Construction and binding analysis of recombinant single-chain TCR derived from tumor-infiltrating lymphocytes and a cytotoxic T lymphocyte clone directed against MAGE-1

D. F. Lake, M. L. Salgaller1, P. van der Bruggen2, R. M. Bernstein3 and J. J. Marchalonis

Department of Microbiology and Immunology, and Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
1 Northwest Biotherapeutics LLC, Seattle, WA, USA
2 Ludwig Institute for Cancer Research, Brussels, Belgium
3 FDA, Rockville, MD, USA

Correspondence to: D. F. Lake


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The TCR is responsible for the specificity of cytotoxic T lymphocytes (CTL) by recognizing peptides presented in the context of MHC. By producing recombinant soluble TCR, it is possible to study this interaction at the molecular level. We generated single-chain TCR (scTCR) from tumor infiltrating lymphocytes (TIL) and one CTL clone directed against melanoma-associated antigen (MAGE)-1. Sixty-eight day anti-MAGE-1 TIL and one cloned anti-MAGE-1 CTL were analyzed by PCR for their V{alpha} and Vß gene usage. The TIL population showed a restriction in V{alpha} and Vß usage with only V{alpha}4 and V{alpha}9 and Vß2 and Vß7 expressed. The anti-MAGE-1 CTL clone demonstrated absolute restriction with only V{alpha}12 and Vß1 expressed. DNA sequence analysis was performed on all V regions. For the TIL, each possible V{alpha}–Vß combination (i.e. V{alpha}4–Vß2, V{alpha}9–Vß2, V{alpha}4–Vß7 and V{alpha}9–Vß7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria. From the anti-MAGE-1 TIL, V{alpha}4–Vß2 scTCR demonstrated binding activity to HLA-A1+ cells pulsed with MAGE-1 peptide. Results obtained from screening a panel of our scTCR constructs on HLA-A1+ cells pulsed with MAGE-1 peptide or irrelevant peptide demonstrated that Vß2 plays a significant role in binding to the MAGE-1 peptide. Amino acid alignment analysis showed that each Vß sequence is distinctly different from the others. These findings demonstrate that soluble TCR in single-chain format have binding activity. Furthermore, the results indicate that in TCR, like antibodies, one chain may contribute a dominant portion of the binding activity.

Keywords: single chain TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Recently many tumor associated peptides have been discovered (reviewed in 1,2). The first to be reported was melanoma-associated antigen (MAGE)-1 (36). The gene encoding MAGE-1 belongs to family of at least 12 closely related genes on chromosome X (7). Although MAGE-1 was discovered as a consequence of cytotoxic T lymphocyte (CTL) recognition of melanoma cells, many other tumor types such as breast carcinomas, non-small cell lung carcinomas and lymphocytic leukemias have been found to express MAGE-1 (4,8,9). The MAGE-1 nonapeptide, EADPTGHSY, is derived from amino acids 161–169 of the MAGE-1 protein and is presented to CD8+ cytolytic T cells in the context of HLA-A1. Since MAGE-1 expression is silent in all tissues except for testis, this marker represents a reasonable target for active-specific and adoptive immunotherapy (2,10).

The {alpha}ßTCR (TCR) recognizes antigenic peptides presented by MHC and is responsible for the specificity of T cells. Both the {alpha} and ß chains of the TCR possess variable (V) and constant domains. The V domains are involved in binding antigenic peptide and the constant domains traverse through the T cell membrane. From crystal structure analysis of TCR, complementarity determining regions (CDR) 3 of both the V{alpha} and Vß chains interact with peptide, while CDRs 1 and 2 interact with MHC (1113). The TCR {alpha}ß heterodimer is closely associated with CD3 proteins, CD4 or CD8, and other adhesion and signal transducing proteins. Binding of antigenic peptide by the TCR V regions triggers T cell activation by signal transduction through the TCR constant domains via CD3 and CD4 or CD8 cytoplasmic proteins.

