SPAS-1 (stimulator of prostatic adenocarcinoma-specific T cells)/SH3GLB2: A prostate tumor antigen identified by CTLA-4 blockade.

Abstract

Discovery of immunologically relevant antigens in prostate cancer forms the basis for developing more potent active immunotherapy. We report here a strategy using the transgenic adenocarcinoma of mouse prostate (TRAMP) model, which allows for the functional identification of immunogenic prostate tumor antigens with relevance for human immunotherapy. Using a combination of active tumor vaccination in the presence of CTL-associated antigen 4 (CTLA-4) in vivo blockade, we elicited tumor-specific T cells used to expression clone the first T cell-defined TRAMP tumor antigen, called Spas-1 (stimulator of prostatic adenocarcinoma specific T cells-1). Spas-1 expression was increased in advanced primary TRAMP tumors. We show that the immunodominant SPAS-1 epitope SNC9-H(8) arose from a point mutation in one allele of the gene in TRAMP tumor cells, and that immunization with dendritic cells pulsed with SNC9-H(8) peptide resulted in protection against TRAMP-C2 tumor challenge. In humans, the Spas-1 ortholog SH3GLB2 has been reported to be overexpressed in prostate cancer metastases. Additionally, we identified a nonmutated HLA-A2-binding epitope in the human ortholog SH3GLB2, which primed T cells from healthy HLA-A2(+) individuals in vitro. Importantly, in vitro-primed T cells also recognized naturally processed and presented SH3GLB2. Our findings demonstrate that our in vivo CTLA-4 blockade-based T cell expression cloning can identify immunogenic cancer antigens with potential relevance for human immunotherapy.

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SPAS-1 (stimulator of prostatic adenocarcinoma-
specific T cells)/SH3GLB2: A prostate tumor
antigen identified by CTLA-4 blockade
Marcella Fasso `*, Rebecca Waitz†‡, Yafei Hou§, Tae Rim*, Norman M. Greenberg¶, Nilabh Shastri†, Lawrence Fong§,
and James P. Allison‡?
Departments of *Urology and§Medicine, University of California, San Francisco, CA 94143;†Department of Molecular and Cell Biology, University of
California, Berkeley, CA 94720;‡Ludwig Center for Cancer Immunotherapy and Howard Hughes Medical Institute, Memorial Sloan–Kettering
Cancer Center, New York, NY 10021; and¶Baylor College of Medicine, Department of Cell Biology, Houston, TX 77030
Contributed by James P. Allison, December 29, 2007 (sent for review November 26, 2007)
Discovery of immunologically relevant antigens in prostate cancer
formsthebasisfordevelopingmorepotentactiveimmunotherapy.
We report here a strategy using the transgenic adenocarcinoma of
mouse prostate (TRAMP) model, which allows for the functional
identification of immunogenic prostate tumor antigens with rele-
vance for human immunotherapy. Using a combination of active
tumor vaccination in the presence of CTL-associated antigen 4
(CTLA-4) in vivo blockade, we elicited tumor-specific T cells used to
expression clone the first T cell-defined TRAMP tumor antigen,
called Spas-1 (stimulator of prostatic adenocarcinoma specific T
cells-1). Spas-1 expression was increased in advanced primary
TRAMP tumors. We show that the immunodominant SPAS-1
epitope SNC9-H8arose from a point mutation in one allele of the
gene in TRAMP tumor cells, and that immunization with dendritic
cells pulsed with SNC9-H8 peptide resulted in protection against
TRAMP-C2 tumor challenge. In humans, the Spas-1 ortholog
SH3GLB2 has been reported to be overexpressed in prostate cancer
metastases. Additionally, we identified a nonmutated HLA-A2-
binding epitope in the human ortholog SH3GLB2, which primed
T cells from healthy HLA-A2?individuals in vitro. Importantly,
in vitro-primed T cells also recognized naturally processed and
presented SH3GLB2. Our findings demonstrate that our in vivo
CTLA-4 blockade-based T cell expression cloning can identify im-
munogenic cancer antigens with potential relevance for human
immunotherapy.
