Download Vaccination with DNA Encoding a Single-Chain

[CANCER RESEARCH 62, 1757–1760, March 15, 2002]
Vaccination with DNA Encoding a Single-Chain TCR Fusion Protein Induces
Anticlonotypic Immunity and Protects against T-Cell Lymphoma1
Stephen M. Thirdborough, Joanna N. Radcliffe, Peter S. Friedmann, and Freda K. Stevenson2
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
The clonotypic T-cell antigen receptor (TCR) provides unique V␣ and
V␤ sequences 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 V␣V␤C␤ sequence. To promote anti-TCR immunity, we
fused a pathogen-derived sequence from tetanus toxin to the 3ⴕ-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 V␣V␤ or
V␤C␤ 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.
effective immunity. The second element necessary for a successful
vaccine is fusion of the V␣V␤C␤ sequence to FrC, without which the
scTCR fails to generate antibody or protective responses.
MATERIALS AND METHODS
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
TCR␣␤, CD3, CD4, and H-2b. The TCR ␣ chain is composed of AV9*04,
AJ56, and the ␤-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 V␤ domains for C6VL
were spliced together with a (Gly4Ser)3 peptide linker to generate the scTCR
sequence V␣V␤ (Fig. 1a) by primer overlap extension, using the paired
primers: VA leader, 5⬘-AAGCTTAGCATGCTCCTGGCACTCCTCCC-3⬘;
JA linker, 5⬘-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGGTAT
AACACTCAGAAC-3⬘; VB linker, 5⬘-GGCGGAGGTGGCTCTGGCGGTGINTRODUCTION
GCGGATCGATCATTACTCAGACACCC-3⬘; and JB reverse, 5⬘-GCTAGCThe structural diversity of the TCR3 ␣ and ␤ chains is generated by TACAACAATGAGCCGGCT-3⬘. To generate V␣V␤C␤ (Fig. 1a), the V␣
somatic recombination of V(D)J gene segments (1). The unique domain was linked to the entire ␤-chain by extending the ␤-chain sequences to
combination of these genetic elements gives rise to Id determinants the residue right before the terminal cysteine, using the CB reverse primer,
that are expressed clonotypically by the T cell. When expressed by 5⬘-GCTAGCGTCTGCTCGGCCCCAGGCC-3⬘. For V␤C␤, 5⬘-AAGCTTAGmalignant or autoreactive T-cell clones, these Id determinants can CATGAACAAGTGGGTTTTCTGC-3⬘ (encompassing the natural leader sequence for the V␤-chain) was paired with the CB reverse primer. Assembled
serve as targets for active immunotherapy. The Id immunoglobulin of
genes were subcloned into the expression vector pcDNA3 (Invitrogen, Leek,
malignant B cells provides a similar molecular target. Vaccination the Netherlands) as HindIII-NheI fragments in-frame and 5⬘ to the coding
strategies against B-cell Id have provided encouraging results in both sequence for FrC (aa 865-1316 of tetanus toxin). Construction of p.FrC with a
preclinical models and human trials (2–5). However, the difficulty of BCL1 VH gene-derived leader sequence has been described previously (8).
preparing individual Id proteins has driven the development of DNA Plasmid DNA was purified for vaccination using a QIAfilter Giga kit (Qiagen,
vaccines that include the encoding V-region genes (6, 7). Although Hilden, Germany). All constructs were sequenced and checked for expression
V-region sequences are weak tumor antigens, when fused to patho- in vitro using the TNT T7 Coupled Reticulocyte Lysate System (Promega,
gen-derived sequences, they can elicit strong protective immunity- Southampton, United Kingdom).
Expression in Vitro. The ability of scTCR-FrC fusion constructs to synmediated primarily through anti-Id antibody (8, 9). Such fusion vacthesize
and export protein was assessed after transient transfection into 293
cines are now in Phase I/II trials for follicular lymphoma and multiple
cells by lipofection (Effectene; Qiagen). Supernatants were collected after
myeloma.