We (15) and others (1622) have constructed both murine and human scTCR, and some have shown binding activity (16,21,22). Here, we report the construction and characterization of seven recombinant single-chain TCR (scTCR) based upon V{alpha} and Vß genes present in an anti-MAGE-1 tumor-infiltrating lymphocytes (TIL) (1277.A) (23) and the 82/30 MAGE-1-specific CTL clone (24).


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
TIL 1277.A and HLA-A1-transfected T2 cells were provided by one of us (M. L. S.) at the National Cancer Institute, Surgery Branch. From a single vial of ~1x107 frozen 1277.A TIL, mRNA was isolated from one-half of the cells while the other half was placed into RPMI 1640 containing 10% FCS supplemented with 100 U/ml IL-2 and 250 U/ml penicillin/streptomycin. The TIL were stimulated every 10 days for 1 month with HLA-A1+, MAGE-1+ melanoma cells (938 mel) treated with mitomycin C. Total RNA from CTL clone 82/30 was provided by Dr Vincent Brichard (Ludwig Institute for Cancer Research Brussels, Belgium).

HLA-A1-transfected T2 cells were cultured in RPMI 1640 containing 10% FCS supplemented with 250 U/ml penicillin/streptomycin and 2.5 mM glutamine in a 5% CO2 humidified incubator.

Peptides
Peptides were synthesized manually using standard Fmoc chemistry. MAGE-1 peptide (EADPTGHSY) and an irrelevant peptide (STEPPMLNY) which binds to HLA-A1 as reported by Rammensee et al. (25) were purified by HPLC using a reverse-phase C18 column.

RT-PCR TCR V{alpha} and Vß screening
Messenger RNA was isolated from ~5x106 TIL 1277.A using the Micro Fast track kit (Invitrogen, San Diego, CA). For the T cell clone 82/30, total RNA was provided by Dr Vincent Brichard. Complementary DNA was then synthesized from 1 µl of RNA with Invitrogen's cDNA cycle kit. PCR was performed using the cDNA as a template with primer pairs designed to specifically amplify TCR variable region genes V{alpha}1-23 (Clontech, Paolo Alto, CA) and Vß1-20 (26). Thirty-five cycles of PCR were performed in an MJ Research (Watertown, MA) minicycler under the following conditions: denature 94°C/20 s, anneal 55°C/25 s, extend 75°C/20 s in a 50 µl reaction volume. Then 15 µl from each PCR was electrophoresed on a 1% agarose gel and visualized with ethidium bromide on a UV light table.

DNA sequence analysis of TCR V{alpha} and Vß
PCR products from amplified V{alpha} and VßTCR genes were cloned into T-vector (Promega, Madison, WI). The Sanger dideoxynucleotide DNA sequencing method (27) was used to determine the sequences of the TCR V{alpha} and Vß genes present in the anti-MAGE-1 TIL and CTL. The sequences were identified as TCR gene segments using BLAST and alignedleft using BCM Search Launcher (28).

Construction of scTCR
Based upon the TCR V{alpha} and Vß PCR screening results, PCR primers were designed to amplify V{alpha}1, 4, 9, 12 and Vß1, 2, 7 and 8 in such a manner to facilitate construction of scTCR genes. The scTCR were constructed as previously described (29). Briefly, each V{alpha} gene was amplified with a 5' sense primer for in-frame expression and a 3' anti-sense primer containing codons for a Gly3–Ser. Each Vß gene was amplified with a 5' primer containing codons for a Gly3–Ser and a 3' primer in-frame for a His6 tag, provided by the pET 21d vector (Novagen, Madison, WI). In a third PCR, the Vß was re-amplified with a 5' universal primer containing codons for (Gly4–Ser)3 and the same Vß 3' primer used above. In the fourth and final PCR, the V{alpha} product containing a 3' Gly3–Ser and the Vß product containing a 5' (Gly4–Ser)3 were combined in a splicing by overlap extension (soeing) reaction to generate the scTCR genes where V{alpha} and Vß genes flank codons corresponding to a (Gly4–Ser)3 linker. The terminal 5' and 3' primers were the 5' V{alpha} and 3' Vß primers used above. Once the scTCR genes were obtained they were sequenced and then cloned into pET 21d for expression of recombinant scTCR proteins.