T cell antigen ? immunotherapy ? TRAMP mice ? prostatic neoplasms
T
eradicate disseminated tumor and to prevent the reoccurrence of
metastases. Until recently, the application of immunotherapy to
prostate cancer has been limited compared with more immuno-
genic tumors such as melanoma or renal cell carcinoma. The
inherent low immunogenicity of prostate cancer has been one
reason that has long prevented the functional identification of
tumor antigens using methods first pioneered by Boon and col-
leagues (1). Although irradiated prostate tumor cells can be and
have been used as a crude source of tumor antigens in prostate
cancer immunotherapy (2), knowledge of the targets of the T cell
response to prostate cancer would allow the development of highly
specific approaches to immunotherapy via specific immunization.
Currently, a number of candidate tumor antigens have been tar-
geted for prostate cancer solely based on their relatively restricted
expression to the prostate or prostate cancer. These include
prostate-specific antigen (PSA) (3), prostatic acid phosphatase
(PAP) (4), prostate-specific membrane antigen (PSMA) (5), and a
few other gene products (6). In these studies, potential relevant T
cell epitopes were identified through a reverse immunology ap-
proach where T cell epitopes are predicted through computer
algorithms according to known HLA-binding motifs. However,
because these antigens are self-proteins, they remain poorly immu-
he goal of immunological approaches to tumor therapy is the
induction of antitumor responses of sufficient strength to
nogenic.Itisonlythroughtherecentdevelopmentofmoreeffective
vaccination strategies that some of the preexisting immune toler-
ance to these self-antigens was overcome. As a result, promising
biologicaloutcomesareemerginginclinicaltrials(6,7).Webelieve,
however, that the efficacy of prostate cancer immunotherapies
couldbefurtherimprovediftargetantigenswereidentifiedthrough
afunctionalapproachratherthanselectedaprioribasedupontissue
expression.
Previously, we have shown that blockade of the inhibitory
signals mediated by the T cell surface molecule CTLA-4 can
greatly enhance T cell responses and, in many cases, result in
tumorrejectioninseveralmousetumorsystems(8,9).Inthecase
of B16 melanoma, tumor rejection through CTLA-4 blockade in
combination with a GM-CSF-producing melanoma tumor cell
vaccine was accompanied by autoimmune depigmentation of
normal melanocytes, evidence that the antitumor T cell response
was also, in part, directed to normal, bona fide self-antigens
shared between the tumor, the vaccine, and melanocytes (10).
One of these antigens was identified as the melanocyte differ-
entiation antigen tyrosinase-related protein 2 (TRP-2) (11).
Significantly, TRP-2 has also been shown to be a major target for
T cells in melanoma patients and is currently in clinical trials as
a melanoma vaccine.
In this study, we describe the use of anti-CTLA-4 therapy in
the TRAMP model (12–14) for the identification of prostate
tumor antigens. In previous studies, we demonstrated that
CTLA-4 blockade by itself or in combination with a GM-CSF-
expressing TRAMP tumor cell vaccine retarded the growth of
transplantable TRAMP prostate tumors (unpublished results)
and primary tumors spontaneously arising in the transgenic
TRAMP mice (13). The same vaccination caused autoimmune
prostatitis in syngeneic C57BL/6 mice, suggesting that TRAMP
tumors may express prostate tissue-specific antigens that could
become potential targets for immunotherapy (13). We hypoth-
esizedthat,asdemonstratedinthemelanomamodel,someofthe
target antigens of the immune prostatitis generated in the mouse
will have human orthologs relevant to human prostate cancer.
Importantly, because these targets are identified by their capac-
ity to stimulate T cells, by definition they would be validated
immunologically, making them ideal targets for immunotherapy.
We applied the vaccination strategy of CTLA-4 in vivo blockade
Author contributions: M.F., L.F., and J.P.A. designed research; M.F., R.W., Y.H., and T.R.
performed research; N.M.G. and N.S. contributed new reagents/analytic tools; M.F. ana-
lyzed data; and M.F. and J.P.A. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. EF676083).
?To whom correspondence should be addressed. E-mail: allisonj@mskcc.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0712269105/DC1.
© 2008 by The National Academy of Sciences of the USA
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in combination with a GM-CSF-expressing TRAMP-C2
(TRAMPC2-GM) cell vaccine to generate a potent anti-
TRAMP tumor response in nontransgenic, syngeneic C57BL/6
mice. The tumor-specific T cells were used to identify the first
immunologically validated prostate cancer rejection antigen. We
refer to this antigenic target in prostate cancer as SPAS antigen
because of its ability to ‘‘stimulate prostatic adenocarcinoma-
specific T cells.’’ We demonstrate that SPAS-1 has a human
ortholog known as SH3GLB2, which is immunogenic in humans
in vitro, making it a potentially attractive candidate target
antigen for the development of antigen-targeted immunothera-
pies in humans.
