Download PT-100, a Small Molecule Dipeptidyl Peptidase

[CANCER RESEARCH 64, 5471–5480, August 1, 2004]
PT-100, a Small Molecule Dipeptidyl Peptidase Inhibitor, Has Potent Antitumor
Effects and Augments Antibody-Mediated Cytotoxicity via a
Novel Immune Mechanism
Sharlene Adams,1 Glenn T. Miller,1 Michael I. Jesson,1 Takeshi Watanabe,2 Barry Jones,1 and Barbara P. Wallner1
1
Point Therapeutics, Inc., Boston, Massachusetts, and 2Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
ABSTRACT
The amino boronic dipeptide, PT-100 (Val-boro-Pro), a dipeptidyl
peptidase (DPP) inhibitor, has been shown to up-regulate gene expression
of certain cytokines in hematopoietic tissue via a high-affinity interaction,
which appears to involve fibroblast activation protein. Because fibroblast
activation protein is also expressed in stroma of lymphoid tissue and
tumors, the effect of PT-100 on tumor growth was studied in mice in vivo.
PT-100 has no direct cytotoxic effect on tumors in vitro. Oral administration of PT-100 to mice slowed growth of syngeneic tumors derived from
fibrosarcoma, lymphoma, melanoma, and mastocytoma cell lines. In
WEHI 164 fibrosarcoma and EL4 and A20/2J lymphoma models, PT-100
caused regression and rejection of tumors. The antitumor effect appeared
to involve tumor-specific CTL and protective immunological memory.
PT-100 treatment of WEHI 164-inoculated mice increased mRNA expression of cytokines and chemokines known to promote T-cell priming and
chemoattraction of T cells and innate effector cells. The role of innate
activity was further implicated by observation of significant, although
reduced, inhibition of WEHI 164 and A20/2J tumors in immunodeficient
mice. PT-100 also demonstrated ability to augment antitumor activity of
rituximab and trastuzumab in xenograft models of human CD20ⴙ B-cell
lymphoma and HER-2ⴙ colon carcinoma where antibody-dependent cytotoxicity can be mediated by innate effector cells responsive to the
cytokines and chemokines up-regulated by PT-100. Although CD26/
DPP-IV is a potential target for PT-100 in the immune system, it appeared
not to be involved because antitumor activity and stimulation of cytokine
and chemokine production was undiminished in CD26⫺/⫺ mice.
INTRODUCTION
In addition to mutations that give rise to malignant transformation
(1), tumor progression requires various responses on behalf of the host
animal. These include the development of a tumor stroma derived
from host fibroblasts (2), vascularization of the tumor (3, 4), and
suppression of an effective immune response against tumor-associated
antigens (5, 6). The growth of solid tumors is accompanied by tissue
remodeling that appears to involve interactions between the tumor
cells and the stroma (2). The reactive stromal fibroblasts are not
transformed, but they are distinguished from normal fibroblasts by
their production of certain extracellular matrix proteins and proteases
(7, 8). Among the latter, fibroblast activation protein (FAP) appears to
be consistently expressed as a cell-surface protein in the stroma of
malignant solid tumors (9). The presence of FAP in two malignant
melanoma cell lines (10, 11) also suggests that FAP expression can be
induced in the cancer cell itself as a result of malignant transformation.
FAP is a serine protease that is closely related to another type II
integral membrane protein, CD26/DPP-IV (12, 13). The catalytic site
in CD26/DPP-IV and FAP contains the characteristic catalytic triad of
Ser630/624, Asp708/702, His740/734 (residues are numbered according to
Received 2/12/04; revised 4/10/04; accepted 5/12/04.
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.
Note: B. Jones and B. Wallner contributed equally to this work.
Requests for reprints: Barry Jones, Point Therapeutics, 125 Summer Street, Boston,
MA 02110. E-mail [email protected].
human CD26/DPP-IV and FAP respectively), and the active serine is
situated in a nucleophile elbow in the sequence Gly-Trp-Ser-Tyr-Gly
(13–15). Both enzymes cleave NH2-terminal dipeptides from
polypeptides with the sequence NH2-Xaa-Pro/Ala, where Xaa can be
any natural amino acid (12–14). In addition, FAP was found to
possess gelatinase activity in vitro, and this observation gave rise to
the notion that the enzyme might play a role in tissue remodeling by
digesting type I collagen of the extracellular matrix (16, 17). Rat
CD26/DPP-IV has also been reported to possess gelatinase activity
(18), and although this activity is seldom attributed to CD26/DPP-IV,
the ability of CD26/DPP-IV to form a complex with FAP at invadopodia of migratory fibroblasts has suggested a possible role for the
coordinated activity of both enzymes (17). The amino boronic dipeptide, Val-boro-Pro (PT-100; Fig. 1), appeared to be an interesting drug
candidate in this context. PT-100 competitively inhibits the dipeptidyl
peptidase (DPP) activity of FAP and CD26/DPP-IV, and there is a
high-affinity interaction with the catalytic site due to the formation of
a complex between Ser630/624 and the boron of PT-100 (11, 19).
The potential of PT-100 as an antitumor agent was intriguing in the
light of our earlier observation that PT-100, apparently through its
interaction with FAP, could stimulate hematopoiesis via the increased
production of cytokines [including granulocyte colony-stimulating
factor (G-CSF)] both in vitro in stromal cell supported human bone
marrow cultures and in vivo in mice (11). The cytokines involved in
the stimulation of hematopoiesis by PT-100 are also known to promote innate as well as T-cell-mediated antitumor responses. In particular, G-CSF gene transfection of the colon adenocarcinoma C-26
cell line has been shown to inhibit tumor take in mice and cause
regression of an established tumor in sublethally irradiated mice
inoculated with the transfected cells (20). The release of G-CSF by the
tumor cells appeared to promote tumor infiltration by polymorphonuclear leukocytes expressing interleukin (IL)-1 and tumor necrosis
factor (TNF)-␣ mRNA followed, sequentially, by macrophages and T
cells.
Here, we report the potent antitumor effect of PT-100 in mice.
PT-100 abrogates tumor growth as a single agent and augments
antibody-dependent cell-mediated cytotoxicity. Similarly to hematopoietic stimulation, the antitumor activity did not require an interaction with CD26. The data suggest that, via the up-regulation of
cytokine and chemokine expression in both the tumor and the draining
lymph nodes, PT-100 can stimulate immune responses against tumors
involving both the innate and adaptive branches of the immune
system.