Expression and refolding of recombinant scTCR proteins
Expression and purification of the recombinant scTCR was performed essentially as described previously (15). After purification of each scTCR in 8 M urea via the C-terminal histidine tag, they were refolded by dialysis against PBS containing 0.01% Tween 20, stepwise, decreasing the urea concentration 2-fold every 2 h until the urea concentration reached 0.25 M. Then the scTCR were dialyzed against 0.01% Tween 20 in PBS for 2 h or overnight. The protein concentration was always kept <200 µg/ml to avoid precipitation of the recombinant protein. All scTCR were refolded at room temperature.

Biotinylation of scTCR proteins
After purification and dialysis of each scTCR in PBS containing 0.01% Tween 20, the protein concentration was determined by measuring optical density at 280 nm. At least 100 µg/ml of scTCR was then dialyzed against 50 mM Na2HCO3 for 4 h. Then the protein concentration was determined in the same manner as above and biotinylated using NHS-LC-biotin according to the manufacturer instructions (Pierce, Rockford, IL).

Flow cytometric binding analyses of scTCR
Flow cytometry was employed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The flow cytometer was calibrated for precision with CaliBRITE beads and for accuracy using commercially available preserved human peripheral blood leukocytes. Lysys II software was used to collect the data and CellQuest software was used to overlay flow diagrams. Data was collected ungated. HLA-A1-transfected T2 cells were pulsed with either 50 µM of (i) MAGE-1 peptide (EADPTGHSY), (ii) irrelevant peptide (an HLA-A1-binding peptide, STEPPMLNY) or (iii) no peptide for 2 h at 37°C. Excess peptide was washed free of cells followed by incubation with biotinylated scTCR.

Flow cytometry was performed using biotinylated scTCR and streptavidin (SA)–FITC to detect binding of scTCR to live MAGE-1 peptide-pulsed cells. To test monomeric scTCR, 100 µl of biotinylated scTCR (25 µl/ml) was added to MAGE-1 peptide-pulsed T2 cells or T2 cells pulsed with control peptide. Cells were incubated on ice for 1 h, washed in ice-cold PBS and then SA–FITC was added. Cells were further incubated on ice for 1 h and then washed free of excess SA–FITC. For multimeric binding experiments, a 4-fold molar excess of biotinylated scTCR was pre-incubated with 0.5 µg SA–FITC for 1 h. Then biotin–scTCR–SA–FITC was added to peptide-pulsed cells for 1 h on ice. Cells were washed 3 times in ice-cold PBS and subjected to flow cytometric analysis.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-MAGE-1 TIL TCR V{alpha} and Vß gene usage
PCR primer pairs which amplified TCR Vß1-22 and Vß1-20 were employed for screening anti-MAGE-1 TIL 1277.A. We observed a restriction in V{alpha} gene usage to V{alpha}4 and V{alpha}9 (Fig. 1AGo) and a restriction in Vß gene usage to Vß2 and Vß7 (Fig. 1BGo). After 1 month in culture and three rounds of stimulation with MAGE-1+ tumor cells, the V{alpha} and Vß TCR gene usage remained constant (23). There was a single V{alpha}12 and a single Vß1 gene present in the 82/30 CTL RNA (Fig. 1C and 1DGoGo) as this result confirms previous findings with the CTL clone published by Romero et al. (24).



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Fig. 1. PCR screening of TCR V{alpha} and Vß families from anti-MAGE-1 1277.A TIL and 82/30 CTL clone. (A) Results of 1277.A TIL TCR V{alpha} screening. (B) Results of 1277.A TIL TCR Vß screening. Lane numbers correspond to individual V region families; M indicates DNA mol. wt markers; H indicates histone controls. Thirty-five cycles of PCR were performed in an MJ Research minicycler under the following conditions: 94°C/25 s, 52.5°C/35 s and 75°C/35 s. PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide and photographed under UV light.

