Download SP-D-GITRL abrogates CD4+CD25+ Treg activity (Fig. 4) and co

Vaccine Adjuvant and Anti-Tumor Effects of a Multimeric Form of
GITR Ligand (GITRL) That Abrogates Treg-Mediated Suppression.
Richard S.
V.
Biotherapeutics, Inc. and
Abstract
Background: The GITR receptor requires clustering in the
plane of the membrane in order to activate downstream
signaling events. To provide this clustering, a multimeric form
of soluble GITRL was produced by genetically fusing the
extracellular domain of GITRL with the body of surfactant
protein D, a self-assembling molecule with 4 trimeric arms
(Stone et al, J. Virol. 80:1762-72, 2006). The resulting
molecule, SP-D-GITRL, is a strong activator of GITR that
reverses Treg suppression of the mixed leukocyte reaction in
vitro (Stone et al., Clin. Vaccine Immunol. 13:1223-1230,
2006).
Methods: For DNA vaccine studies, mice were vaccinated
i.m. with a plasmid for HIV Gag antigen every 2 weeks X 3.
Plasmids for multimeric forms of GITRL (SP-D-GITRL) or
CD40L (SP-D-CD40L) were added to the injections as
adjuvants. Two weeks later, T cell and antibody responses
were measured. For tumor immunotherapy studies,
established A20 lymphoma tumors > 4 mm in diameter were
injected peritumorally every other day X 5 with plasmids
encoding multimeric GITRL or CD40L.
Results: When used as an adjuvant in a DNA vaccine,
multimeric GITRL enhanced CD8+ T cell responses,
particularly CD8+CD62Lhi central memory cells. Multimeric
GITRL also enhanced CD4+ T cell proliferative responses to
the vaccine antigen and was a strong adjuvant for IgG
antibody responses. When used for tumor immunotherapy,
peritumoral injections of DNA encoding either multimeric
GITRL or CD40L cured mice of A20 lymphoma tumors.
Conclusions: Multimeric GITRL, which has been shown to
obviate Treg-mediated immunosuppression in vitro, can be
used as an adjuvant for CD4+ T cell, CD8+ T cell, and antibody
responses to DNA vaccination. Peritumoral injections of
plasmid DNA encoding multimeric GITRL cured mice with A20
lymphoma, a tumor known to be rich in intratumoral Tregs.
These data indicate that multimeric GITRL has significant
potential as a vaccine adjuvant and tumor immunotherapy
agent.
2
Snarsky ,
2UCSD,
S.
2
Barzee ,
La Jolla, CA; and
Molecular Design of SP-D-GITRL
C.
3Miller
2
Santucci ,
B.
2
Tran ,
120
Intratumoral injections of SP-D-GITRL
DNA have antitumor effects
pScGag
+ pSP-D-CD40L
100
80
pScGag
+ pSP-D-GITRL
60
40
pScGag
20
0
0
.
Fig. 3. To provide a receptor-clustering multimeric form of
soluble GITRL , the extracellular domain of GITRL was
genetically fused with the body of surfactant protein D, a selfassembling molecule with 4 trimeric arms to make SP-D-GITRL
(Stone et al, J. Virol. 80:1762-72, 2006). In addition to forming
an X-shaped molecule, the disulfide bonds in the center of the
molecule stack in the Z-axis to form wheel-like “astral bodies.”
These many trimer forms of GITRL are strong activators of
GITR.
SP-D-GITRL abrogates Treg
suppression of MLR in vitro
20
40
60
E:T Ratio
Fig. 6. The addition of SP-D-GITRL to a DNA vaccine for HIV-1
Gag significantly increased anti-Gag CTLs in BALB/c mice.
However, these CD8+ T cell responses were not as strong as
those produced using SP-D-CD40L as a molecular adjuvant.
DNA vaccination methods: A plasmid for secreted, codonoptimized HIV Gag (pScGag) was used as the test antigen.
BALB/c mice were injected i.m. in both quadriceps every other
week X 3 with a combination of pScGag (80 μg) plus GITRL or
CD40L plasmid or empty control vector (20 μg). Two weeks
after the last vaccination, splenocyte CD8+ T cell activity was
determined by CTL activity using peptide-pulsed P815 cells
using the H-2Kd immunodominant peptide, AMQMLKETI.
