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Vaccination with DNA Encoding a Single-Chain TCR Fusion Protein Induces Anticlonotypic Immunity and Protects against T-Cell Lymphoma¹

Molecular Immunology Group, Tenovus Laboratory [S. M. T., J. N. R., F. K. S.] and Dermatopharmacology [P. S. F.], Southampton University Hospitals Trust, Southampton SO16 6YD, United Kingdom

	ABSTRACT

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The clonotypic T-cell antigen receptor (TCR) provides unique V and Vßsequences with potential as idiotypic targets for immunoregulation. For T-cell malignancies, vaccination with the TCR could induce therapeutic anti-idiotypic responses. To facilitate this approach, we have developed DNA vaccines that include the genes encoding TCR sequences from a T-cell lymphoma (TCL). To combine requirements for stable folding with a simple minimized single-chain construction, we used a three-domain VVßCß sequence. To promote anti-TCR immunity, we fused a pathogen-derived sequence from tetanus toxin to the 3'-end of the single-chain TCR. The fusion gene vaccine induced anti-idiotypic antibodies and generated protection against the TCL. The critical requirement for the conformational integrity of the delivered TCR antigen was highlighted by the observation that DNA fusion vaccines containing either VVß or VßCß sequences failed to generate antibodies reactive with the native TCR or provide protection. This is the first report of a DNA vaccine able to induce anti-idiotypic immunity against TCL, and it presents a simple strategy for selectively eliminating T-cell clones in vivo.

	INTRODUCTION

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The structural diversity of the TCR³ and ß chains is generated by somatic recombination of V(D)J gene segments (1) . The unique combination of these genetic elements gives rise to Id determinants that are expressed clonotypically by the T cell. When expressed by malignant or autoreactive T-cell clones, these Id determinants can serve as targets for active immunotherapy. The Id immunoglobulin of malignant B cells provides a similar molecular target. Vaccination strategies against B-cell Id have provided encouraging results in both preclinical models and human trials (2, 3, 4, 5) . However, the difficulty of preparing individual Id proteins has driven the development of DNA vaccines that include the encoding V-region genes (6 , 7) . Although V-region sequences are weak tumor antigens, when fused to pathogen-derived sequences, they can elicit strong protective immunity-mediated primarily through anti-Id antibody (8 , 9) . Such fusion vaccines are now in Phase I/II trials for follicular lymphoma and multiple myeloma.

The greater difficulty of producing Id protein vaccines from the TCR has slowed investigation of this approach. However, recombinant TCR protein vaccines have been shown to prevent experimental autoimmune diseases and the development of a murine TCL (10, 11, 12) . Here we extend the concept of DNA fusion gene vaccination to T-cell malignancies. We delineate two critical elements required for the vaccine, including a minimal VVßCß sequence able to encode a scTCR with conformational integrity as predicted from crystallographic studies (13, 14, 15) . Our data reveal that products of VVß or VßCß DNA constructs have conformational deficits and fail to induce effective immunity. The second element necessary for a successful vaccine is fusion of the VVßCß sequence to FrC, without which the scTCR fails to generate antibody or protective responses.

	MATERIALS AND METHODS

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Cell Lines.
C6VL, a radiation-induced thymoma/lymphoma derived from the C57BL/Ka mouse strain (16) , was kindly provided by Craig Okada (Stanford, CA). C6VL cells have a mature T-cell phenotype expressing TCRß, CD3, CD4, and H-2b. The TCR chain is composed of AV9*04, AJ56, and the ß-chain of BV19*01, BD1*01, and BJ1–3*01. TCL-1 arose spontaneously in a C57BL/Ka mouse bred in house and served as a control in tumor challenge experiments. 293 cells were purchased from the European Collection of Animal Cell Cultures (Salisbury, United Kingdom).