Results
GenerationofTRAMP-ReactiveTCellHybridomasandIdentificationof
Spas-1.TRAMP-specificCD8?Tcelllineswereestablishedfrom
spleens andlymph nodes
TRAMPC2-GM in combination with anti-CTLA-4 antibody
treatment. We have shown that this vaccination protocol was
sufficient to lead to complete rejection of transplantable
TRAMP tumors (unpublished results). The function and spec-
ificity of the T cell line were assessed by using standard assays for
IFN-? production (Fig. 1A) and cytotoxicity (Fig. 1B) in re-
sponse to incubation with a panel of syngeneic C57BL/6-derived
tumors of different cellular origins. In both assays, the T cell line
recognized TRAMP-C2 cells but not other tumors, including a
melanoma (B16BL6), a colon adenocarcinoma (MC38), and a
lymphoma (EL4) (Fig. 1A). To facilitate expression cloning of
antigens responsible for stimulating the CD8?T cell lines, we
establishedTcellhybridomasbyfusingtheCD8?Tcellswiththe
LacZ-inducible fusion partner BWZ.36 (15). Eight BTZ
(C57BL/6-derived, TRAMP-reactive, LacZ inducible) hybrid-
oma clones were generated and tested for retention of reactivity
by measuring induction of LacZ activity after incubation with
tumor cells [supporting information (SI) Fig. 7A]. Incubation
with anti-Dbbut not anti-Kbblocking monoclonal antibodies
resulted in partial inhibition of T cell activation, suggesting that
eight of eight BTZs tested were specific for a TRAMP-C2
antigen presented by H-2DbMHC molecules (SI Fig. 7B). Next,
we screened a cDNA library generated from TRAMP-C2 tumor
cells with one of the T cell hybridoma subclones, BTZ5.65. A
single cDNA clone was isolated from the library that was capable
of stimulating BTZ5.65 and the remaining panel of T cell
hybridomas (data not shown). The 1.0-kb cDNA insert con-
tained 462 nt of an open-reading frame followed by an untrans-
lated 3? region (3?UTR) of 509 nt including a polyA tail. A
ofmice vaccinated with
full-length transcript was isolated that encoded a predicted
protein of 395 aa (SI Fig. 8). We named this gene Spas-1, for
stimulator of prostatic adenocarcinoma-specific T cells (Gen-
Bank accession no. EF676083). Search of the GenBank database
revealed the existence of a human ortholog of mouse SPAS-1 of
unknown function designated SH3-domain, GRB2-like, en-
dophilin B2 (SH3GLB2, GenBank accession no. AF257319).
Sequence alignment showed that the human and mouse genes
share90%identityatthenucleotideleveland96%identityatthe
amino acid level.
Tissue Distribution of Murine Spas-1. Real-time quantitative RT-
PCR was performed to assess the expression of Spas-1 in normal
mouse tissues. Spas-1 expression was not limited to the prostate
but was found in other tissues, with the highest level of expres-
sion in the heart (Fig. 2A). To determine whether Spas-1
expression levels changed during tumorigenesis, real-time RT-
PCR was performed on prostate tissues from age-matched
C57BL/6 wild-type and TRAMP mice. Based on previous stud-
ies, TRAMP mice start developing severe intraepithelial hyper-
plasia of the prostate ?20 weeks of age, progressing to full
neoplasia by the time they are 24–30 weeks old (16). We
therefore chose to determine levels of Spas-1 expression in
prostate tumors from both 21- and 27-week-old TRAMP mice
to determine expression changes during tumor progression. The
expression levels of Spas-1 were significantly increased in pros-
tate tumor tissues from 27-week-old TRAMP mice compared
with normal prostate tissues from nontransgenic mice or pros-
tate tissues from 21-week-old TRAMP mice (Fig. 2B).
Identification of the SPAS-1 T Cell Epitope. To identify the antigenic
peptiderecognizedbytheBTZ1.4Tcellhybridoma,welimitedour
search to the last 152 C-terminal amino acids of the protein, which
Fig. 1.