 
Sequence analysis of V{alpha} and Vß genes
DNA sequencing was performed on V{alpha}4 and V{alpha}9 and Vß2 and Vß7 genes present in the 1277.A TIL population. V{alpha}12 and Vß1 from CTL 82/30 were also sequenced. Amino acid sequences deduced from the DNA sequences are shown alignedleft with each other in Fig. 2Go.



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Fig. 2. Anti-MAGE-1 TCR V{alpha} and Vß amino acid alignmeleftnts. (A) V{alpha}4, V{alpha}9 and V{alpha}12 alignmeleftnts. V{alpha}1 and V{alpha}9 are from TIL 1227.A while V{alpha}12 is from CTL clone 82/30. (B) Vß1, Vß2 and Vß7 alignmeleftnts. Alignments were performed using BCM search launcher, ClustalW 1.6 using default settings. CDRs are labeled over hatched bars. Shaded residues indicate sequence identity. Dashes indicate gaps introduced so that the sequences align properly.

 
Twenty-seven percent amino acid identity exists between V{alpha}4 and V{alpha}9. Twenty-one percent amino acid identity exists between V{alpha}4 and Vß12, and 31% amino acid identity exists between V{alpha}9 and Vß12. Only two amino acid identities were observed in the CDR3s of V{alpha}4, V{alpha}9 and V{alpha}12, while only one identity was observed among Vß1, Vß2 and Vß7. Interestingly in V{alpha}4, there was a 54 bp deletion from residue 84 in framework region (FR) 3 to residue 98 in CDR3 (Fig. 3Go) [numbering according to Kabat et al. (30)].



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Fig. 3. Comparison of 1277.A V{alpha}4 to HAVP08 V{alpha}4 and V{alpha}1 from Jurkat cells. CDRs are labeled over hatched bars. Shaded residues indicate sequence identity. Dashes indicate no amino acid at that position.

 
Twenty-five percent amino acid identity exists between Vß1 and Vß2, 37% identity exists between Vß1 and Vß7, and 28% amino acid identity exists between Vß2 and Vß7. For the CDR3s, Vß1 and Vß2 share 2 amino acid identities, Vß1 and Vß7 share 3 identities, and Vß2 and Vß7 share 3 identities (Fig. 2BGo). It is clear from these alignmeleftnts that the V{alpha} and Vß V regions found in both the anti-MAGE TIL and CTL are different from each other. Binding analysis of the scTCR, shown in the following section elucidates which V{alpha} and Vß are biologically active.

Binding analysis
To determine which chain, V{alpha} or Vß, was most important in binding to MAGE-1 peptide in the context of HLA-A1, different scTCR were constructed using our novel splicing by the overlap extension technique (29). scTCR proteins based upon TIL1277.A and 82/30 anti-MAGE-1 CTL V{alpha} and Vß genes were expressed in bacteria, purified, biotinylated, and then tested in monomeric and multimeric format for binding to peptide-pulsed HLA-A1-transfected T2 cells. Initial experiments with monomeric scTCR suggested weak binding of MAGE-1-pulsed T2 cells by those scTCR containing Vß2. Since the initial experiments with monomeric scTCR gave marginal results, we decided to form multimeric scTCR using SA–FITC as described in Methods. In flow cytometric analysis, we found that multimeric scTCR containing Vß2 bound to MAGE-1 peptide-pulsed HLA-A1-transfected T2 cells (Fig. 4Go). Other scTCR that did not contain Vß2 did not bind to MAGE-1 peptide-pulsed HLA-A1-transfected T2 cells. Multimeric Vß2-containing scTCR did not bind to HLA-A1-transfected T2 cells pulsed with the irrelevant peptide, STEPPMLNY (25).



