In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer
cells can originate from viral proteins, normal self-proteins regulated during differentiation, or
altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector
CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to
design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor
peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor
antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo
applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.
Key words:
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Introduction |
Cytotoxic T lymphocytes (CTLs) can play a central role
in rejecting tumors (1). Tumor antigens recognized
by CTL generally originate from three sources: (a) viruses;
(b) self-proteins expressed during development or differentiation; and (c) mutant or aberrantly expressed proteins (1).
Because many, if not most, tumor antigens are products of
normal or altered cellular genes, they are typically not efficient at initiating immune responses. Thus, a central problem in cancer immunotherapy is how to efficiently prime
CTLs against poorly immunogenic tumor antigens.
CTLs recognize target antigens in the form of short intracellularly processed peptides, presented by self-MHC-
encoded class I molecules (pep:class I). Upon binding of the
antigen-specific TCRs on a CTL to its cognate peptide-
MHC complex on the tumor cell, the target cell is lysed and
the tumor eliminated. To develop into effector CTLs capable of tumor lysis, naive precursor CTLs (pCTLs)1 have to
be activated. This pCTL activation requires two signals: the
first, or stimulatory signal (signal 1), transmitted via the TCR-CD3 complex, and the second, or costimulatory signal (signal 2), delivered by professional APCs (2). In the
thymus, a strong signal 1 will induce negative selection of
immature thymocytes, regardless of signal 2 (5, 6). In the
periphery, the same strong signal 1 will induce immunity
(including pCTL
CTL differentiation) or anergy, depending on the presence or absence of signal 2 (2). By
contrast, even a weak signal 1 without signal 2 can be sufficient for target cell lysis by differentiated CTLs (7). This
means that a whole class of antigenic peptides exists that, although poorly immunogenic (i.e., unable to induce CTL
immunity), can readily serve as molecular targets for lysis by
differentiated effector CTLs. Such antigenic, but poorly immunogenic, peptides remain invisible to the naive pCTL.
One strategy to exploit the presence of such poorly immunogenic or nonimmunogenic peptides at the surface of
tumor cells is to design immunogenic variants of these peptides that would prime CTL response that cross-reacts to
the original targeting peptide. If successful, this strategy
could be attractive, since the relative invisibility of poorly
immunogenic self-peptides to the immune system could be
advantageous. Namely, unlike self-peptides that provide
strong signal 1 (8), poorly immunogenic peptides would most likely fail to induce tolerance, and the T cell repertoire reactive against them should be intact and available
for activation with immunogenic peptide variants. According to the nomenclature used for variants of a pigeon cytochrome C peptide (9), peptides of higher biological potency than the original peptide were called heteroclitic.
Crystal structure analysis revealed that, of the 8-10 amino
acid residues of a class I-bound peptide, roughly half point
into the solvent and can interact directly with the TCR via
their side chains (10). The other half are buried by class I and are not directly accessible to the TCR (10). Heterocliticity has been achieved by substituting amino acids
that contact class I, the TCR, or both (13, 14).
The aim of this study was to produce heteroclitic immunogens that elicit antitumor CTL responses that cross-react
to the original poorly immunogenic antigens. Therefore, to
design peptides that are heteroclitic for polyclonal CTL responses, one would like to optimize pep:class I binding,
since this property correlates with immunogenicity (7, 15).
At the same time, the peptide:TCR contact should not be
disturbed, to maximize the potential crossreactivity between the heteroclitic and the original, nonimmunogenic
targeting peptide. We sought to test whether this strategy
could induce antitumor CTLs reactive to nonimmunogenic targeting peptides. We demonstrate successful in vivo
induction of cross-reactive CTLs in two tumor models, using an engineered viral peptide variant expressed as a tumor
antigen (a model of a tumor antigen of viral origin) and a
self-antigen expressed in melanomas (a model of nonmutated, differentiation antigen). Induced CTLs were biologically active in vivo, and were able to effect rejection of
both newly implanted and established day 3 tumors.
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Materials and Methods |
Mice.
Female C57BL/6 (B6) mice were purchased from the
National Cancer Institute breeding program (Frederick, MD).