The tumor specificity of the T cell line was determined by specific IFN-?
production in response to TRAMP-C2, MC38, B16BL6, or EL4 target cells.
Standard deviations of triplicate cultures are shown. (B) The tumor specificity
of the T cell line was determined by cytotoxicity using a standard JAM assay.
Tcellswereculturedatvariedeffectortotargetcellratios(E:T)withTRAMP-C2
or MC38 EL4 target cells.
Generation of TRAMP-reactive CD8?T cell lines and hybridomas. (A)
Fig. 2.
older TRAMP tumors. (A) Mouse Spas-1 expression in normal tissues: quanti-
tative real-time PCR was performed on two individual panels of cDNA pre-
pared from normal mouse tissues obtained from two adult C57BL/6 mice.
Representative data from three experiments are shown. The error bars rep-
resent the standard deviation between two different mouse cDNA samples
analyzed in the same experiment. (B) Mouse Spas-1 expression in normal
versus tumor prostate: the level of Spas-1 expression was determined in a
panel of normal prostates and primary TRAMP tumors from 21- and 27-week-
old mice. Representative data from two experiments are shown. The P value
(unpaired Student’s t test) was ?0.01 in both experiments.
Spas-1 expression is not restricted to the prostate but is increased in
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are encoded by the original SPAS-1 cDNA clone. We used the
BIMAS computer algorithm to predict possible H-2Db-binding
peptide motifs in this region. Eight synthetic peptides with the
highest predicted binding scores (H-2Db-1 to H-2Db-8) were tested
in vitro for their ability to stimulate BTZ1.4. Although the H-2Db-3
peptide (THVNHLHCL) led to activation of BTZ1.4, this activa-
tion appeared to be very weak, because even at a 100 nM peptide
concentration, the activation plateau had not been reached (SI Fig.
9A). Through testing of deletion mutants, the antigenic region of
SPAS-1 was narrowed down to the first 78 nt of the cDNA insert,
thesameregionthatalsoencodedtheH-2Db-3peptide(SIFig.9B).
Toascertainthiswasindeedthecorrectantigenicpeptide,wetested
a set of overlapping minigenes spanning the predicted region for
expression and presentation of the correct peptide to BTZ1.4 (SI
Fig. 9C). Interestingly, the minigene coding for the predicted
H-2Db-3 peptide did not lead to any T cell activation. The actual
minimal peptide that led to the highest T cell activation was not
THVNHLHCL, but rather STHVNHLHC (SNC9-H8), which was
five logs more potent than THVNHLHCL in stimulating BTZ1.4
(SI Fig. 9D). That SNC9-H8was indeed the naturally processed
epitope of TRAMP-C2 cells was confirmed by the fact that the
stimulatory activity of the synthetic peptide, and that of naturally
processed tumor peptides extracted from TRAMP-C2 cells coe-
luted upon fractionation by reverse-phase HPLC (SI Fig. 9E).
Antitumor Effect of SPAS-1 Peptide Vaccination. Next, we tested
whether prophylactic vaccination with the SNC9-H8 peptide
protects against TRAMP-C2 tumor growth. C57BL/6 mice were
given four i.v. injections, 3 weeks apart, of bone-marrow-derived
dendriticcells(BM-DC)pulsedeitherwiththeSNC9-H8peptide
or the irrelevant ovalbumin peptide SIINFEKL (SL8) before
challenge with the transplantable TRAMP-C2 cells. As a posi-
tive control, mice received four s.c. injections of irradiated
TRAMPC2-GM cells, which have been shown to have a pro-
tective effect against TRAMP-C2 tumor growth when given
prophylactically (unpublished results). As expected, all of the
untreated mice developed palpable tumors by day 19 post-tumor
challenge, whereas tumor growth in mice that received the
TRAMPC2-GM cell vaccine was delayed by at least 28 days (Fig.
3B). Vaccination of mice with SNC9-H8-pulsed BM-DC resulted
in a statistically significant delay in tumor growth (medium time
of 41.5 days) when compared with untreated mice or mice
vaccinated with BM-DC pulsed with the irrelevant peptide SL8
(Fig. 3B). Despite the well documented nonspecific protective
effect conferred by vaccination with BM-DC alone (17–20),
targeting the response to SPAS-1 (SNC9-H8) led to a signifi-
cantly greater delay in tumor growth rate compared with vac-
cination with SL8-pulsed BM-DC (P ? 0.05) (Fig. 3A), confirm-
ing that SNC9-H8was indeed a target of the anti-TRAMP tumor
T cell response in vivo.