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Fig. 4. sc TCR binding assay. Flow cytometry results of multimeric scTCR with HLA-A1 transfected T2 cells pulsed with control peptide (dotted line) or MAGE-1 peptide (dark, solid line). Multimeric sc TCR were prepared as described in Methods and incubated with HLA-A1-transfected T2 cells pulsed with either MAGE-1 peptide of HLA-A1-binding control peptide.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Many groups have reported restricted use of TCR V-region genes in TIL populations (24,31,32). Although this information is potentially useful, it has become clear that absolute V{alpha}–Vß restrictions are rare and one can only speculate about which V{alpha}–Vß pair in a given TIL population is responsible for binding to the tumor. To address this problem, we constructed several permutations of recombinant scTCR from TCR V{alpha}–Vß genes in an anti-MAGE-1 TIL population and one anti-MAGE-1 CTL clone. Our results indicate that Vß2 from the TIL population is primarily responsible for binding to MAGE-1 peptide presented in the context of HLA-A1. In addition we were also able to show binding of the CTL clone-derived Vß12–Vß1 to MAGE-1 peptide-pulsed HLA-A1-transfected T2 cells.

Sequence analysis of anti-MAGE-1 TCR V{alpha} and Vß
Amino acid alignment of anti-MAGE-1 V{alpha}4, V{alpha}9 and V{alpha}12 was performed. The percent identities between the three are low, suggesting that the capacity to bind to MAGE-1 peptide is not a property restricted to a unique V sequence. It was interesting that the V{alpha}4 sequence obtained from TIL 1277.A lacked at least 16 residues, 10 from the end of FR3 and six from the first half of CDR3. This V{alpha}4 does not contain a `signature' cysteine at the end of FR3, which is necessary for intra-domain disulfide bonding in all Ig-like molecules. Without this cysteine, it seems doubtful that this V{alpha} could fold properly. Alternatively, the FR3–CDR3 deletion may play a role in binding to the MAGE-1 peptide when paired with Vß2. Further study is necessary to determine if this FR3–CDR3 deletion is a germline deletion or a product of aberrant joining between V{alpha}4 and J{alpha}.

Vß sequence alignment was also performed. Although Vß2 from the TIL and Vß1 from the anti-MAGE clone are from TCR with binding activity, it is very clear that Vß1 and Vß7 are more similar to each other (38%) than they are with Vß2 (25 and 27% respectively). CDR3 has been suggested to be the region in the V{alpha} or Vß that is largely involved in peptide binding. This suggestion was supported by recent crystal structures (1113). In the present study, it was not possible to imply that CDR3 in any of the V{alpha}s or Vßs, including Vß2, is responsible for MAGE-1 peptide binding because they were all significantly different. The underlying reasons for performing sequence analysis on the TIL was to determine whether any similarities existed between TCR V{alpha}s and Vßs in the TIL population. Since they were not similar and only scTCR constructs containing Vß2 demonstrated binding to MAGE-1 peptide, it seems logical to suggest that of the two CTL in this particular TIL population, only the one containing Vß2 was responsible for binding MAGE-1.

Mutational analysis of CDRs in future studies might suggest which specific residues of the TCR are involved in binding to the MAGE-1 peptide. A salient conclusion is that TCR, like antibodies, show a degeneracy in their capacity to bind antigens, and there is apparently no unique sequence of V{alpha} and Vß FRs and CDRs required for recognition of MAGE-1 peptide presented by HLA-A1. It is clear in this study, however, that Vß2 plays a dominant role in binding to MAGE-1 peptide in the context of MHC class I.

scTCR binding analysis
As a result of T cell maturation in the thymus, TCR require peptide to be presented in the context of host MHC for proper immune recognition of antigenic peptide. However, other proteins, such as CD3, CD4/8 CD28, LFA and CD2, on the surface of a T cell play a crucial role in intracellular signaling and adherence of the T cell to its target. Prior to this study we did not know whether soluble, monomeric, recombinant TCR composed only of V regions in scTCR format would bind to peptide–MHC. Others have reported that three-domain scTCR of the form V{alpha}–Vß–Cß possess binding activity (22). Since single-chain antibodies (scFv), composed of Ig V regions have been shown to retain the specificity of the parent antibody, we decided to determine whether soluble scTCR bound to peptide–MHC.