B6.C-H-2bm8 (bm8) mice were bred in the MSKCC vivarium
from a breeding stock obtained from The Jackson Laboratory
(Bar Harbor, ME) via Dr. J. Sprent (The Scripps Research Institute, La Jolla, CA). All mice entered the study between 7 and 10 wk of age.
Antibodies, In Vivo CD8 Depletion, Flow Cytometry and Class I
Stabilization Assays.
The anti-CD8 mAb, 53.6.7 (rat IgG) and
the anti-Kb mAb, Y3 (mouse IgG2b), both obtained from the
American Type Culture Collection (Rockville, MD) were produced as ascitic fluid in our lab. For in vivo CD8 depletion, 100 µl
of ascitic fluid was injected intraperitoneally on days
7 and
3
relative to tumor challenge, which was denoted as day 0. PE-conjugated anti-mouse IgG2b was purchased from Fisher Biotech
(Malvern, PA). Flow cytometry and the class I stabilization assays
were performed exactly as previously described (16) using a FACScan® instrument equipped with Lysys II software (Becton Dickinson, Mountain View, CA).
Construction of Minigenes.
Inserts coding for the endoplasmic
reticulum (ER) insertion sequence (17, 18) (amino acid sequence:
MRYMILGLLALAAVCSA) followed by the peptides SEI
(SEIEFARL) and SSI (SSIEFARL), based on the immunodominant sequence 498-505 of the Herpes simplex virus glycoprotein
B, or peptides TWH (TWHRYHLL) or TAY (TAYRYHLL), based upon the sequence 222-229 of the melanoma gp75 protein, were produced by multistep PCR. All oligonucleotides
were purchased from Retrogen (San Diego, CA). For the construction of pERIS-SSI and pERIS-SEI minigenes, the PCR reactions were done with two common oligomers, C1 (GGG AAG
CTT ACC ATG AGA TAC ATG ATC CTG GGC CTG
CTG), C2 (GGC CTG CTG GCC CTG GCC GCC GTG
TGC AGC GCT GCC AGC), and the specific oligomers SSI
(TTT CTC GAG TCA CAG CCT GGC GAA CTC GAT
GCT GCT GGC AGC) or SEI (TTT CTC GAG TCA CAG CCT GGC GAA CTC GAT CGA GCT GGC AGC). C2 and
SSI or SEI were first joined in 50-µl reactions consisting of 300 µM
dNTPs and 20 µg/ml of primers for 30 cycles at 95°C for 30 s,
30°C for 60 s, and 72°C for 30 s. 5 µl of the product was added
to 45 µl of C1 and either SSI or SEI at the same primer and
dNTP concentrations, and another PCR was performed for 10 cycles as above, followed by 30 cycles at 95°C for 1 min and
72°C for 2 min. For the construction of pERIS-TAY and
pERIS-TWH, the common oligomers C1.1 (GGG AAG CTT
ACC ATG AGA TAC ATG ATC CTG GGC CTG CTG GCC
CTG GCC GC) and C2.1 (GGC CTG CTG GCC CTG GCC
GCC GTG TGC AGC GCT GCT) were used with the specific
oligomers TAY (TTT CTC GAG TCA CAG CAG GTG GTA TCT GTA GGC GGT GGC AGC GCT) or TWH (TTT CTC
GAG TCA CAG CAG GTG GTA TCT GTG CCA GGT
GGT AGC GCT). The first step was exactly as described above.
However, the second step was done for 40 cycles at 94°C for 20 s,
followed by 60°C for 20 s and 72°C for 45 s. Products thus engineered contain the HindIII and XhoI sites, which were used for
cloning into a LacZ-containing pCR2 cloning vector (Invitrogen, San Diego, CA). Clones that scored positive by blue/white screening were digested by HindIII and XhoI, and the inserts
were recloned into pCDNA3 to obtain the appropriate expression constructs, pcERIS-SSI, pcERIS-SEI, pcERIS-TWH, and
pcERIS-TAY. The transfer was confirmed by sequencing.
Cell Transfection.
10 µg of linearized plasmid DNA was electroporated into 107 RMA-S cells in 500 µl of Optimem (GIBCO
BRL, Gaithersburg, MD) containing 5% FCS, using a Gene
Pulser (Bio-Rad, Hercules, CA) set at 220 V and 960 µF. 48 h
later, the cells were incubated in the presence of 600 µg/ml of
G418 (GIBCO BRL). G418-resistant clones were selected from
the 96-well plates with <30% positive wells.