SPAS-1 Epitope Mapping from Different Tissues Reveals a G to A
Substitution in Position P8 of the T Cell Epitope. Having determined
the genetic region in the Spas-1 gene that codes for the antigenic
epitope, we compared the sequence of that region in transcripts
isolated from the TRAMP-C2 cells with that of the Spas-1
transcripts obtained by RT-PCR from normal tissues, such as
liver and prostate. As shown in Fig. 4A, the Spas-1 sequence
obtained through expression cloning of the TRAMP-C2 cDNA
library differed from that of normal tissues by a single nucleotide
substitution (G to A), a change that translated into a histidine
rather than an arginine at the eighth position (P8) of the
antigenic epitope. This suggested two possibilities: (i) the anti-
genic epitope arose as a result of a point mutation in the tumor,
or (ii) it resulted from genetic differences between the TRAMP
mouse from which the TRAMP-C2 tumor line was derived and
the C57BL/6 substrain we vaccinated to generate our TRAMP-
specific CD8?T cell line. The latter was ruled out by the fact that
Fig. 3.
werevaccinatedprophylacticallyfourtimes,21daysapart,witheitherSPAS-1
peptide (SNC9-H8), OVA peptide (SL8)-pulsed BM-DC, or with 1.5 ? 106irra-
diated TRAMPC2-GM tumor cells before TRAMP-C2 tumor challenge. (A)
TRAMP-C2 tumor growth is shown for each vaccinated group. Statistics (un-
paired Student’s t test P value) are given for the SPAS-1 peptide (SNC9-H8)
vaccinatedgroup.(B)TRAMP-C2tumorincidenceisshownforeachvaccinated
group.Mediantimetotumordetection(med)andPvalues(Mantel–Haenszel
test, GraphPad PRIZM.4) are indicated. Representative results of three inde-
pendent experiments are shown.
Antitumor effect of SPAS-1 peptide vaccination. Male C57BL/6 mice
Fig. 4.
sequences from different tissues reveal a G to A substitution in position P8 of
the T cell epitope: Sequence of the genetic region encoding the amino acid
residueP8intheSPAS-1Tcellepitopeisshownfordifferenttissuesandtumor
cell lines. (B) BTZ1.4 response to titrated amounts of synthetic peptide corre-
spondingtoeitherthewild-type(SNC9-R8)ormutated(SNC9-H8)SPAS-1Tcell
epitope pulsed on H-2Db-expressing L cells. Standard deviations of triplicate
cultures are shown.
SPAS-1 epitope in normal and tumor tissues. (A) SPAS-1 epitope
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the Spas-1 sequence was identical in normal tissues of both
C57BL/6 mice and TRAMP mice. We also ruled out the
occurrence of a polymorphism in our C57BL/6 substrain by
showing that the Spas-1 sequence was identical in C57BL/6
strains from three independent vendors (data not shown).
Sequence analysis of genomic DNA derived from three different
TRAMP transplantable tumor sublines revealed in each case a
mixed sequence of G and A at the relevant position, whereas
sequencing of the transplantable melanoma cell line B16BL6 or
of the colon adenocarcinoma MC38 yielded only G at that
position (Fig. 4A).
To determine how the wild-type peptide, which contains an
arginine at position 8 of the T cell epitope (SNC9-R8) would
affect T cell recognition, we pulsed H-2Db-expressing L cells
with synthetic peptides corresponding to either the mutated
(SNC9-H8)orthewild-type(SNC9-R8)epitopesanddetermined
their ability to stimulate BTZ1.4 (Fig. 4B). Although the T cell
hybridoma recognized both the mutated SNC9-H8 and the
wild-type SNC9-R8 peptides, the response to the wild-type
peptide SNC9-R8 was 4 logarithms less potent than to its
mutated counterpart.