48 h, and the secreted protein captured onto ELISA plates with a monoclonal
The greater difficulty of producing Id protein vaccines from the anti-FrC antibody (E6C, in house), coated at 5 ␮g/ml. Bound protein was
TCR has slowed investigation of this approach. However, recombi- detected with biotinylated mAb 124-40 (C6VL V␣ specific; kindly provided
nant TCR protein vaccines have been shown to prevent experimental by Ron Levy, Stanford, CA), RR4 –7 (VB6 specific; Becton Dickinson, Oxautoimmune diseases and the development of a murine TCL (10 –12). ford, United Kingdom), or polyclonal mouse anti-FrC antibody, followed by
Here we extend the concept of DNA fusion gene vaccination to T-cell Streptavidin-horseradish peroxidase (Cambridge Bioscience, Cambridge,
malignancies. We delineate two critical elements required for the United Kingdom).
Production of Recombinant scTCR. Recombinant V␣V␤C␤ protein, bivaccine, including a minimal V␣V␤C␤ sequence able to encode a
scTCR with conformational integrity as predicted from crystallo- otinylated at the COOH terminus in vivo, was expressed in Drosophila melagraphic studies (13–15). Our data reveal that products of V␣V␤ or nogaster S2 cells. Two synthetic oligonucleotides encoding a biotin-acceptor
sequence (17) were annealed and then ligated directly into pMT/Bip/V5-His
V␤C␤ DNA constructs have conformational deficits and fail to induce
(Invitrogen) as a NotI-AgeI fragment, generating the construct pMT-BA.
V␣V␤C␤ rDNA was then subcloned into pMT-BA as a SpeI-NotI fragment. A
Received 10/25/01; accepted 1/15/02.
PCR product encompassing Escherichia coli biotin ligase was amplified from
The costs of publication of this article were defrayed in part by the payment of page
the plasmid pACYC184 (Avidity, Denver, CO) and subcloned into pMT/Bip/
charges. This article must therefore be hereby marked advertisement in accordance with
V5-His as an NcoI-NotI fragment. The V␣V␤C␤ and biotin ligase constructs
18 U.S.C. Section 1734 solely to indicate this fact.
1
were cotransfected into S2 cells (Invitrogen) by calcium phosphate precipitaSupported by the Leukaemia Research Fund Grant 9955.
2
To whom requests for reprints should be addressed, at Molecular Immunology
tion together with pCoHYGRO (Invitrogen). Stable transfectants, derived by
Group, Tenovus Laboratory, Southampton University Hospitals Trust, Southampton SO16
selection in Schneider Drosophila expression medium containing 300 ␮g/ml
6YD, United Kingdom.
3
hygromycin-B, were expanded in protein-free medium (Insect Xpress; BioThe abbreviations used are: TCR, T-cell antigen receptor; FrC, fragment C of tetanus
whittaker, Wokingham, United Kingdom), supplemented with 50 ␮M D-biotin.
toxin; Id, idiotype; mAb, monoclonal antibody; sc, single-chain; TCL, T-cell lymphoma.
1757
DNA VACCINES AGAINST T-CELL LYMPHOMA
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 V␤
domain or to V␤C␤. 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 V␤-specific mAbs.
Protein expression was induced over a period of 72 h by the addition of copper
sulfate to a final concentration of 0.7 ␮M. 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 ␮g 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 ␮g 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 V␣V␤C␤
protein was captured onto streptavidin-coated plates at 0.5 ␮g/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 V␣V␤C␤-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 ␮g of total IgG, followed
by an i.v. challenge with 1000 C6VL cells.
in-frame and 5⬘ to the coding sequence for FrC. The V␣ domain was
spliced via a (Gly4Ser)3 flexible linker to either the V␤ domain alone
or to the entire ␤-chain (minus the transmembrane/cytoplasmic regions), generating the constructs V␣V␤-FrC and V␣V␤C␤-FrC, respectively (Fig. 1a). To assess whether V␣ was required to stabilize
V␤ folding, a V␤C␤-FrC fusion construct was also assembled
(Fig. 1a).