MATERIALS AND METHODS
Animals and Cell Lines. BALB/c, C56BL/6, DBA/2, and BALB/cnu/nu
mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and
Taconic (Germantown, NY). NOD.CB17-Prkdcscid/J and B6.129S7Rag1tm1Mom mice were from The Jackson Laboratory, and C.129S6 (B6)Rag2tmFwa mice were from Taconic. B6, BALB/c CD26⫹/⫺ mice from the
laboratory of Takeshi Watanabe were back-crossed to the BALB/c strain for
seven generations to provide BALB/c CD26⫺/⫺ mice. The CD26 mutation in
these mice was identified as described previously (11, 21). WEHI 164,
MM45T.Sp, MM52.T, EL4, A20/2J, B16-F10, P815, LL/2, Namalwa, and
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ANTITUMOR EFFECT OF DPP INHIBITOR PT-100
Fig. 1. Structure of PT-100.
LS180 cell lines (American Type Culture Collection, Manassas, VA) were
propagated in vitro by serial culture.
PT-100. The amino boronic dipeptide PT-100 was synthesized at ⬎95%
purity as previously described (22), by Ash Stevens, Inc. (Riverview, MI), and
Bristol-Myers Squibb (Princeton, NJ).
Assays of PT-100 Antitumor Activity in Vivo. Mice were injected s.c.
with tumor cells in a shaved flank as follows: 8 ⫻ 105 B16-F10 cells or
4 ⫻ 106 EL4 cells in C57BL/6 mice; 4 ⫻ 106 WEHI 164, MM52.T, and
MM45T.Sp cells or 6 ⫻ 106 A20/2J cells in BALB/c mice; and 4 ⫻ 106 P815
cells in DBA/2 mice. A total of 1 ⫻ 107 Namalwa or 4 ⫻ 106 LS180 human
cells was inoculated into nonobese diabetic (NOD) scid mice. Tumor-inoculated mice were given varying doses (5– 40 ␮g) of PT-100 or saline vehicle by
oral gavage, twice daily at ⬃8-h intervals. The time of tumor inoculation was
defined as day 0, and PT-100 treatment was started on either day 2 or day 7 and
continued daily. To study the effect of day 2-initiated PT-100 treatment in
combination with monoclonal antibodies (mAb) specific for tumor-associated
antigens, mice inoculated with Namalwa cells received (i.p.) 75 mg/kg rituximab or normal human IgG (Sigma) on days 3, 5, and 7, and mice inoculated
with LS180 cells received (i.p.) 10 mg/kg trastuzumab or rituximab (both from
Genentech, San Francisco, CA) on days 4, 7, 10, 13, and 16. Tumor growth
was monitored by measurement of the length (L) and the width (W) with
Vernier calipers. Tumor volume was calculated by the formula: volume
(c.c.) ⫽ W2 ⫻ 0.5L. Experiments were performed at least twice, using groups
of 5, 10, or 20 mice/treatment regimen as indicated in “Results.”
Tumor Challenge of Tumor-Free Mice. BALB/c mice that rejected
WEHI 164 tumors as a result of PT-100 treatment were given a second
inoculation of either WEHI 164 cells or the unrelated A20/2J-, MM45T.Sp-, or
MM52.T BALB/c-derived tumor cells. Likewise, C57BL/6 mice that rejected
EL4 tumors received second inoculations of EL4, LL/2, or B16-F10 tumor
cells. Mice were challenged by s.c. injection ⱖ30 days after the loss of the
primary tumor. Mice received no treatment with PT-100 after a secondary
challenge. As a control for tumorigenicity of cell lines, naı̈ve mice received
injections of the same cells contemporaneously.
Assays of PT-100 Cytotoxicity in Vitro. Tumor cell lines were seeded in
96-well plates at initial cell densities of 0.5 ⫻ 103/ml and 103/ml and cultured
in the presence or absence of PT-100 for 54 h. PT-100 was added to cultures
at concentrations of 30 ␮g/ml to 30 pg/ml in duplicate wells. Standard
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (23)
were performed.
In Vitro CTL Assays. Twenty-two days after EL4 tumor cell inoculation,
spleen cells were harvested from pools of 5 C57BL/6 mice/treatment group
and restimulated in vitro by incubation (3 ⫻ 106/ml) with X-irradiated (20,000
rad) EL4 cells (3 ⫻ 105/ml) in RPMI 1640 (Life Technologies, Inc., Rockville,
MD) supplemented with 10% heat-inactivated fetal bovine serum, 10 mM
HEPES, 1 mM glutamine, 100 units/ml penicillin, 100 ␮g/ml streptomycin (all
from Life Technologies, Inc.), and 0.05 mM 2-mercaptoethanol (Sigma, St.
Louis, MO). After 6 days of culture, cytotoxicity of restimulated lymphocytes
was determined by a 6-h 51Cr-release assay (24) in triplicate wells. Spontaneous 51Cr release was 10 –15% of maximum release. Antibody inhibition was
investigated by addition of 8 ␮g of anti-CD8 (53– 6.7), anti-CD4 (GK1.5), or
isotype-matched rat IgG2a,␬ (R35–95) and IgG2b,␬ (A95–1) mAb (all from
BD Biosciences PharMingen, San Diego, CA) to the effector lymphocytes
before the 51Cr release assay.
Flow Cytometry. Cell-surface expression of HER2/neu in the LS180 cells
was determined by flow cytometry after immunofluorescent staining with
human Fc receptor blocking (3G8, 3D3, 10.1) mAb and trastuzumab or normal
human IgG (Sigma) followed by phycoerythrin-labeled antihuman IgG (G18145). Likewise, CD26 expression in the EL4 cells was determined by staining
with mouse Fc receptor blocking (2.4G2) mAb and phycoerythrin-conjugated
rat IgG2a,␬ (R35-95) or anti-CD26 mAb (H194-112). mAb were from PharMingen. Data were collected from 104 viable cells and analyzed with Winlist
3D (Verity Software, Topsham, ME) software.
Gene Chip Analysis. WEHI 164 tumor-inoculated mice were treated with
PT-100 or saline from day 2 onward. Total RNA was purified by Trizol (Life
Technologies, Inc.) extraction of lymph nodes and tumors harvested 2 h after
administration of the first dose of PT-100 on day 4. cDNA was synthesized
using T7-(dT)24 primers (Amitof Biotech, Brighton, MA) and a Superscript
cDNA kit, processed into cRNA by in vitro transcription (Invitrogen, Carlsbad,
CA), and analyzed on Affymetrix (Santa Clara, CA) U74 gene chips according
to the manufacturer’s instructions. Data were processed using Affymetrix
Microarray Suite 5 and expressed as log2 signal ratios (PT-100 treatment/saline
treatment) of mRNA expression.