TRAMP-Tumor Cell Vaccination in Combination with CTLA-4 Blockade
Leads to T Cell Responses Against Both Wild-Type and Mutated SPAS-1
Epitopes in Vivo. Because the TRAMP tumor cell lines expressed
both the wild-type and the mutated form of SPAS-1, we ques-
tioned whether mice injected with the TRAMPC2-GM tumor
cell vaccine in combination with CTLA-4 blockade could raise
T cell responses not only to the mutated but also the wild-type
SPAS-1 antigen. C57BL/6 mice were immunized with the
TRAMPC2-GM tumor cell vaccine in combination with block-
ing antibodies to CTLA-4, followed by five vaccination boosts
with the TRAMPC2-GM cell vaccine alone. Six days after the
last vaccination, spleens were removed and assayed for antigen-
specific IFN-? production in response to SNC9-H8and SNC9-R8
peptides. As expected, a strong response against the mutated
SNC9-H8peptide was detected, which titrated down into nano-
molar peptide concentration. In addition, a significant T cell
response was also observed at higher antigen dose in response to
the wild-type peptide SNC9-R8(Fig. 5). This response was 3–4
logarithms weaker compared with its mutated counterpart but
showed that vaccination with the TRAMPC2-GM cell vaccine in
combination with CTLA-4 blockade is capable of eliciting
self-reactive SNC9-R8-specific T cells.
In Vitro SPAS-1/SH3GLB2 T Cell Responses in Human Peripheral Blood
Lymphocytes. To assess the immunogenicity of SPAS-1/SH3GLB2
in humans, we examined whether SPAS-1/SH3GLB2-reactive T
cells could be elicited from peripheral blood lymphocytes of
HLA-A2?individuals. We used computer algorithms SYF-
PEITHI, BIMAS, and nHLApred to predict HLA-A2-binding
epitopes in the human SPAS-1/SH3GLB2 protein. Five nonamer
peptides (P1–P5) were synthesized that had high binding scores
according to all three algorithms: P1, (LV-9); P2, (YL-9); P3,
(LT-9); P4, (FL-9); and P5, (IL-9). Four of the five peptides bound
to HLA-A2, as demonstrated by stabilization of surface HLA-A2
expression in a conventional T2-binding assay (SI Fig. 10).
Next,wedeterminedwhetherthesepeptidesareimmunogenic
in vitro. T cell priming cultures were set up for each individual
peptide with T cells from three healthy HLA-A2?donors. For
each individual HLA-A2?donor, significant IFN-? secretion
was induced only in the culture restimulated with peptide P4,
FLTPLRNFL (FL-9) (Fig. 6A), indicating that T cells specific to
this SPAS-1/SH3GLB2 peptide can be induced in humans.
Finally, to determine whether the endogenously processed
SPAS-1/SH3GLB2peptidesthemselvesareimmunogenic,weover-
expressed the SPAS-1/SH3GLB2 cDNA in the HLA-A2?human
monocytic leukemia-derived cell line THP-1 and asked whether it
couldactivateFL-9-specificTcells.Inagreementwithourprevious
findings,specificactivationoftheFL-9-specificTcellswasobserved
in response to titrated doses of the FL-9 but not the LV-9 peptide
pulsed onto THP-1 cells (Fig. 6B). More importantly, FL-9-specific
CD8?T cells also recognized endogenously processed and pre-
sented SPAS-1/SH3GLB2 peptide, as demonstrated by the robust
T cell activation in response to titrated numbers of THP-1 cells
over-expressing SPAS-1/SH3GLB2 (Fig. 6C) compared with
Fig. 5.
leads to T cell responses against both wild-type and mutated SPAS-1 epitopes
in vivo. Three C57BL/6 mice received the TRAMPC2-GM/anti-CTLA-4 vaccina-
tion regimen described in Methods. After the fifth and last boost with the
tumor cell vaccine, their splenocytes were tested for specific IFN-? production
in response to titrated doses of either the mutated (SNC9-H8) or the wild-type
(SNC9-R8)SPAS-1peptidesinanintracellularcytokineassay(A)andanELISPOT
assay (B). Ten micromolars of the OVA-SL8 peptide was used as irrelevant
antigen in the ELISPOT assay. Standard deviations of triplicate cultures are
shown. These are representative results of three independent experiments.
TRAMP-tumor cell vaccination in combination with CTLA-4 blockade
Fig.6.