Construction of DNA Vaccines.
The TCR V and Vß domains for C6VL were spliced together with a (Gly₄Ser)₃ peptide linker to generate the scTCR sequence VVß (Fig. 1a) by primer overlap extension, using the paired primers: VA leader, 5'-AAGCTTAGCATGCTCCTGGCACTCCTCCC-3'; JA linker, 5'-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGGTAT AACACTCAGAAC-3'; VB linker, 5'-GGCGGAGGTGGCTCTGGCGGTGGCGGATCGATCATTACTCAGACACCC-3'; and JB reverse, 5'-GCTAGCTACAACAATGAGCCGGCT-3'. To generate VVßCß (Fig. 1a) , the V domain was linked to the entire ß-chain by extending the ß-chain sequences to the residue right before the terminal cysteine, using the CB reverse primer, 5'-GCTAGCGTCTGCTCGGCCCCAGGCC-3'. For VßCß, 5'-AAGCTTAGCATGAACAAGTGGGTTTTCTGC-3' (encompassing the natural leader sequence for the Vß-chain) was paired with the CB reverse primer. Assembled genes were subcloned into the expression vector pcDNA3 (Invitrogen, Leek, the Netherlands) as HindIII-NheI fragments in-frame and 5' to the coding sequence for FrC (aa 865-1316 of tetanus toxin). Construction of p.FrC with a BCL1 V_H gene-derived leader sequence has been described previously (8) . Plasmid DNA was purified for vaccination using a QIAfilter Giga kit (Qiagen, Hilden, Germany). All constructs were sequenced and checked for expression in vitro using the TNT T7 Coupled Reticulocyte Lysate System (Promega, Southampton, United Kingdom).

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Structural design and conformational integrity of scTCR-FrC fusion products. In a, the TCR V domain was spliced via a (Gly4Ser)3 flexible linker to either the Vß domain or to VßCß. FrC from tetanus toxin was fused to the 3' end of the scTCR sequence via a (GlyPro)2 linker, and the complete open reading frame was subcloned into pcDNA3. All constructs produced protein of predicted size in an in vitro transcription/translation assay (data not shown). In b, to evaluate the conformational integrity of the FrC-expressing proteins, soluble scTCR from 293 transfectants was tested by ELISA. Protein was captured by immobilized anti-FrC mAb and then assessed for reactivity with conformation-dependent V and Vß-specific mAbs.

Production of Recombinant scTCR.
Recombinant VVßCß protein, biotinylated at the COOH terminus in vivo, was expressed in Drosophila melanogaster S2 cells. Two synthetic oligonucleotides encoding a biotin-acceptor sequence (17) were annealed and then ligated directly into pMT/Bip/V5-His (Invitrogen) as a NotI-AgeI fragment, generating the construct pMT-BA. VVßCß rDNA was then subcloned into pMT-BA as a SpeI-NotI fragment. A PCR product encompassing Escherichia coli biotin ligase was amplified from the plasmid pACYC184 (Avidity, Denver, CO) and subcloned into pMT/Bip/V5-His as an NcoI-NotI fragment. The VVßCß and biotin ligase constructs were cotransfected into S2 cells (Invitrogen) by calcium phosphate precipitation together with pCoHYGRO (Invitrogen). Stable transfectants, derived by selection in Schneider Drosophila expression medium containing 300 µg/ml hygromycin-B, were expanded in protein-free medium (Insect Xpress; Biowhittaker, Wokingham, United Kingdom), supplemented with 50 µM D-biotin. Protein expression was induced over a period of 72 h by the addition of copper sulfate to a final concentration of 0.7 µM. Supernatants were concentrated by centrifugal filtration (Millipore, Watford, United Kingdom) and then dialyzed against PBS. Biotin-tagged scTCR was then purified on a Softlink monomeric avidin column as recommended by the manufacturer (Promega).

Vaccination and Tumor Challenge.
C57BL/Ka mice, bred in house, were vaccinated at 8–10 weeks of age with a total of 50 µg of DNA in normal saline injected into two sites in the quadriceps. Injections were on days 0, 21, and 42. To assess antibody responses, tail bleeds were taken on days 35 and 56. Three weeks after the last immunization, mice were challenged with an i.v. injection of 750 C6VL cells. Cell depletion experiments were performed in vivo by i.p. injection of 100 µg of rat antimouse CD8 mAb (clone YTS 169.4.2.1, kindly provided by Dr. S. Cobbold, Sir William Dunn School of Pathology, Oxford, United Kingdom) or an isotype control. Mice were injected every 2–3 days over a fortnightly period beginning 1 week before tumor challenge.

Measurement of Antibody Responses.
Anti-FrC antibodies were measured by ELISA as described previously (8) . For the measurement of antibody responses against the TCR of C6VL, biotin-tagged recombinant VVßCß protein was captured onto streptavidin-coated plates at 0.5 µg/ml. Mouse immune serum was titered over four wells in 4-fold dilutions. A pooled terminal serum was assigned an arbitrary value of 200 units/ml to act as a comparative standard. Bound mouse IgG was detected with horseradish peroxidase sheep antimouse Fc (The Binding Site, Birmingham, United Kingdom).