Peptide and Gene Gun Immunization, In Vitro CTL Restimulation, and CTL Assays.
The five peptides used in this study,
HIV-10 (RGPGRAFVTI), SSI (SSIEFARL), SEI (SEIEFARL),
TWH (TWHRYHLL), and TAY (TAYRYHLL), were synthesized by the Memorial Sloan Kettering Cancer Center Microchemistry Core Facility, and were HPLC purified to >98% purity. Peptide immunization using the synthetic immune adjuvant
TiterMax (CytRx Inc., Norcross, GA), referred to as pep/TM in
the text, CTL restimulation, and 51Cr-release assays were performed as previously described (19). In brief, mice were immunized in the footpad with 10 µl of the pep/TM emulsion (mixed
according to the manufacturer's instructions) containing 5 µg of the
indicated peptide. 7 d later, spleen cells from the immunized mice
were restimulated in vitro with syngeneic, irradiated (30 Gy),
peptide-coated (1 µg/ml, 2 ml/spleen, 1 h at 37°C followed by
three washes) cells. 5 d later, cytolytic activity was assessed in a
standard 51Cr-release assay. Genetic immunization using DNA-coated gold particles was performed exactly as previously described using a gene gun provided by Powderject, Inc. (Middleton, WI) (20). 100 µg of DNA from the plasmids described in the
previous section was mixed with 0.95-2.6-µm diameter gold
particles, in the presence of 0.05-0.1 µM spermidine. CaCl2 (1.5 mM) was added in a dropwise fashion to this mixture during vortexing. After precipitation, the gold plasmid DNA complex was
washed three times in 100% ethanol and 7 ml of ethanol was
added to achieve a bead-loading rate of 0.5 mg of gold to 1.0 µg
plasmid DNA per injection. This solution was instilled into plastic Tefzel tubing, the ethanol was gently drawn off, and the tube
was purged under nitrogen gas at 400 ml/min for drying. The
tube was then cut into 0.5-inch bullets. The gold particles in the
"bullets" were injected into the skin of anesthetized mice using a
helium-driven gene gun (Powderject, Inc.). The skin was shaved
and depilated before injection (20). Four injections at 400 pounds/square inch were delivered to each mouse, one to each
of the abdominal quadrants, for a total of 4 µg of plasmid DNA per mouse. 7 d later, spleen cells were restimulated and
CTL activity was determined as described for pep/TM.
Tumor Challenge and Follow Up.
5 × 105 RS-H2E or RS-Null cells or 105 B16F10LM3 (designated B16 in the text) melanoma cells (derived from B16F10 melanoma cells, a gift of Isaiah
Fidler, MD Anderson Cancer Center, Houston, TX) were injected into the shaved left flank of the mice. Mice were then
monitored three times/wk for tumor growth, initially by palpation and subsequently, when tumor growth was manifest, using
Vernier calipers. Measurements were achieved by obtaining the
maximum diameter of the tumor and the diameter perpendicular
to the maximum, which were then multiplied, and the product of
these two values was reported as tumor size. Tumor growth
curves are shown for individual mice per experiment. Mice surviving tumor challenge were followed for a minimum of 60-90 d.
The mice were killed if the maximum tumor diameter exceeded
10 mm, or if the tumor became ulcerated.
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Results |
Heteroclitic Vaccination in an Engineered Lymphoma Model.
To establish the principle of heteroclitic immunization
against tumors, we used an engineered peptide based on the
sequence of the Herpes simplex virus glycoprotein B498-505
peptide, which also served as a model of a tumor antigen of
viral origin. This peptide, called SEI (sequence SEIEFARL),
binds poorly to the murine MHC I molecule H-2Kb (Kb)
due to the electrostatic repulsion between the negatively
charged glutamic acid residues at the buried position 2 of
the peptide (P2E) and the adjacent position 24 (MHC24E)
of Kb (16). Due to poor binding, this peptide cannot elicit a
CTL response in Kb-bearing C57BL/6 (B6) mice after immunization with peptide/adjuvant (Fig. 1 A, circles, and Table 1) or with genetic immunization using DNA delivered
by particle bombardment (Table 1). However, SEI was a
good immunogen in B6.C-H-2.bm8 (bm8) mice (Fig. 1 B,
circles), which express a natural Kb variant, Kbm8. This class I
molecule has an E24
S mutation that enables strong SEI
binding (16). These results demonstrated that the immunogenicity of the above peptide correlated directly to peptide
binding, and that the absence of a response in B6 was not a
result of deficient vaccine formulation.