C␤ Is Required to Stabilize V␤ 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, V␤6-specific mAb RR4-7
for detection (18), we demonstrated clear reactivity with expressed
V␣V␤C␤-FrC (Fig. 1b). There was also partial reactivity with V␤C␤FrC, indicating limited V␤-folding in the absence of the V␣ domain.
V␣V␤-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 V␣V␤C␤-FrC (Fig. 1b). These
findings confirm the premise that C␤ is required to stabilize V␤folding (19, 20).
V␣V␤C␤-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 ␮g 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 V␣V␤C␤-FrC were able to induce
antibody reactive with C6VL cells (Fig. 2). An absolute requirement
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 V␣V␤C␤-FrC induced antibody
reactive with native TCR.
Table 1 Serum antibody responses induced by DNA vaccination
Target antigen
Vaccine
FrC
V␣V␤-FrC
V␤C␤-FrC
V␣V␤C␤-FrC
V ␣V␤C␤
RESULTS AND DISCUSSION
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
rFrC
rV␣V␤C␤a
C6VL cells
24/24
24/24
24/24
24/24
0/24
0/24
18/24
23/24
24/24
0/24
0/24
0/24
0/24
10/24
0/24
b
a
Determined by ELISA.
Serum samples were collected on day 56, after vaccination with the constructs on
days 0, 21, and 42.
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b
DNA VACCINES AGAINST T-CELL LYMPHOMA
for the scTCR to maintain a native conformation was highlighted by
the failure of V␣V␤-FrC or V␤C␤-FrC to induce anti-C6VL antibodies. The essential role of the FrC sequence was shown by the failure
of the V␣V␤C␤ vaccine to induce anti-C6VL antibody (Fig. 2).
Importantly, immune serum raised by vaccination with V␣V␤C␤-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 TH1/TH2-dominated response.
The V␣V␤C␤-FrC vaccine induced antibody recognizing C6VL
cells in only 42% of mice (Table 1). In contrast, sera from all mice
recognized recombinant V␣V␤C␤ protein by ELISA. Surprisingly,
however, immune sera from mice vaccinated with V␣V␤-FrC or
V␤C␤-FrC constructs, known to produce mainly misfolded protein,
also recognized recombinant V␣V␤C␤ protein (Table 1). The antibody response was specific for C6VL, with no reactivity against
V␣V␤C␤ protein prepared from an unrelated T-cell line (data not
shown). This indicates that the recombinant V␣V␤C␤ protein contains some misfolded molecules, possibly a common feature of recombinant proteins. It points to a requirement to confirm reactivity of
apparent anti-Id antibodies with native cell-expressed Id determinants.
V␣V␤C␤ 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 V␣V␤C␤-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
V␣V␤-FrC, V␤C␤-FrC, or V␣V␤C␤ 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
V␣V␤C␤-FrC ranging from 50 to 80%, whereas the other constructs
were ineffective at prolonging survival.
DNA Vaccination Does Not Perturb the Normal T-cell Repertoire. One potential problem with this approach is that the DNA
vaccine could raise an autoimmune response capable of eliminating
normal T cells expressing the same V␣ or V␤ framework determinants. To assess if vaccination with V␣V␤C␤-FrC caused a change in
TCR usage, peripheral lymphocytes from immunized mice were processed 3 weeks after the final vaccination and stained with RR4 –7.
Cells expressing V␤6 were still detectable at levels comparable with
those found in nonvaccinated mice or mice vaccinated with FrC alone,
indicating no autoimmune effect (data not shown).
Tumor Protection Is Independent of CD8ⴙ 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 (JH ⫺/⫺) 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
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 V␣V␤C␤-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, naı̈ve mice were
injected i.p. with purified IgG from mice immunized with V␣V␤C␤-FrC before challenge
with C6VL cells. All mice were protected, whereas control IgG had no effect.
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 V␣V␤C␤-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
1759
DNA VACCINES AGAINST T-CELL LYMPHOMA
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 V␣V␤ protein used in this study (11). Similarly, recombinant V␣V␤ 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
C␤ 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.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS
17.
We thank Kerry Cox, Sam Martin, and Lisa Davis for invaluable technical
assistance.
18.
19.
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