Reverse Transcription-PCR Analysis. Total RNA extracted from cells,
tumors or tissues with Trizol was used to synthesize cDNA with Superscript
First-Strand Synthesis System for reverse transcription-PCR (Invitrogen) according to the manufacturer’s instructions. cDNA was amplified with the
following forward and reverse primers: FAP, 5⬘-CTCTGGCGATATTCATACACAGCGACATAC-3⬘ and 5⬘-GAGAATTCAGCTAACTTGCAAAACATC-3⬘; CD26, 5⬘-TCAAGGAGCCGCCCGACCATGC-3⬘ and 5⬘-AGTTCTGAATCCTCCTGAGCCAC-3⬘; and glyceraldehyde-3-phosphate dehydrogenase primers (Clontech, Palo Alto, CA). PCR reactions were performed in a
PTC-100 thermal cycler (MJ Research, Watertown, MA). Amplification
(30 –35 cycles) conditions with Platinum PCR Supermix (Invitrogen) according to the manufacturer’s instructions were as follows: denaturing, 94°C/15 s;
annealing, 55°C/30 s; and extension, 72°C/30 s. Products were analyzed by
agarose gel electrophoresis, ethidium bromide staining, and imaging with a
Typhoon 9210 Imager (Amersham Pharmacia Biotechnology, Piscataway, NJ).
ELISA. Serum cytokine and chemokine concentrations were determined
by Quantikine ELISA (R&D Systems, Minneapolis, MN).
Statistical Analysis. Standard errors and significance by Student’s t test
were calculated using Microsoft Excel software (Redmond, WA).
RESULTS
Inhibition of Tumor Growth by PT-100 in Mice in Vivo. To
screen mouse tumors for sensitivity to PT-100, mice received s.c.
injections of syngeneic tumor cells and were given saline or PT-100
twice daily from day 2 after tumor inoculation and onward. Comparison of tumor sizes between control and PT-100-treated mice on days
15–20 indicated that PT-100 significantly (P ⬍ 0.005) inhibited the
growth of WEHI 164, MM45T.Sp, and MM52.T fibrosarcomas, EL4
and A20/2J lymphoid tumors, B16-F10 melanoma, and P815 mastocytoma (Table 1). A potent antitumor effect was indicated by test/
control ratios of ⬍0.4, corresponding to a ⬎60% reduction in tumor
size. Potential direct cytotoxicity of PT-100 was investigated in
in vitro cultures of the tumor cell lines that were tested in mice.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
of cell viability indicated that PT-100 had no effect on tumor cell
growth in vitro at concentrations of 30 pg/ml to 30 ␮g/ml (data not
shown).
Growth Inhibition of Established Tumors by PT-100 in Vivo.
The effect of PT-100 on established tumors was investigated in mice
inoculated with B16-F10, WEHI 164, and EL4 tumor cells by delaying PT-100 administration (p.o.) until palpable tumors were detectable
in all of the mice on day 7 or 8 after s.c. injection of tumor cells.
Additional growth of these tumors was significantly (P ⬍ 0.005)
inhibited by treatment with PT-100 in all three models (Fig. 2). In
addition to slowing tumor growth, treatment of EL4 tumor-bearing
mice with PT-100 caused a dose-dependent regression in mean tumor
size in all mice (Fig. 2C) and a loss of detectable tumors in some of
the mice. This complete regression of EL4 tumors occurred by day 20
after tumor inoculation in 5 of 10, 4 of 10, and 3 of 10 mice receiving
20-, 30-, and 40-␮g doses of PT-100, respectively; however, these
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ANTITUMOR EFFECT OF DPP INHIBITOR PT-100
Table 1 PT-100 inhibition of mouse tumors in vivoa
Test/control volume ratio
PT-100 (␮g)
WEHI 164
EL4
A20/2J
B16-F10
P815
MM45T.Sp
MM52.T
5
10
20
40
0.10
0.04
0.04
0.09
0.42
0.36
0.17
0.17
0.10
0.25
0.04
NTb
0.50
0.30
0.15
0.23
0.50
0.66
0.28
0.38
0.56
0.46
0.42
0.39
0.73
0.73
0.59
0.71
a
Tumors were injected s.c. into syngeneic mice, and the indicated doses of PT-100 were administered p.o. and twice daily from day 2 after tumor inoculation onward. Control mice
received saline. Means (n ⫽ 10) of tumor volumes recorded between days 15 and 20 were used to calculate the test/control volume ratios. Test/control volume ratios corresponding
to significant (P ⬍ 0.005) inhibition of tumor growth by PT-100 are indicated in bold type.
b
Not tested.
mice relapsed at various times after day 30 when PT-100 treatment
was stopped on day 19 or 24. Tumors reappeared either at the site of
s.c. injection or as metastases in the axial or inguinal lymph nodes.
Tumor Rejection by Early PT-100 Treatment in Vivo. To investigate whether early treatment of tumors might achieve long-term
survival, mice inoculated with WEHI 164 or EL4 tumor cells were
given PT-100 p.o. twice daily from days 2 to 20. WEHI 164 tumorinoculated mice were treated with a 5-␮g dose of PT-100, and EL4
tumor-inoculated mice were treated with a 20-␮g dose because previous investigation of dose responses indicated these were the optimal
doses (data not shown). WEHI 164 and EL4 tumors grew progressively in saline-treated mice resulting in a control tumor incidence of
100%. Tumors also grew initially in PT-100-treated mice and reached
a palpable size by day 7 after inoculation. Tumors persisted in
PT-100-treated mice with minimal increase in size until day 15, at
which time, mean tumor volumes of 0.08 ⫾ 0.03 and 0.29 ⫾ 0.04 c.c.
were recorded for WEHI 164 and EL4 tumors, respectively. Thereafter, tumor regression leading to apparent rejection between days 15
and 30 was observed in 7 of 10 WEHI 164 tumor-inoculated mice and
13 of 20 EL4 tumor-inoculated mice (Fig. 3). In PT-100-treated mice
that did not reject their tumors, tumor growth was significantly inhibited (WEHI 164, P ⬍ 0.005; EL4, P ⬍ 0.000005). Data from replicate
experiments in which the loss of tumors was scored between days 15
and 30 indicated that PT-100 administration caused apparent rejection
Fig. 2. Effect of PT-100 on growth of mouse
tumors established in syngeneic mice. Mice were
inoculated s.c. with B16-F10 melanoma cells,
WEHI 164 fibrosarcoma cells, or EL4 lymphoma
cells. When palpable tumors were apparent at 7 or
8 days after inoculation, mice received the following treatments. A, B16-F10 tumor-bearing C57BL/
6mice given saline (E), 10 ␮g of PT-100 (Œ), or 40
␮g of PT-100 (F) from days 8 to 14 after tumor
inoculation. B, WEHI 164 tumor-bearing BALB/c
mice given saline (E), 5 ␮g of PT-100 (䡺), 10 ␮g
of PT-100 (Œ), or 40 ␮g of PT-100 (F) from days
7 to 24 after tumor inoculation. C, EL4 tumorbearing C57BL/6 mice were given saline (E), 5 ␮g
of PT-100 (f) or 20 ␮g of PT-100 (‚) from days
7 to 19, or 30 ␮g of PT-100 (⫻) or 40 of ␮g PT-100
(F) from days 7 to 24 after tumor inoculation.