Immune Sera Transfer.
Mice were immunized with VVßCß-FrC or A31 scFv-FrC as described above. On day 56, mice were sacrificed, and serum was collected and pooled. Total IgG was purified on a protein G column (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) and dialyzed against PBS. Mice were injected i.p. with 100 µg of total IgG, followed by an i.v. challenge with 1000 C6VL cells.

	RESULTS AND DISCUSSION

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Construction of DNA Vaccines.
To investigate DNA vaccination against T-cell malignancies, V-region sequences from the murine TCL cell line C6VL were assembled as scTCR by PCR and cloned in-frame and 5' to the coding sequence for FrC. The V domain was spliced via a (Gly₄Ser)₃ flexible linker to either the Vß domain alone or to the entire ß-chain (minus the transmembrane/cytoplasmic regions), generating the constructs VVß-FrC and VVßCß-FrC, respectively (Fig. 1a) . To assess whether V was required to stabilize Vß folding, a VßCß-FrC fusion construct was also assembled (Fig. 1a) .

Cß Is Required to Stabilize Vß Folding and Pairing with V.
We initially evaluated the structural integrity of the scTCR DNA vaccine products by transfecting the constructs into 293 cells. All supernatants were found to contain levels of FrC protein similar to those produced from a plasmid containing the FrC gene alone (Fig. 1b) . Using a sandwich ELISA with an anti-FrC mAb as capturing antibody and the conformation-dependent, Vß6-specific mAb RR4-7 for detection (18) , we demonstrated clear reactivity with expressed VVßCß-FrC (Fig. 1b) . There was also partial reactivity with VßCß-FrC, indicating limited Vß-folding in the absence of the V domain. VVß-FrC protein failed to react with RR4–7 (Fig. 1b) , suggesting inappropriate folding of this molecule. Reactivity with mAb 124-40, which recognizes a clonotypic determinant on the C6VL V chain (16) , could only be demonstrated to VVßCß-FrC (Fig. 1b) . These findings confirm the premise that Cß is required to stabilize Vß-folding (19 , 20) .

VVßCß-FrC Induces Antibody Reactive with Native C6VL TCR.
We next investigated whether the scTCR-FrC fusion constructs would induce antibody reactive with the C6VL lymphoma cells. Mice were vaccinated i.m. with 50 µg of plasmid DNA on days 0, 21, and 42. Serum samples were collected on day 56 and pooled and analyzed for anti-C6VL antibodies by flow cytometry (Fig. 2) . Although all FrC-containing constructs generated antibodies against FrC (Table 1) , only those animals vaccinated with VVßCß-FrC were able to induce antibody reactive with C6VL cells (Fig. 2) . An absolute requirement for the scTCR to maintain a native conformation was highlighted by the failure of VVß-FrC or VßCß-FrC to induce anti-C6VL antibodies. The essential role of the FrC sequence was shown by the failure of the VVßCß vaccine to induce anti-C6VL antibody (Fig. 2) . Importantly, immune serum raised by vaccination with VVßCß-FrC was unable to stain peripheral lymphocytes from nonimmunized mice (data not shown), suggesting that the antibody response was directed against private Id determinants. Analysis of pooled sera for immunoglobulin subclasses showed comparable levels of IgG1 and IgG2a (0.8:1), consistent with a mixed T_H1/T_H2-dominated response.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Assessment of ability of vaccine constructs to induce antibody against native TCR. C6VL cells were stained with pooled sera (1:20 dilution) taken on day 56 from mice immunized with the scTCR-FrC fusion constructs on days 0, 21, and 42. Bound antibody was detected with FITC goat antimouse IgG. Only VVßCß-FrC induced antibody reactive with native TCR.