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Fig. 1.
Efficacy of CTL priming by SSI and SEI peptides. (A) CTL
responses to peptide priming of B6 mice by SEI (circles) and SSI (squares).
Three mice per group were vaccinated with indicated peptides in adjuvant (pep/TM). 7 d later, spleen cells were restimulated in vitro and CTL
responses of individual mice tested in a 51Cr-release assay using Kb-
expressing EL-4 target cells pulsed with 10 µM of the immunizing peptide, as previously described (19). The lysis of unpulsed EL-4 cells (always
<10%) was subtracted, and results are shown for individual mice at indicated effector/target ratios. Results are representative of >45 mice tested
in at least 10 independent experiments. Indistinguishable results were obtained using DNA immunization by particle bombardment (Table 1). (B)
Peptide immunogenicity correlates to peptide binding. bm8 mice respond
to peptide priming by SSI (squares) and SEI (circles), both of which bind
well to Kbm8 (16). Results are representative of at least 25 mice/strain
tested in at least six independent experiments. Methods and data representation were as described in A.
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The natural viral peptide from which H2E was derived,
SSI (SSIEFARL, also referred to as HSV-8), differs from
SEI by having a serine (P2S) instead of the glutamic acid
(P2E) in position 2. SSI would be predicted to remove the
electrostatic repulsion between the peptide and Kb. Indeed,
SSI bound 100-fold better than SEI to Kb (16) and was
strongly immunogenic for B6 CTLs (Fig. 1 A, squares, and
Table 1). We next asked whether SSI-induced CTLs could
lyse cells bearing the SEI peptide in a cross-reactive fashion,
and found that this was the case when target cells were
coated with high concentrations of SEI (>10 µM) in vitro
(Dyall, R., unpublished data).
Although these in vitro results were encouraging, their
in vivo relevance for tumor immunity was obscure. In particular, it was unclear whether intracellularly expressed
weak MHC binders, such as SEI, would be processed and
presented efficiently enough for target cell lysis. To address
that issue, we expressed SEI in a B cell lymphoma, RMA-S
(21). RMA-S has a chemically induced deletion of one of
its transporter associated with peptide processing (TAP)
genes, Tap-2. This deletion prevents the vast majority of cytosolically processed peptides from entering the ER and
binding to empty class I molecules, which leads to decreased expression of stable class I molecules at the surface
of RMA-S cells. The TAP defect was circumvented using
a minigene encoding an ER insertion sequence (ERIS)
(17) followed by the SEI peptide. Fusion proteins encoded
by such ERIS-containing minigenes have been shown previously to insert the attached class I binding peptides into
the ER, thereby bypassing the TAP defect and partially restoring the surface expression of pep:class I (17, 18). Indeed, the experimental tumor line, RS-SEI (RMA-S cells
transfected with the pERIS-SEI plasmid), had a higher surface level of Kb than RS-Null cells (transfected with the
"empty" control plasmid, pcDNA3), as measured by flow
cytometry. The mean relative Kb fluorescence intensity for
RS-SEI was 123 as compared to only 79 for RS-null (Dyall,
R., unpublished data). These observations were consistent
with the efficient ERIS-mediated import of SEI into the
ER. The fact that RS-SEI was lysed by anti-SSI CTL line, whereas RS-Null was not (Fig. 2), not only confirmed this
conclusion, but also demonstrated that CTLs induced by
the heteroclitic vaccine cross-reacted on the SEI:Kb expressed by the minigene-transfected cells.

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Fig. 2.
SSI is a heteroclitic
immunogen for the antigenic,
but not immunogenic, SEI peptide. Three anti-SSI CTL lines,
derived from individual B6 mice
by peptide immunization, were
tested for the ability to lyse the
Kb-expressing target cell lines
RS-SEI (closed squares) and RS-Null (open squares), transfected
with SEI-encoding and control
plasmids, respectively, in a standard 51Cr-release assay. Six more
lines were tested and gave identical results.