Saline and PT-100 were administered p.o., twice
daily. Data represent, means ⫾ SE (n ⫽ 10) of
tumor volumes, and in C, tumor volumes in individual mice on day 20 are shown. At all doses
tested, PT-100 significantly inhibited the growth of
the three tumors: B16-F10, P ⬍ 0.0005 (day 15);
WEHI 164, P ⬍ 0.005 (day 25); and EL4,
P ⬍ 0.0005 (day 20). A representative experiment
of at least two is shown for each tumor model. For
the EL4 model, the responses to saline, 5 and 20 ␮g
of PT-100, and the responses to 30 and 40 ␮g of
PT-100 were determined in two independent experiments in which tumor growth in saline-treated
control mice was similar.
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ANTITUMOR EFFECT OF DPP INHIBITOR PT-100
Fig. 3. Effect of early PT-100 treatment on
growth of WEHI 164 and EL4 tumors in syngeneic
mice. Mice were injected s.c. with tumor cells as
described in Fig. 2. From days 2 to 20 after tumor
inoculation, WEHI 164 (A) and EL4 (B) tumors
were treated by twice daily p.o. administration of 5and 20-␮g doses of PT-100 (F), respectively. Control groups of tumor-inoculated mice received saline (E). In left panels, data represent means ⫾ SE
(A, n ⫽ 10; B, n ⫽ 20) of tumor volumes and
percentage of tumor incidence in PT-100-treated
mice (f). In right panels, tumor volumes in individual mice are shown on day 20. Tumor incidence
in saline-treated mice was 100%, and these mice
were sacrificed on day 20 as were PT-100-treated
mice that failed to reject tumors. Compared with
controls on day 20, mean tumor volumes were
significantly (P ⬍ 0.000005) smaller in PT-100treated mice. The experiments shown are representative of multiple similar experiments as described
in the text.
of 65 ⫾ 16% (mean ⫾ SD of percentage of tumor rejection in each of
12 independent experiments) of WEHI 164 tumors in BALB/c mice
and 51 ⫾ 13% (mean ⫾ SD, 5 independent experiments) of EL4
tumors in C57BL/6 mice. In the WEHI 164 model, tumor regrowth
did not occur during an additional 60-day period of observation. In the
EL4 model, however, tumors reappeared, either at the site of inoculation or as lymph node metastases, in 35 of 90 mice that had earlier
appeared to be tumor free, resulting in a 22% frequency of stable
rejection. Thus, early PT-100 treatment resulted in tumor rejection
and long-term survival of mice inoculated with WEHI 164 or EL4
tumors. In a third model, day 2-initiated treatment of B16-F10 tumors
in C57BL/6 mice with 40-␮g PT-100 doses caused significant inhibition of tumor growth but did not achieve tumor rejection (data not
shown).
PT-100 Induces Specific Immunity to Tumors. The ability of
PT-100 to stimulate a tumor-specific CTL response was investigated
in mice that received s.c. injections of EL4 tumor cells and were given
20 ␮g of PT-100 p.o. twice daily from days 2 to 20. On day 22,
specific CTLs were readily detectable after in vitro restimulation of
spleen cells from PT-100-treated mice, whereas activity was inappreciable in spleen cells from tumor-inoculated control mice (Fig. 4A).
Tumor cytolysis appeared to be due to classical MHC class-I restricted CTL because the activity was strongly inhibited by addition of
anti-CD8 but not anti-CD4 mAbs to the in vitro cytotoxicity assays
(Fig. 4B).
Mice that had rejected WEHI 164 and EL4 tumors because of
PT-100 treatment from days 2 to 20 were completely resistant to
growth of the primary tumor cell type as indicated by the lack of
tumor formation (Fig. 4, C and D) and survival of the mice over a
30-day observation period. Tumor resistance appeared to be immunologically specific because inoculation of different BALB/c-derived
and C57BL/6-derived tumor cells resulted in equally rapid tumor
growth regardless of whether the mice were naı̈ve or had previously
rejected primary WEHI 164 or EL4 tumors as a result of PT-100
treatment.
PT-100 Has Limited Efficacy in Immunodeficient Mice. The
role of adaptive immunity in the antitumor effect of PT-100 was
additionally investigated by comparing activity in immunocompetent
versus immunodeficient mice. PT-100 had an insignificant effect upon
EL4 and B16-F10 tumor growth in C57BL/6J Rag1⫺/⫺ mice but did
significantly inhibit the growth of WEHI 164 and A20/2J tumors in
BALB/cnu/nu and Rag2⫺/⫺ mice (Fig. 5). Similar activity of PT-100
against WEHI 164 tumors was also observed in Rag mutant mice
(data not shown). In marked contrast to immunocompetent mice,
BALB/cnu/nu mice failed to reject WEHI 164 tumors. Similarly,
A20/2J tumor rejection was observed in 60% of BALB/c mice treated
with PT-100, but rejection did not occur in Rag2⫺/⫺ mice. The data
suggest that, although PT-100 can have significant activity against
certain mouse tumors in the absence of adaptive immunity, functional
T cells are required for tumor rejection.