Mo-DC from HLA-A2?healthy donors were pulsed with each of the five
candidatepeptidesP1(LV-9);P2(YL-9);P3(LT-9);P4(FL-9);orP5(IL-9)andthen
incubated separately with autologous PBMC for 10 days. After one restimu-
lation, T cell cultures were assessed for their capacity to produce IFN-? in
responsetothecorrespondingpeptide,pulsedontotheBlymphoblastoidcell
line721.221transfectedwithHLA-A2.(B)HumanTcellcultureswereassessed
fortheircapacitytoproduceIFN-?inresponsetotitrateddosesofFL-9,pulsed
onto the HLA-A2 expressing cell line THP-1. (C) Responsiveness of the FL-9-
reactiveCD8?TcelllinetoendogenouslyprocessedSPAS-1/SH3GLB2peptides
was assessed by coculturing 100,000 CD8?T cells with titrated numbers of
THP-1 cells transduced with either a retroviral vector encoding full-length
human SPAS-1/SH3GLB2 DNA or with an empty retroviral vector. Results are
representative of T cell cultures from three separate healthy donors. Repre-
sentativeresultsoffourindependentexperimentsandstandarddeviationsof
triplicate cultures are shown.
SPAS-1/SH3GLB2-derivedpeptideFL-9isimmunogenicinhumans.(A)
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THP-1 cells transfected with an empty vector. This higher response
was statistically significant over four independent experiments
(unpaired Student’s t test P value ?0.05). The low level expression
of endogenous SPAS-1/SH3GLB2 in vector control-transfected
THP-1 cells (SI Fig. 11) might have contributed to the background
activation of FL-9-specific T cells.
Discussion
We describe in these studies an approach for the functional
identification of immunogenic tumor antigens in the TRAMP
model of prostate cancer. Because these tumor antigens are
defined by their capacity to stimulate T cell responses, our
working hypothesis is that their human orthologs will be immu-
nogenic as well and might become appealing candidate targets
for future antigen-directed immunotherapies for prostate can-
cer. We name these immunogenic targets SPAS antigens, for
‘‘stimulators of prostatic adenocarcinoma-specific’’ T cells. By
using T cells from mice immunized with a GM-CSF-expressing
TRAMP cell vaccine in combination with in vivo CTLA-4
blockade, we have identified the first of these SPAS tumor
antigens, SPAS-1, involved in T cell-mediated rejection of
TRAMP tumors. The mouse Spas-1 gene, like its human or-
tholog SH3GLB2, encodes a protein of unknown function. We
show that immunization with SPAS-1 peptide-pulsed dendritic
cells results in protection against TRAMP-C2 tumor challenge.
Because the immunodominant SPAS-1 epitope displays a point
mutation that is found in all three available TRAMP-C tumor
sublines, it seems likely that the mutation occurred in vivo,
because all three sublines were derived from the same mouse but
subcultured soon after excision of the primary tumor (12). To
determine the frequency by which this mutation occurs in
primary tumors, we sequenced the Spas-1 gene from 20 primary
TRAMP tumors that were also used for expression analysis (see
Fig. 2B) but were unable to detect that particular mutation again
(data not shown). It is conceivable that this mutation might have
been masked in our sequencing analysis by a larger pool of
nonmutated cDNA derived from a heterogeneous cell popula-
tion in the TRAMP tumor.
Recent published work has documented expression of human
SPAS-1/SH3GLB2 in human prostate cancer and possibly in
othertumortypes.Inonestudy,anextensiveexpressionprofiling
not only allowed discrimination between normal prostate and
tumor tissues, but also delineated three subgroups of prostate
cancer that correlate with biological and clinical behavior.
Interestingly, SPAS-1/SH3GLB2 appeared to be most highly
expressed in the lymph node metastases of the most clinically
aggressive subgroup III (21). These findings are corroborated by
another published report that shows SPAS-1/SH3GLB2 to be the
third most-abundant transcript in androgen-stimulated LNCap
cells, a prototypic prostate cancer cell line derived from lymph
node metastases (22). Finally, SPAS-1/SH3GLB2 was identified
among the 40 most significantly up-regulated genes in malignant
granular odontogenic tumors (GCOT) (23). Taken together,
these findings are in agreement with our own expression data in
mice, where increased expression of Spas-1 is observed in more
advanced TRAMP tumors.