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To test the potency of SSI as a heteroclitic vaccine in
vivo, mice were immunized with either the heteroclitic or
the parental peptide as previously described (19) and challenged with RS-SEI or RS-Null tumor lines. Tumor
growth was then assessed for 90 d. Tumor growth curves
among the different groups of challenged mice for a typical
experiment are shown in Fig. 2 A and results from all experiments are shown in Table 2. The only group protected was the one vaccinated with the heteroclitic vaccine (SSI)
and challenged with the tumor line expressing SEI. Numbers shown above each figure show cumulative tumor survival for all mice within the indicated experiment. These
results clearly demonstrate the antigenic specificity of the
response, and confirm the in vitro findings that SEI cannot
induce a protective immune response in B6 mice (Fig. 3).
Heteroclitic protection was dependent on CD8+ cells, because mice depleted of CD8+ cells by antibody treatment
were not protected (Fig. 3, Table 2). Identical results were
obtained with genetic immunization (Table 2). The ability
of the heteroclitic immunogen to induce rejection of 3-d
tumors was next investigated, using both peptide (19) and
genetic (20) vaccination. Successful rejection was achieved
by genetic immunization only (Fig. 4 and Table 2), consistent with our previous findings that genetic vaccination
may be more potent than peptide vaccination (20). These
results establish the in vivo relevance of heteroclitic immunization.

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Fig. 3.
In vivo ability of the
heteroclitic vaccine SSI to protect mice against a transplantable
tumor expressing SEI. 10 B6
mice per group were vaccinated
with peptides SSI, SEI, or control PBS (19) emulsified in TM.
7 d later, animals were challenged with 5 × 105 RS-SEI or
RS-Null cells subcutaneously.
Nodules were palpable 3 d after
challenge. Numbers on figures
show numbers of tumor-free
mice at 90 d. A seventh group
also received SSI and was challenged with RS-SEI, but the animals were depleted of CD8+
cells by administration of an anti-CD8 mAb before the challenge.
Tumors were measured as described in Materials and Methods,
and results are shown as tumor
growth curves. All tumor-free
mice remained free of tumors for
>90 d. DNA vaccination yielded
identical results (Table 2).
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Fig. 4.
DNA vaccination with a heteroclitic immunogen eradicates
3-d tumors in mice. B6 mice were injected with the RS-SEI tumor, as
described in the legend of Fig. 2 A. 3 d later, when palpable tumors appeared (2-3 mm in diameter), the mice were injected with DNA constructs (indicated in the figure) and tumor growth was scored. Tumors
were followed and data shown exactly as in Fig. 3, with the figure showing one experiment and the numbers depicting tumor-free animals after
90 d of observation. Together with another similar experiment, this data
is also shown in Table 2.
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Heteroclitic Vaccination against a Tyrosinase Family Epitope of
a Melanoma.
We next sought to test the applicability of
this approach using naturally occurring tumor antigens. As
a model, we selected the brown locus product, also known
as the tyrosinase-related protein 1, or gp75. This glycoprotein is a lineage-specific self-antigen, present in melanocytes and expressed in melanomas (22, 23). The product of
the brown locus is a relevant cancer antigen, recognized by
both antibodies and T cells in patients with melanoma (22- 24). In mice, passive and active immunization against gp75
results in both melanoma rejection and manifestations of
autoimmunity (25, 26). However, in the mouse model,
CTL immunity against gp75-expressing melanoma cells
was not induced by immunization with tumor cells plus
adjuvant, nor tumor cells engineered to express cytokines,
or with purified gp75 protein (26).
Using the canonical Kb-binding motif (27) to scan the
amino acid sequence of gp 75 (28), five potential epitopes
were identified. Synthetic peptides corresponding to these
epitopes were used in both CTL inhibition and class I stabilization assays to determine their ability to bind to Kb
(Moroi, Y., and R. Dyall, unpublished data). Peptide TWH,
corresponding to gp75 residues 222-229 (TWHRYHLL),
exhibited similar binding characteristics to SEI and was selected for further experiments. Since this peptide has two
bulky side chains not commonly found at the buried,
MHC-contacting positions 2 and 3 (P2W and P3H), we
suspected that they might sterically impair pep-Kb binding.