Increased Cytokine and Chemokine Expression in TumorBearing Mice Treated with PT-100. PT-100 was previously shown
to stimulate cytokine (11) and chemokine (G. T. Miller, unpublished
data) production by cultured bone marrow stromal cells. It was therefore anticipated that the antitumor immune responses observed in
PT-100-treated mice were induced via the up-regulation of certain
cytokines and chemokines. Affymetrix gene arrays were used to
compare cytokine and chemokine mRNA expression in WEHI 164
tumors and draining lymph nodes between control mice and mice
given 5 ␮g of PT-100 twice daily from day 2 after tumor inoculation
and onward. Examination of cytokine mRNA levels 2 h after the first
administration of PT-100 on the third day of treatment (day 4)
revealed that expression of IL-1␣, IL-1␤, IL-6, IFN-␤, G-CSF, IL1RA, and thrombospondin-1 mRNA was increased in lymph nodes of
PT-100-treated mice, and expression of IL-1␤, IL-6, and G-CSF
mRNA was also increased in the tumors (Fig. 6A). PT-100 did not
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ANTITUMOR EFFECT OF DPP INHIBITOR PT-100
Fig. 4. Generation of CTL and resistance of PT-100treated mice to tumor rechallenge. Syngeneic mice were
injected s.c. with WEHI 164 or EL4 cells and treated with
PT-100 from days 2 to 20 after tumor inoculation as described in Fig. 3. On day 22, spleen cells pooled from five
EL4-inoculated mice/treatment group were cocultured with
irradiated EL4 cells and assayed for cytotoxicity as described in “Materials and Methods.” A, cytotoxic activity of
spleen cells from PT-100 (filled symbols) or saline-treated
mice (open symbols) against EL4 (F, E), B16-F10 (f, 䡺),
or Lewis lung tumor cells (Œ, ‚). B, effect of anti-CD4 (F),
anti-CD8 (Œ), or appropriate isotype control monoclonal
antibodies (open symbols) on CTL from EL4-inoculated,
PT-100-treated mice. Data represent means ⫾ SD of percentage of lysis (corrected for spontaneous lysis) of target
cells in triplicate assays at the indicated E:T cell ratios. The
experiment in A was repeated twice. Forty to 60 days after
tumor rejection, the BALB/c mice that rejected WEHI 164
tumors (C) were rechallenged with WEHI 164 (F), A20/2J
(Œ), MM45T.Sp (f), or MM52.T (⽧) BALB/c tumor cells,
and the C57BL/6 mice that rejected EL4 tumors (D) were
rechallenged with EL4 (F), Lewis lung (Œ), or B16-F10 (f)
C57BL/6 tumor cells by s.c. injection. Naı̈ve BALB/c (C)
and C57BL/6 (D) mice were similarly injected with tumor
cell lines (open symbols). Data represent means ⫾ SE
(n ⫽ 10) of tumor volumes and are representative of two
experiments with each tumor model.
appear to affect the expression levels of GM-CSF, lymphotoxin, IL-2,
IL-3, IL-4, IL-9, IL-12, IFN-␥, IFN-␣, IL-18, TNF-␣, TNF-␤, TGF-␤,
and IL-15 mRNA at the time point investigated because the changes
in mRNA levels in PT-100-treated versus control mice were ⬍2-fold
(data not shown). PT-100 treatment also increased mRNA expression
of a wide array of chemokines in lymph nodes and a smaller subset in
tumors (Fig. 6A). Of the chemokine mRNA levels evaluated, only
those of SDF-1/CXCL12, Angie/CXCL13, MRP-2/CCL9/10, TECK/
CCL5, and PF4/CXCL4 appeared not to be increased by PT-100
treatment. These data indicate that PT-100 treatment increases the
expression of mRNA for chemokines that collectively chemoattract
and activate multiple immune effector cells (25, 26).
PT-100 administration to either normal or myelosuppressed mice
was previously shown to increase serum levels of G-CSF (11); therefore, it seemed likely that the increased expression of cytokine and
chemokine mRNA observed in the present study would be paralleled
by increases in the serum levels of the corresponding proteins. Although it was not feasible to investigate serum levels of all of the
cytokines and chemokines implicated in the PT-100 response by the
mRNA analysis, ELISA of G-CSF, eotaxin/CCL11, macrophage inflammatory protein (MIP)-1␤/CCL4, thymus and activation-regulated
chemokine (TARC)/CCL17, and KC/CXCL1 indicated that PT-100
treatment caused highly significant increases (Fig. 6B).
PT-100 Enhances the Activity of Tumor-Specific Antibodies
against Human Tumor Xenografts. The known biological activities
(25, 26) of the cytokines and chemokines up-regulated by PT-100 in
vivo suggested that PT-100 treatment might promote the antitumor
activity of neutrophils, macrophages, or natural killer cells participating in antibody-dependent cell-mediated cytotoxicity mediated by the
CD20-specific mAb, rituximab, and the HER2-specific mAb, trastuzumab (27). The antitumor effect of PT-100 was therefore investigated in combination with each antibody in NOD scid mice inoculated
s.c. with human Namalwa CD20⫹ B-cell lymphoma or LS180
HER2⫹ colon carcinoma cells.
In both Namalwa and LS180 xenograft models, tumor growth did
not differ significantly between untreated mice and mice receiving
control antibodies (s.c.) and saline (p.o), and the tumor growth curves
in saline-treated mice have been omitted in Fig. 7 for simplicity. As
previously reported (28), growth of the human CD20⫹ B-cell lymphoma line, Namalwa, in NOD scid mice was inhibited by a 75-mg/kg
dose rituximab injected i.p. on days 3, 5, and 7 after tumor inoculation
(Fig. 7A), and increasing the dose to 150 or 300 mg/kg did not result
in any greater inhibition of tumor growth (data not shown). The effect
on tumor growth of a 5-␮g dose of PT-100 administered from day 2
onward was similar to that of rituximab. When administered in combination, PT-100 and rituximab resulted in an ⬃2-fold greater reduction in tumor growth, and this represented a significant (P ⬍ 0.05)
improvement over the antitumor effect of either agent alone (Fig. 7A).
Cells of the LS180 human colon carcinoma cell line were shown to
express HER-2 by cell-surface immunofluorescent staining with trastuzumab and phycoerythrin-labeled antihuman IgG: 49.7 versus 2.5
linear mean fluorescence units in cells stained with phycoerythrinanti-IgG alone. In NOD scid mice, LS180 tumors were quite resistant
to treatment with 1– 60 mg/kg doses of trastuzumab (data not shown),
and 10 mg/kg had optimal, albeit weak, activity. LS180-inoculated
NOD scid mice were given five injections of a 10-mg/kg dose of
trastuzumab (i.p.) at 3-day intervals from days 4 to 16, and 5 ␮g of
PT-100 were administered (p.o.) twice daily from day 2 onward.
Neither treatment with trastuzumab nor PT-100 alone caused significant inhibition of tumor growth; however, the combination resulted in
significantly reduced (P ⬍ 0.005) tumor sizes on days 15, 18, and 21
(Fig. 7B).
Analysis of the Molecular Targets of PT-100 in Tumor-Bearing
Mice. Because DPP-IV/CD26 and FAP are both inhibited by PT-100
(11), their expression was investigated by reverse transcription-PCR
in tumors and lymphoid organs where a biological response to PT-100
was indicated by increases in cytokine and chemokine mRNA levels.