SP-D-GITRL vaccine adjuvant induces
CM > EM memory CD8+ T cells
Fig. 9. A20 lymphoma tumors were established s.c. in
BALB/c mice and allowed to grow to > 4 mm in mean
diameter before beginning treatment. Then, 50 μg DNA was
injected intratumorally every other day X 5 and the mice were
observed for 3 months. Compared to PBS control injections,
plasmid DNA expressing natural membrane CD40L
(pMemCD40L) was inactive. However, DNA for multimeric
soluble CD40L (pSP-D-CD40L) significantly slowed tumor
growth and cured most of the mice, where cure is defined as
tumor-free survival 90 days after treatment. Similarly, DNA
for multimeric soluble GITRL (pSP-D-GITRL) was very active
against this tumor (p<0.05) and cured about 60% of mice.
Conclusions
Introduction
Fig. 4. An in vitro human MLR was set up using irradiated Tcell depleted stimulator cells from subject 1 and co-cultured
with CD4+CD25- effector responder cells from subject 2 in the
presence of sub-optimal plate-bound anti-CD3 antibody.
Some cultures also contained CD4+CD25+ Tregs from subject
2. SP-D-GITRL (produced from 293 cells using sequences
from Rhesus macaques) or control 293 supernatant were
added as shown and the cultures were incubated for 5 days,
with a pulse of 3H-thymidine during the last 18 hours. As
shown, Tregs suppressed the MLR, but the addition of SP-DGITRL
as
a
DNA
Vaccine
Adjuvant
GITRL largely restored MLR-induced cell proliferation.
Fig. 7. DNA vaccinated mice were rested for 3 months following
which their splenocytes were stained for Gag peptide/H-2Kd
tetramer and for CD62L. As shown, SP-D-GITRL adjuvant favored
CD8+CD62L+ central memory cells (CM) over CD8+CD62Leffector memory (EM) T cell responses. In contrast, SP-D-CD40L
adjuvant favored EM > CM CD8+ T cell memory responses..
SP-D-GITRL vaccine adjuvant
promotes antibody responses
SP-D-GITRL co-stimulates CD4+ T
cell proliferation in vitro
O.D. A650
0.6
pScGag + pSP-D-GITRL
pScGag
pScGag + pSP-D-CD40L
pcDNA3.1
0.5
0.4
0.3
0.2
► SP-D-GITRL abrogates
CD4+CD25+ Treg activity (Fig. 4) and
co-stimulates CD4+ T cell
proliferation in vitro (Fig. 5)
► As an DNA vaccine adjuvant, pSPD-GITRL augments cytotoxic CD8+ T
cell responses (Fig. 6), promotes the
development of CD8+CD62L+ central
memory T cells (Fig. 7), and
enhances antibody responses (Fig.
8).
►pSP-D-GITRL DNA intratumoral
DNA injections cure established A20
lymphoma tumors in mice (Fig. 9).
0.1
Fig. 2. GITRL dimer (left) stimulates a weak signal, 1-trimer
GITRL (middle) stimulates a modest signal, but 4-trimer GITRL
(right) stimulates a strong signal. Zhou, PNAS 105:5465-5470,
2008.
10
00
00
0
10
00
00
10
00
0
10
00
10
0.0
10
0
Fig. 1. Many receptors for TNF SuperFamily (TNFSF) ligands,
including GITR, require clustering in the plane of the membrane
in order to activate downstream signaling events.
and G.W.
3
Stone .
School of Medicine, University of Miami, Miami, FL.
SP-D-GITRL adjuvants CTL responses
to DNA vaccination
% Specific Lysis
1Multimeric
1,2
Kornbluth ,
1/serum dilution
Fig. 5. Human CD4+ T cells were cultured with sub-optimal
plate-bound anti-CD3 antibody along with increasing amounts of
macaque SP-D-GITRL produced in 293 cells (solid) or with
control 293 supernatants (open bars). Proliferation was
measured by 3H-thymidine incorporation and expressed as the
mean CPM + SEM.
Fig. 8. Sera from DNA vaccinated mice were tested for anti-Gag
IgG by ELISA. As shown, SP-D-GITRL augmented antibody
production almost 30-fold. In contrast, SP-D-CD40L was inactive
as an adjuvant for antibody responses.
Contact information:
Richard S. Kornbluth, M.D., Ph.D.
Email: [email protected]