Based on the available sequence/motif information for Kb-binding peptides, and on the crystallographic data on the
pep:Kb structures (10, 27), we changed these two residues to A and Y, respectively. The variant designed in this
manner, named TAY (TAYRYHLL), turned out to be an
excellent Kb binder, comparable to SSI (Fig. 5).

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Fig. 5.
The TAY peptide is
an excellent binder to Kb. An
RMA-S stabilization assay was
performed and results are shown
as previously described (16), using peptides SSI (open diamonds,
positive control), HIV-10, a
Dd-binding peptide, (RGPGRAFVTI, filled squares, negative
control), TWH (filled circles), and
its heteroclitic variant, TAY
(filled triangles) at indicated concentrations. In this type of assay,
the percentage of maximal stabilization provides a direct correlate of peptide binding (16). Experiment is representative of
three such assays.
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We next immunized B6 mice with the TAY and TWH
peptides using both pep/TM and genetic methods. For
both immunization protocols, successful CTL priming was
obtained only with the engineered, and not the native,
peptide (Table 1). Importantly, the anti-TAY CTLs lysed
target cells pulsed with TWH in vitro, revealing that TAY
exhibits the heteroclitic properties for TWH (Fig. 6 A).
The well-characterized melanoma, B16 (29), and its radiation-induced gp75 loss mutant, B78H.1 (29), were next
used as in vitro targets for the anti-TAY CTL lines (Fig. 6
B). Despite comparable surface expression of Kb after induction with IFN-
(data not shown), B16 (Fig. 6 B, closed squares), but not B78H.1 (Fig. 6 B, open circles), was efficiently lysed. Importantly, TWH-peptide-sensitized B78H.1
cells were also efficiently lysed, showing that such cells expressed sufficient levels of MHC class I molecules for CTL
lysis. These results show that: (a) peptide priming was antigen specific; (b) gp75 TWH peptide was naturally processed in the class I pathway; and (c) the TWH-Kb complexes were expressed at high enough levels on the
melanoma cell surface to serve as targets for CTL attack.

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Fig. 6.
The TAY peptide is a heteroclitic immunogen for the native
gp75 melanoma peptide, TWH. (A) TAY-induced CTLs lyse Kb-
expressing target cells pulsed with TWH. CTL activity of anti-TAY
CTLs against Kb-expressing target cells pulsed with 1 µM TAY (closed
squares) or TWH (open squares). Lysis of control target cells (<10% at any
point) was subtracted from the shown values. Target cells were pulsed
with peptides and a Cr-release assay was performed as described (19). Cumulative results from several experiments of this type are shown in Table
1. (B) TWH is naturally processed in vivo, and can serve as a target for
anti-TAY CTLs. The gp75-positive B16 melanoma line, but not its gp75
negative variant (B78.H1), is efficiently lysed by anti-TAY CTLs. B16
(filled squares), B78.H1 (open circles), or B78H.1 pulsed with 10 µM of the
TWH peptide (open squares) were used as targets in a standard Cr-release
assay after a 24-h incubation with 10 U/ml of IFN- to induce MHC
class I expression. The extent of class I induction was confirmed by flow
cytometry, and was similar for both tumor lines (data not shown). Similar
results were obtained with the ex vivo explanted B16 melanoma (these
cells express high levels of MHC class I molecules owing to class I upregulation in vivo). Results are shown for three independent CTL lines, each
one depicted by a different type of line, and are representative of six lines
tested thus far.
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We next investigated the potential of TAY as a heteroclitic gp75 vaccine to protect against tumor challenge. Fig.
7 and Table 2 show tumor growth and incidence in mice
vaccinated with TWH or TAY and challenged with B16.
Of the mice that were vaccinated with the heteroclitic vaccine, TAY, 100% were protected in that experiment (Fig.
7), and 90% were protected in two experiments (Table 2).