In BALB/c and C57BL/6 mice bearing WEHI 164 and B16-F10
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CD26 in the antitumor effect of PT-100 was therefore investigated in
BALB/c CD26⫺/⫺ mice inoculated with WEHI 164 tumor cells. As
described above for CD26-expressing wild-type mice, administration
of PT-100 starting on day 2 after tumor inoculation resulted in tumor
rejection, and this was apparent in both CD26⫺/⫺ and CD26⫹/⫹ mice
(Fig. 9A). Comparison of the tumor rejection frequency, 9 of 10 in
CD26⫺/⫺ mice versus 5/10 in CD26⫹/⫹ mice, indicated that the
interaction of PT-100 with CD26 was not required for the antitumor
effect. Because the activity of PT-100 appeared to involve the upregulation of cytokines and chemokines, this response was compared
between BALB/c CD26⫺/⫺ and CD26⫹/⫹ mice by ELISA of serum
levels of G-CSF, KC/CXCL1, Eotaxin, MIP-1␤, and TARC. Again,
CD26 did not appear to be required for the cytokine and chemokine
response to PT-100, and interestingly, the G-CSF, KC/CXCL1,
eotaxin/CCL11, and TARC/CCL17 dose response curves suggested
that CD26⫺/⫺mutant mice were significantly more sensitive to PT100 (Fig. 9B).
DISCUSSION
Fig. 5. Involvement of the adaptive immune system in the antitumor effect of PT-100.
BALB/c (F, E) and BALB/cnu/nu (Œ, ‚) mice received s.c. injections of WEHI 164 cells
(A). C57BL/6J Rag1⫹/⫹ (F, 䡬) and Rag1⫺/⫺ (Œ, ‚) mice received s.c. injections of EL4
(B) or B16-F10 (C) cells. BALB/c Rag2⫹/⫹ (F, E) and Rag2⫺/⫺ (Œ, ‚) mice received
s.c. injections of A20/2J cells (D). Groups of 5–10 mice were treated p.o. with optimal
doses of PT-100 (filled symbols and solid lines: WEHI 164, 5 ␮g; B16-F10, 40 ␮g; EL4,
20 ␮g; and A20/2J, 5 ␮g) or saline (open symbols and broken lines) twice daily from day
2 after tumor inoculation and onward. Data represent means ⫾ SE (n ⫽ 5–10) of tumor
volumes. In normal mice, the antitumor effect of PT-100 was significant: WEHI 164 and
B16-F10, P ⬍ 0.00005; EL4, P ⬍ 0.000005; and A20/2J, P ⬍ 0.0005. In immunodeficient
mice, inhibition of WEHI 164 (A) and A20/2J (D) tumor growth by PT-100 was
significant (P ⬍ 0.005 and P ⬍ 0.00005, respectively, on day 20), but the effect of PT-100
on B16-F10 (C) and EL4 (B) tumors was insignificant (P ⬎ 0.05).
tumors, respectively, FAP and CD26 mRNAs were found to be
coexpressed in tumors, draining lymph nodes, and spleens (Fig. 8A).
In C57BL/6 mice bearing EL4 tumors, FAP and DPP-IV/CD26
mRNAs were also coexpressed in lymphoid tissue, but only FAP
mRNA appeared to be present in the tumors. These findings, together
with the determination that FAP mRNA was not expressed by WEHI
164, B16-F10, or EL4 tumor cells propagated in tissue culture (Fig.
8B), were consistent with previous studies that FAP expression is
induced in the reactive fibroblasts of tumor stroma by the growth of
malignant tumors that do not themselves express the protein (29). In
agreement with previous studies of restricted expression of FAP in
normal tissues (9, 11), FAP mRNA was detected by reverse transcription-PCR in whole embryonic tissue and bone marrow but not in testis
and stomach (Fig. 8C). In addition, FAP mRNA was found to be
expressed in normal lymph nodes. Similarly, CD26 mRNA was found
not to be expressed in these tumor cells in vitro. Even EL4 lymphoma
cells, which due to their T-cell lineage might have been expected to
express CD26 (30), did not do so, as confirmed by immunofluorescence with CD26-specific mAb H194-112 (data not shown).
Data previously obtained in CD26-deficient mice suggested that
PT-100 might exert its effect on hematopoietic tissue through an
interaction with FAP rather than CD26 (11). The involvement of
PT-100 had a potent antitumor effect in mice inoculated with
syngeneic tumor cell lines derived from fibrosarcoma, lymphoma, or
melanoma. The antitumor effect differed from that of cytotoxic agents
such as the taxanes (31) or cisplatin (32) in that PT-100 had no effect
on tumor cell growth or viability in in vitro cell cultures, even at
concentrations that exceeded those achievable in vivo by active doses.
PT-100 instead exerts its antitumor activities through the induction of
cytokines and chemokines that are known to promote innate and
adaptive immune responses against the tumor in vivo.
The establishment of specific immunological memory in mice that
had rejected WEHI 164 or EL4 tumors as a result of PT-100 treatment
and the increased priming of tumor-specific CTL elicited by PT-100
in the EL4 model implicate T-cell immunity in the antitumor effect.
This hypothesis is supported by the observation that antitumor activity
was reduced or abolished, depending upon the tumor model, in
congenitally immunodeficient mice. Knowledge of the biological
activities of the cytokines and chemokines up-regulated by PT-100 in
tumors and draining lymph nodes suggests that the stimulation of
tumor-specific T-cell immunity results from increased efficiency of
antigen presentation by dendritic cells and greater clonal expansion
of antigen-specific T cells (33, 34). The cytokines IL-1, IFN-␤, and
IL-6 are known to increase the efficiency of the interactions between
T cells and dendritic cells leading to T-cell responses. IL-1 and IFN-␤
can promote B7 costimulation of T cells via paracrine pathways that
trigger dendritic cell maturation (35, 36), and IL-6 has recently been
shown to abrogate the suppressive effects of CD4⫹ CD25⫹ regulatory
T cells on antigen-specific T-cell activation (37). The development of
tumor immunity also requires efficient trafficking of antigen-presenting cells and primed T cells between the tumor and draining lymphoid
tissue (34). The up-regulation of monocyte chemotactic protein
(MCP)-1, MCP-2, and MCP-3, interferon-␥ inducible protein (IP)-10,
and monokine induced by interferon-␥ (MIG) by PT-100 in tumor
tissue demonstrated here was highly relevant in this regard because it
had been shown previously that the MCP chemokines target monocytes and CD4⫹ and CD8⫹ T cells via the CCR3 receptor, and IP-10
and MIG target activated T cells via the CXCR3 receptor (25, 38).
