mismatching was avoided seemed surprising, with only approximately 5% of the introduced chains appearing to be in TCR complexes that failed to respond to antigen targeted by the TCR. This high efficiency made us question if the introduction of cysteine modifications not only enhanced the formation and export of matched pairs but also interfered with the formation of mismatched pairs. Our results support this hypothesis, as, in distinction to the higher expression observed with introduction of 2 Cys-modified chains, introduction of TCR chains in which only a single chain was Cys modified was not a neutral event but rather reproducibly resulted in decreased expression of the introduced chains. Such reduced expression of pairs of TCR chains in which 1 has a novel cysteine might reflect abnormal folding and degradation of these pairs as a consequence of formation of improper disulfide bonds with other cysteines naturally present in the other chain; indeed incorrect disulfide formation has been shown to reduce the folding capacity of TCR chains in other settings.28 Thus, Cys-modified TCR chains are not only more likely to form matched pairs but also appear less capable of forming productive pairs with chains that lack the complementary cysteine. However, cysteine modification did not appear to completely prevent pairing with endogenous TCR chains, and strategies that can further reduce mismatching would still be desirable. In summary, our results demonstrate that introducing artificial cysteines in the constant region of the chains of a TCR has multiple beneficial effects for the strategy of imparting T-cell specificity by TCR transfer, including enhanced expression of matched pairs, reduced expression of mismatched pairs, and increased proportional expression of the introduced TCR chains in the total TCR pool. These changes result in greater avidity for target cells expressing the recognized antigen and reduced risk of producing a T cell expressing sufficient levels of an undesirable TCR formed from an unpredictable pair of an endogenous and exogenous chain to mediate autoimmune injury. Thus, this approach should improve both the efficacy and safety of the ongoing efforts to use TCR transfer as a strategy to generate T cells that can be used for therapy of malignant diseases.
Contribution: J.K. designed, performed, and analyzed experiments. M.L.D. and W.Y.H. cloned T cells and T-cell receptors. M.W., R.-H.V., and C.F. designed and analyzed experiments. P.D.G. analyzed experiments. J.K. and P.D.G. wrote the manuscript. All authors edited and approved the written manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Jüurgen Kuball, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Thomas Building, D3-100, Seattle, WA 98109; e-mail: jkuball{at}fhcrc.org; or Philip D. Greenberg, University of Washington, BB1325 Health Sciences Building, Box 356527, Seattle, WA 98195; e-mail: pgreen{at}u.washington.edu.
This work was supported by grants from the Leukemia & Lymphoma Society (LLS 7040-03) and the National Institutes of Health (P01 CA18029, R37 CA33084). J.K. and M.W. are fellows of the Deutsche Krebshilfe (Germany) and M.L.D. was supported by a Poncin Scholarship. We gratefully appreciate expert technical contributions by Jianhong Cao and Hieu N. Nguyen. Current address for W.Y.H.: Genentech Inc, 1 DNA Way, MS-88, South San Francisco, CA 94080-4918.
Submitted May 12, 2006; accepted October 28, 2006.
Prepublished online as Blood First Edition Paper, November 2, 2006
DOI: 10.1182/blood-2006-05-023069
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Top
Abstract
Introduction
–APC, annexin V–PE (BD Pharmingen, San Diego, CA); CFSE (Molecular Probes, Eugene, OR); and anti-HA and anti–mouse IgG-PE (Sigma-Aldrich, St Louis, MO) were commercially obtained. PE- or APC-labeled WT1126-134 multimeric HLA-A2 complexes (WT1126-134 multimer-PE) were synthesized as described
Cys and residue 57 of the Cß region from Ser
), or v
), were simultaneously stained with WT1126-134 multimer-PE and the anti-vß21–FITC antibody. MFI of the anti-vß21–FITC signal was plotted against the MFI of the WT1126-134 multimer-PE signal. (B) Transduced cells were sorted based on equivalent levels of low (left panel) or high (right panel) vß21 TCR chain, (C) equivalent levels of WT1126-134 multimer binding, or (D) equivalent levels of v
) or v
) were selected for lower (denoted as L: MFI 16) or higher (denoted as H: MFI 28) vß21 TCR-chain expression and then were either not treated or stimulated with 
, 
, 
, 
) in a 5-hour 51chromium-release assay. The assays were performed in the presence of excess (20:1) cold target T2 cells pulsed with either the relevant (open symbols, thin lines) or irrelevant peptide (closed symbols, bold lines).
T cells,