The broad expression pattern of Spas-1 found in mice raises
the question of possible autoimmune side effects in the setting
of active immunotherapy. It remains to be determined whether
a similar expression pattern in normal tissue will be found in
humans. Nonetheless, it has been shown that for many tumor
antigens, including the PSMA (24), the antiapoptotic protein
ML-IAP (25), the human telomerase reverse transcriptase
hTERT (26), the proteinase-3 derived peptide PR1 (27), and the
epidermal growth factor receptor HER2/neu (28) an antitumor
T cell response can be elicited after vaccination without toxicity
toward normal tissues. A proposed mechanism is that the
epitopes processed and presented by normal tissues are below
the threshold level for T cell recognition, whereas overexpres-
sion in tumor cells triggers an anticancer response by breaking
previously established tolerance. Therefore, in the event that
SPAS-1/SH3GLB2 is measurably overexpressed in cancer com-
pared with normal tissues, it is conceivable that a T cell
response can primarily be directed to the site with highest
protein expression. Expression studies would need to be done
at the protein rather than at the RNA level to evaluate this
possibility.
Regarding the issue of immunological tolerance, we provide
evidence that tolerance toward the self-antigen SPAS-1/
SH3GLB2 is not complete in humans, as demonstrated by our
ability to elicit T cells specific for the SPAS-1/SH3GLB2 peptide
FL-9 in all healthy individuals tested. Importantly, we also show
this reactivity can be directed to an endogenously processed and
presented SPAS-1/SH3GLB2 epitope. Similarly, in the mouse,
we demonstrate that T cell tolerance to SPAS-1 is not absolute,
becausevaccinationwiththeTRAMPC2-GMtumorcellvaccine
leadstoTcellresponsestobothmutatedSNC9-H8andwild-type
SNC9-R8SPAS-1 epitopes. To fully address the potential use-
fulness of SPAS-1/SH3GLB2 as either an immunologic target or
immune marker, the status of SPAS-1/SH3GLB2-specific T cell
responses in prostate cancer patients will need to be determined.
From a preclinical aspect, the SPAS-1 SNC9-H8 epitope
represents the first-identified tumor rejection antigen on
TRAMP tumor cells. Although several studies have described
the expression of other prostate cancer antigens in TRAMP
tumor cells, such as PAP, prostate stem cell antigen, and PSMA
(29–31),noneofthesehavebeenshowntobetargetsforimmune
rejection in this model. Alternative approaches have been the
generation of TRAMP cells expressing artificial antigens such as
Flu-HA (32). We believe that the identification of SPAS-1 as the
first-described endogenous T cell TRAMP tumor-specific anti-
gen might facilitate the evaluation of vaccines in this mouse
model for prostate cancer.
Our experimental strategy for the identification of T cell
targets in the TRAMP model was based on the rationale that
identified mouse antigens have human orthologs with potential
relevance for immunotherapy in human prostate cancer. Our
finding that one can elicit specific T cell responses to wild-type
SPAS-1/SH3GLB2 in vitro from the blood of healthy individuals,
together with the reported overexpression in advanced forms of
human prostate cancer, supports the validity of our strategic
approach.WeproposethatthestrategyusedtoclonetheSPAS-1
antigen can be broadly applied as a strategy for identifying T cell
targetsspecifictoavarietyoftumorsand,mostimportantly,with
relevance to human cancers.
Methods
For additional details, see SI Methods.
Animals. C57BL/6 mice were purchased from The Jackson Laboratory. Males
from 5 to 7 weeks of age were used.
Tumor Cell Lines. The TRAMP-C1, TRAMP-C2, TRAMP-C3, GM-CSF-expressing
TRAMP-C2 (TRAMPC2-GM), and B7-expressing TRAMP-C2 (B7-TRAMP-C2) cell
lines have all been described (12–14).
Immunizations and Generation of T Cell Lines and Hybridomas. Five-week-old
C57BL/6 mice received s.c. injections of 1.5 ? 106irradiated (12 krads) GM-
TRAMP-C2 cells, six times, 3 weeks apart, and 3 i.p. injections of 100 ?g of
anti-CTLA-4 antibody [clone 9H10 (33)] on days 1, 4, and 7 after the first
vaccination. CD8?T cell lines were generated by restimulating splenocytes on
mitomycin C-treated B7-TRAMP-C2. T cell hybridomas were generated and
tested for specific responses by the production of ?-galactosidase activity as
described (15).
MouseTCellActivationAssays.Specific T cell responses against peptide/MHC
were measured by the production of IFN-? by either ELISA, ELISPOT, or
Fasso ` et al.
PNAS ?
March 4, 2008 ?
vol. 105 ?
no. 9 ?
3513
IMMUNOLOGY