The cytokines and chemokines up-regulated by PT-100 in vivo can
also promote tumor cytotoxicity and tumor infiltration by the effector
cells of innate responses (25, 38). Innate effector cells such as natural
killer cells, macrophages, neutrophils, and eosinophils could therefore
contribute to the activity of PT-100 observed in immunodeficient
mice. In addition, PT-100 induced the expression of thrombospondin-1, which is a potentially angiostatic agent (39), in lymph
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Fig. 6. PT-100 increases cytokine and chemokine expression during treatment of WEHI 164
tumors. Mice received injections of tumor cells and
were given (p.o) a 5-␮g dose of PT-100 or saline
twice daily, starting on day 2 after tumor inoculation as described in Fig. 3. Two h after the first
administration of PT-100 on day 4 after tumor
inoculation, tumors and their draining lymph nodes
were excised from the mice, and serum samples
were collected. A, polyA mRNA was isolated from
tumor and lymph node tissue and used to produce
cRNA for analysis with Affymetrix U74 gene
chips. Data represent changes in expression levels
of lymph node (䡺) and tumor (f) mRNA encoding
cytokines and chemokines expressed as the log
signal ratios (PT-100 treatment/saline treatment).
B, serum levels of the cytokines and chemokines
indicated on the ordinate were determined by
ELISA of sera from individual saline (䡺) or PT100 (f)-treated mice. Data represent means ⫾ SE
(n ⫽ 10), and PT-100-stimulated increases were
significant [granulocyte colony-stimulating factor
(G-CSF), KC, TARC, and eotaxin: P ⬍ 0.000005;
MIP-1␤: P ⬍ 0.05).
nodes and the chemokines IP-10/CXCL10 and MIG/CXCL9 in tumor
tissue, where they have the potential to cause apoptotic death of
vascular endothelial cells via an interaction with an alternatively
spliced variant of CXCR3 (40). The degree to which the potential
antiangiogenic activity of cytokines and chemokines contributes to the
antitumor effect of PT-100 in vivo is currently uncertain. Although
IP-10/CXCL10 and MIG/CXCL9 belong to the angiostatic subset of
CXC chemokines characterized by the absence of the Glu-Leu-Arg
motif, other CXC chemokines induced by PT-100 possess the motif
and have been shown to promote angiogenesis (41). Analysis of
chemokine mRNA expression in WEHI 164 tumors indicated that
PT-100-induced expression of proangiogenic KC/CXCL1, MIP-2/
CXCL2, and ENA 78/CXCL5 predominated over expression of antiangiogenic IP-10/CXCL10 and MIG/CXCL9. Because WEHI 164
tumors were extremely sensitive to PT-100, the data suggest that
angiogenic effects may not play a major role in the antitumor activity
of PT-100 in this particular tumor model. However, the net effect of
PT-100 on angiogenesis could vary depending on tumor type. To
address this question, the cytokine and chemokine responses to PT100 will need to be analyzed and correlated with markers of angiogenesis across a panel of different tumor types in vivo.
The potential of PT-100 to stimulate both innate and antigenspecific T-cell responses suggested that the antitumor effect involves
an initial attack on the tumor by innate effector cells followed by the
development of T-cell immunity. Such a mechanism is attractive,
given the role chemokines up-regulated by PT-100 appear to play in
linking innate and adaptive immunity (34) and the role of innate
effector cells such as natural killer cells in promoting tumor-specific
immunity (42). In addition, the observation that PT-100 augmented
the effect of rituximab and trastuzumab on the growth of CD-20⫹
Namalwa B-cell lymphoma and HER2⫹ LS180 carcinoma cell lines
in NOD scid mice supports the hypothesis of a link between the
PT-100 effect and the antitumor responses of macrophages, neutrophils, and natural killer cells that contribute to antibody-dependent
cell-mediated cytotoxicity (27). It should be noted, however, that
PT-100 could have a strong antitumor effect even when innate activity
was not apparent. This was indicated by highly significant activity of
PT-100 against EL4 and B16 tumors in immunocompetent mice
compared with an insignificant effect in immunodeficient mice. The
data in these two tumor models support the notion that PT-100 can
directly stimulate T-cell immunity and are in agreement with the
recent observation that PT-100 can stimulate CTL responses against
synthetic peptide antigens (G. Cole, unpublished data).
PT-100 treatment resulted in tumor rejection in 50 –70% of mice
inoculated with WEHI 164, EL4, or A20 tumor cells when administration was started before establishment of a palpable tumor mass.
Although tumor growth inhibition was highly significant in all of the
mice treated, rejection frequencies and the duration of tumor-free
survival varied between tumor models. Stochastic events clearly influence the efficacy of PT-100 treatment. The greater magnitude of
the antitumor effect obtained when PT-100 was administered earlier
rather than later in the course of tumor progression probably reflects
a balance between the rate of tumor growth and the time required for
PT-100 to induce optimal immune effector mechanisms. It is also
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relatively increased or decreased production of certain cytokines or
chemokines constitutes a recognizable phenotype across a broad range
of immune responses in CD26-deficient mice. Independently of any
potential immunological role, it has been suggested that CD26/
DPP-IV can act as a tumor suppressor. Transfection of human melanoma and non-small cell lung cancer cell lines with CD26/DPP-IV
was found to reverse the malignant cell phenotype—most notably,
tumorigenicity in athymic nude mice (46, 47). At first sight, these
results would seem to conflict with the present data; however, because
CD26/DPP-IV was not expressed by the mouse tumor cells studied
but instead appeared to be expressed by cellular constituents of the
tumor mass derived from the host, the PT-100 experiments did not
address the role of the proteolytic activity of CD26/DPP-IV in malignant cells. Furthermore, it is uncertain whether enzymatic activity
is required for the tumor suppressor activity of CD26/DPP-IV observed in the transfection experiments because mutant CD26/DPP-IV
in which Ser630 was substituted by Ala appeared to be as effective as
wild-type protein in reversing the malignant phenotype (46).