By contrast, minimal (if any) protection was conferred upon the mice vaccinated with the wild-type peptide
TWH (Fig. 7 and Table 2). In day 3 tumors, the same
priming regimen did not result in tumor eradication (Table
2), possibly owing to differences in tumor biology between
the melanoma and a lymphoma, differences in inherent immunogenicity of the tumors, and other factors. Together
with previous studies in vitro (30), the above results
confirm the principles and establish the applicability of rationally designed heteroclitic vaccination to tumor immunity in vivo.

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Fig. 7.
In vivo efficacy of a heteroclitic antimelanoma vaccine. 10 mice per group were vaccinated with TAY/TM or TWH/TM, as previously described (19). 7 d later, they were challenged with 105 B16 melanoma
cells per mouse, subcutaneously in the flank. Tumor measurements, number of experiments and result presentation was as in Figs. 3 and 4. The tumors typically became palpable after 9-15 d. Numbers represent the ratio
of tumor-free mice to total mice challenged in each group over a period
of >90 d. Another experiment yielded comparable results (Table 2).
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Discussion |
These results demonstrate the applicability and the in
vivo efficacy of heteroclitic CTL vaccines. The approach to
substitute the buried, MHC-contacting residues of a poorly
binding peptide, but leave the solvent-exposed TCR-contacting residues intact, was previously used to generate
CTLs that killed virally infected (30) or tumor (31, 32) targets expressing poorly immunogenic peptides in vitro. Furthermore, heteroclitic peptides of the human tyrosinase
were shown to be more effective in stimulating CD4+ cells
in vitro (31). Most recently, in a heterologous vaccination model, a fortuitous presence of a peptide with heteroclitic
properties primed T cells that exhibited antitumor activity
upon in vivo adoptive transfer (33). Using rational epitope
identification and engineering and in vivo tumor challenge,
we now demonstrate the in vivo potency of this strategy.
The amino acid sequences of tumor antigens can be easily
examined to select candidate MHC-binding peptides that
can target the tumor for CTL attack. The increasing wealth
of the available structural data about MHC-peptide and MHC-peptide-TCR interactions (10, 27) can then be
used to rationally design candidates for heteroclitic vaccines. Once the best candidates are identified, they can be
used individually or in a cocktail vaccine (reference 19 and
Dyall, R., and L. Weber, unpublished data). The latter
would maximize the odds of success and minimize the risk
of tumor escape by epitope mutation. Of course, similar
principles can be applied equally well against intracellular
pathogens.
Our data also touch upon the issue of self-tolerance and
tumor immunotherapy. As recently discussed (8), it is now
clear that many self-differentiation antigens do not induce
complete tolerance through deletion of self-reactive lymphocytes from the immune repertoire. In that regard, an
important advantage of poor MHC binders derived from
self-proteins may be that they are unlikely to tolerize T
cells (as the signal 1 they provide is not strong enough),
sparing a precursor CTL population that can be activated by an appropriate heteroclitic vaccine. Our results with the
gp75 peptide TWH lend experimental support to this
view, suggesting that this class of weak differentiation antigens could become a potential target for tumor therapy. Of
interest, in the experiments described here, we did not notice any overt signs of autoimmunity (including depigmentation) that one might expect if a melanocyte antigen, such
as gp75, is used to target the tumor for lysis by heteroclitic
CTLs. Indeed, such autoimmune manifestations frequently accompany productive antimelanoma immunity in mice
and have been suggested in humans (26 and the references
contained therein). At present, it is unclear whether CTLs
induced by heteroclitic vaccination are less prone to induce
autoimmunity or whether the results observed were due to
the particular peptide chosen. This issue is currently being
addressed experimentally.
The authors wish to thank Dr. Y. Takechi for help with peptide binding assays; Dr. S. Vukmanovic for perusing the manuscript; Ms. D. Nikoli
- Z
gi' c for flow cytometry; and CytRx Inc. and Powderject, Inc. for
generously providing the immune adjuvant Titermax and the gene gun, respectively.
This work was supported in part by the Byrne Fund and the DeWitt Wallace Fund awards (to J. Nikoli' c- Z
gi' c), Swim Across America, the Louis and Anne Abrons Foundation, the Milstein Family Fund, the Lymphoma Foundation, and US Public Health Service grants CA56821, CA59350 (to A.N. Houghton), and
CA08253 (to the Memorial Sloan-Kettering Cancer Center) from the National Institutes of Health.
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