Binding of scTCR to MAGE-1 peptide-pulsed T2 cells was observed. Our results showed that only multimeric scTCR containing Vß2 bound to MAGE-1 peptide-pulsed HLA-1-transfected T2 cells. Multimeric scTCR bound MAGE-1 peptide-pulsed T2 cells, while monomeric scTCR binding was very weak. Since our biotinylation process results in at least 14 molecules of biotin per scTCR protein, it is possible that `tetrameric' scTCR was multimeric, increasing avidity of scTCR for peptide–MHC. It is not surprising that multimeric scTCR bound peptide–MHC because the primary recognition of native, cell surface TCR with peptide–MHC is thought to be weak (33,34) and other adhesion molecules help increase the avidity of the cells' interactions. In one respect, the binding by multimers of low-affinity Fab-like TCR constructs parallels the situation in polymeric IgM where individual single-site binding is likewise of low affinity.

It is possible that the other scTCR which did not show binding activity were not folded correctly, but all scTCR were refolded under the same conditions. We have previously reported that our refolding protocol results in scTCR with the appropriate ß-pleated sheet structure as determined by circular dichroism analysis (35). However, the most sensitive assay for correct folding is a functional recognition assay which we have employed here. Since the V{alpha}4–Vß2 scTCR combination shows the highest binding activity, we suggest that the V{alpha}4 and Vß2 gene products were expressed in the same CTL and formed the effective TCR that mediated recognition of MAGE-1 tumor targets. However, it is certainly possible that another V{alpha}–Vß combination was used.

Compared to scFvs constructed from mAb, a high concentration (between 25 and 50 µg/ml) of scTCR was necessary for binding to MAGE-1 peptide in the context of HLA-A1. Although we did not determine the affinity of the scTCR for peptide–MHC, we suggest that binding is weak because multimeric scTCR was needed to demonstrate binding to MAGE-1 peptide presented in the context of MHC. Because multimers were required to observe binding of scTCR to MAGE-1 peptide-pulsed cells, measurement of affinity would be difficult. Avidity measurements, however, with multimeric scTCR could be done in future studies.

Like VH in antibodies, binding of this particular set of scTCR appears to depend on the presence of Vß2, as those scTCR without Vß2 did not bind. Kabat and Wu reported that antibodies with different specificities can have identical VL, but different VH, suggesting VH is most important for antigen binding (36). Since antibody VH and TCR Vß are both similar in their structures and genetics, we suggest they are also similar in function. TCR Vß2 was present in every case where multimeric scTCR bound MAGE-1 peptide and absent from scTCR which did not bind MAGE-1.

Identification and cloning of TCR V-region genes utilized by lymphocytes infiltrating tumors and autopathogenic lymphocytes which infiltrate normal self tissue in autoimmune diseases is important because it allows construction and analysis of recombinant TCR. These receptors can then be characterized for their biological properties, such as binding analysis, spectrum and even identification of antigen. It is unclear at this time whether monomeric or multimeric scTCR could be therapeutically or diagnostically useful. Multimeric scTCR would probably be too large and immunogenic, but monomeric scTCR could be useful if their affinities were sufficiently high. Since the natural affinity of TCR for MHC–peptide is thought to be low (33,34), perhaps mutagenesis of V regions to increase affinity to peptide and/or MHC might enhance the clinical utility of recombinant TCR.

Another application arising from identification and cloning of tumor antigen-specific TCR is transfection of the DNA encoding them into non-specific effector cells, producing tumor-specific killers. This has already been done in another system with scFv (37), generating an unrestricted T cell. However, more work must be done at the molecular level to understand how TCR recognizes peptide–MHC and what type of TCR recognition is necessary to activate a T cell once interaction with it's target has occurred.


    Acknowledgments
 
Supported by Arizona Disease Control Research Commission grant no. 9831 to J. J. M. and by internal funding from the Arizona Cancer Center to D. F. L.


    Abbreviations
 
CDRcomplementarity determining region
FRframework region
MAGEmelanoma associated antigen
SAstreptavidin
scTCRsingle-chain TCR
TILtumor-infiltrating lymphocyte

    Notes
 
Transmitting editor: G. Klein

Received 11 November 1998, accepted 27 January 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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