Other inhibitors of CD26/DPP-IV have previously been reported
to suppress immune responses in allograft rejection (48), experimental arthritis (49), and autoimmune encephalomyelitis (50). In
contrast to these observations, the present studies demonstrate that
PT-100 can stimulate T-cell immunity and, in the EL4 model, elicit
CTL priming. Because these PT-100-associated effects occur via a
CD26/DPP-IV-independent pathway, the data raise the question of
whether CD26/DPP-IV and FAP, the putative molecular target of
PT-100 involved in the antitumor effect, play opposing roles in
Fig. 7. Effect of PT-100 and tumor-specific antibodies on growth of human tumor cell
xenografts in nonobese diabetic scid mice. Mice received s.c. injections of Namalwa
B-cell lymphoma cells (A) or LS180 colon carcinoma cells (B) and were given control
antibody plus saline (⫻), control antibody plus PT-100 (f), antitumor antibody plus saline
(E), or antitumor antibody plus PT-100 (F). Treatment (p.o.) with 5 ␮g of PT-100 or
saline was started on day 2 after tumor inoculation and continued twice daily until the end
of the experiment. Tumor-specific antibody treatments (i.p. as indicated by Œ) were
rituximab (75 mg/kg) on days 3, 5, and 7 (A) or trastuzumab (10 mg/kg) on days 4, 7, 10,
13, and 16 (B). Equivalent doses of control antibodies, either normal human IgG (A) or
rituximab (B), were administered according to the same schedules. Data represent
means ⫾ SE (A, n ⫽ 5; B, n ⫽ 10) of tumor volumes. In each experiment, tumors treated
with antitumor antibody plus PT-100 were significantly (A, P ⬍ 0.00005 to P ⬍ 0.005;
B, P ⬍ 0.05 to P ⬍ 0.0005, depending on time of measurement) smaller than tumors
treated with control antibody plus saline at all time points. Treatment of Namalwa tumors
with rituximab plus PT-100 resulted in significantly (P ⬍ 0.05) greater inhibition of tumor
growth from day 11 onward than treatment with either rituximab plus saline or normal IgG
plus PT-100. Likewise, treatment of LS180 tumors with trastuzumab plus PT-100 resulted
in significantly (P ⬍ 0.005) greater inhibition from day 15 onward than treatment with
either trastuzumab plus saline or rituximab plus PT-100. For each xenograft model, data
are representative of two experiments.
possible that the failure to achieve complete rejection of tumors is due
to the outgrowth in vivo of tumor cell variants that have either lost or
reduced antigen expression.
PT-100 is a potent inhibitor of FAP, as well as CD26/DPP-IV
proteolytic activities (11). Notwithstanding the postulated role of
CD26/DPP-IV in T-cell costimulation, proteolysis of chemokines, and
adenosine deaminase binding (30, 43, 44), its involvement in the
antitumor activity of PT-100 has been excluded by the undiminished
activity of PT-100 in CD26⫺/⫺ mice. In fact, PT-100 appeared to have
a greater effect in the absence of CD26. CD26⫺/⫺ mice have recently
been shown to secrete less IL-4 and IL-2 in response to pokeweed
mitogen in vivo (45). It would be of interest to determine whether
Fig. 8. Expression of DPP-IV/CD26 and fibroblast activation protein (FAP) mRNAs in
mouse tumors and tissues. A, syngeneic mice received s.c. injections of WEHI 164,
B16-F10, or EL4 tumor cells, and 5 days later, tumors, draining lymph nodes, and spleens
were excised for analysis of DPP-IV/CD26 and FAP mRNA expression by reverse
transcription-PCR as described in “Materials and Methods.” Expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was assayed as a control for RNA integrity.
B, reverse transcription-PCR analysis of the tumor cells propagated in vitro. C, day 17
whole mouse embryo (E17), bone marrow (BM), lymph node (LN), testis (Te), and
stomach (Stm) from normal mice without tumors were analyzed by reverse transcriptionPCR.
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Fig. 9. Activity of PT-100 in vivo in CD26deficient mice. A, BALB/c CD26⫹/⫹ (F, E) and
CD26⫺/⫺ (Œ, ‚) mice received s.c. injections of
WEHI 164 cells and were treated with 5 ␮g of
PT-100 (filled symbols) or saline (open symbols),
starting on day 2 after tumor inoculation as described in Fig. 3. B, BALB/c CD26⫹/⫹ (F) and
CD26⫺/⫺ (Œ) mice were given single doses of
PT-100 as indicated on abscissae. Serum levels of
G-CSF and chemokines were determined by
ELISA either at 8 h (G-CSF and TARC) or 2 h (KC
and MIP-1␤) after PT-100 administration. Data
represent tumor incidence in groups of 10 mice (A)
and means ⫾ SE (n ⫽ 5) of serum G-CSF or
chemokine concentrations determined in individual
mice (B). Significantly increased serum levels
of G-CSF or chemokines in CD26⫺/⫺ versus
CD26⫹/⫹ mice are indicated by P values.
immune regulation. In the present study, FAP mRNA expression
was shown to be constitutive in lymphoid tissue and bone marrow
in vivo and to be induced in PT-100-sensitive solid tumors derived
from mouse tumor cell lines that did not express the protein in in
vitro cell cultures. The hypothesis that FAP is actively involved in
tumor progression is additionally supported by a previous study
indicating that FAP expression in transfected HEK293 cells could
increase their tumorigenicity in scid mice (15). Polyclonal antiFAP antibodies that could inhibit the DPP activity of FAP in vitro
were also found to suppress HT-29 adenocarcinoma growth in
vivo, thereby supporting the involvement of the proteolytic activity
of FAP in tumor progression (15). The antitumor activity of
PT-100 additionally supports a role for DPP activity in tumor
growth. The up-regulation of cytokines and chemokines early
during the course of PT-100 administration in vivo suggests that, in
addition to the digestion of extracellular matrix (16, 17), DPPs
such as FAP might also play a role in suppressing cytokine and
chemokine production in tumor or lymphoid tissue. This could
occur via NH2-terminal cleavage that modulates the biologically
active half-life of polypeptide signaling molecules that might regulate cytokine and chemokine production in either an autocrine or
paracrine fashion. For example, the potential biological activities
of CD26/DPP-IV include the regulation of immunity by the cleav-
age of certain chemokines and the regulation of blood glucose by
cleavage of glucagon-like peptide-1 or glucose-dependent insulinotropic polypeptide (21, 44). It will be important to understand
the role of FAP in terms of its biologically relevant substrates.
The in vivo antitumor activity of orally administered PT-100 reported here provides the rationale for clinical development of the
compound in oncology. Acting via the stimulation of innate and
adaptive immunity, PT-100 appears to be uniquely suited as a cancer
therapeutic, either as a single agent or in combination with other
anticancer agents. Mouse data support clinical investigation of the
ability of PT-100 to augment the activity of antibodies that engage
effector cells in antibody-dependant cytotoxicity, and the clinical need
to improve the efficacy of antibody-based cancer therapies is well
recognized (51).
ACKNOWLEDGMENTS
We thank Herman Eisen, Richard Benjamin, Paul Allen, Richard Flavell,
and George Demetri for helpful advice throughout the course of these
studies.
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PT-100, a Small Molecule Dipeptidyl Peptidase Inhibitor, Has
Potent Antitumor Effects and Augments Antibody-Mediated
Cytotoxicity via a Novel Immune Mechanism
Sharlene Adams, Glenn T. Miller, Michael I. Jesson, et al.
Cancer Res 2004;64:5471-5480.
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