VVßCß Induces Protective Immunity against C6VL Lymphoma.
The relevance of conformational integrity for induction of protective immunity against C6VL lymphoma was then addressed. C6VL is a highly aggressive tumor, with as few as 50 cells killing 100% of recipient mice (data not shown). Vaccination with the DNA construct VVßCß-FrC induced strong protection against challenge with C6VL (Fig. 3a) , compared with nonvaccinated control mice and mice vaccinated with FrC alone (P < 0.001). Vaccinations with VVß-FrC, VßCß-FrC, or VVßCß alone were also completely ineffective. The protective response generated was specific for C6VL, with no protection induced against TCL-1, an unrelated T-cell tumor (data not shown). The pattern of protection has been confirmed in two subsequent experiments, with survival rates of mice vaccinated with VVßCß-FrC ranging from 50 to 80%, whereas the other constructs were ineffective at prolonging survival.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Protective mechanisms against C6VL lymphoma induced by DNA scTCR vaccines. In a, mice (n = 8) were vaccinated at days 0, 21, and 42 with each of the constructs. Three weeks after the final vaccination, they were challenged with C6VL cells and monitored for survival. Only the VVßCß-FrC construct induced protection (P < 0.001). In b, vaccinated mice were treated in vivo with a CD8+ T cell-depleting mAb before tumor challenge, with no significant effect on protection. In c, naïve mice were injected i.p. with purified IgG from mice immunized with VVßCß-FrC before challenge with C6VL cells. All mice were protected, whereas control IgG had no effect.

Tumor Protection Is Independent of CD8⁺ Cells.
Previous studies using a TCR protein vaccine against C6VL lymphoma had indicated a role for CD8⁺ T cells in mediating suppression of tumor growth (12 , 21) . We investigated this effector pathway by in vivo depletion of CD8⁺ cells with mAb YTS 169.4.2.1. Flow cytometric analysis of peripheral lymphocytes 1 day before tumor challenge confirmed >98% depletion of CD8⁺ T cells (data not shown). However, depletion did not compromise protection against tumor (Fig. 3b) . Thus, despite the known efficacy of DNA vaccines in inducing CD8⁺ T-cell responses, there was no evidence for involvement of this pathway with TCR as antigen. The critical requirement for native folding of the scTCR points to B cells being involved in the protective response. This was also suggested for TCR protein vaccines, which failed to induce immunity in B cell-deficient (J_H^-/-) mice (12 , 21) . However, B cells could be involved in antigen presentation, as well as antibody production. In our vaccinated mice, there was no clear correlation between anti-C6VL antibody levels and survival, and some mice with no detectable anti-Id were protected. This raises the possibility of a role for anti-Id CD4⁺ T cells, known to be capable of mediating protection against B-cell malignancies (22 , 23) . Parallel depletion of CD4⁺ T lymphocytes could not be performed because of expression of CD4 by C6VL cells.

Antibody Transfer Can Provide Protection.
The ability of immune serum to mediate tumor protection was assessed in vivo by passive antibody transfer. The efficacy of antibody was revealed by the fact that 90% of naive recipients given purified IgG from mice immunized with VVßCß-FrC were protected against a subsequent lethal challenge with C6VL cells (Fig. 3c) . Mice similarly treated with total IgG from mice immunized with an irrelevant fusion vaccine were not protected (Fig. 3c) . Therefore, it appears that antibody alone can mediate protection but that it may not be the only mechanism. Evidence from the use of recombinant scTCR as antigen to suppress autoimmune T lymphocytes has implicated CD4⁺ regulatory cells as effectors (11) . Consistent with our findings, no antibody was produced by the VVß protein used in this study (11) . Similarly, recombinant VVß scTCR protein from C6VL did not induce antibody or protective immunity (12) , again supporting a requirement for conformational integrity. One interpretation is that anti-Id antibody is a highly effective mechanism for attacking T-cell clones and that recombinant protein may be an inefficient inducer because of inappropriate folding. For DNA vaccines where levels of protein expression are low, folding must be optimized, and the presence of the stabilizing Cß domain is essential. There are likely to be additional cellular mechanisms capable of attacking T-cell targets, and these may be induced by less well-folded protein. However, our analysis reveals the power of anti-Id antibody and points to inclusion of this weapon in our attack against T-cell tumors.

These results reveal a strategy for selectively eliminating T-cell clones by DNA vaccination. A combination of optimally folded scTCR sequence with a pathogen-derived sequence induces antibody and generates protection against TCL. The range of application is wide but may be especially attractive for cutaneous TCLs, where clinical need and a feasible clinical setting are evident.

	ACKNOWLEDGMENTS

 
We thank Kerry Cox, Sam Martin, and Lisa Davis for invaluable technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the Leukaemia Research Fund Grant 9955.

² To whom requests for reprints should be addressed, at Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals Trust, Southampton SO16 6YD, United Kingdom.

³ The abbreviations used are: TCR, T-cell antigen receptor; FrC, fragment C of tetanus toxin; Id, idiotype; mAb, monoclonal antibody; sc, single-chain; TCL, T-cell lymphoma.

Received 10/25/01. Accepted 1/15/02.

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