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AACR Annual Meeting • 6-10 April 2013 • Abstract #2855
Anti-PSMA x Anti-CD3 ADAPTIR™ Molecule, ES414, Inhibits Tumor Growth In Vivo in Mouse Models of Prostate CancerRobert E. Miller, Hang Fang, Jessica Lambie, Megan Aguilar, Robert Bader, Ruth A Chenault, John Kumer, Padma Ravikumar, Toddy Sewell, Jennifer Wiens, Paul A Algate, David Bienvenue, Gabriela Hernandez-Hoyos, Catherine McMahan, Jane Gross, and John W. Blankenship Emergent Product Development Seattle LLC, Seattle, WA, USA
Introduction
Prostate cancer is the most common cancer in men. Although screening, radiation, surgery and hormone ablation therapy have greatly improved the detection and treatment of early stage prostate cancer, few options exist to treat metastatic, castrate-re-sistant prostate cancer (CRPC). We have developed a therapeutic approach that uses a bispecific ADAPTIR™ (modular protein technology) molecule, ES414, that redirects T cell cytotoxicity against cells expressing a common prostate cancer antigen, PSMA (Prostate Specific Membrane Antigen). PSMA was selected as the target tumor antigen because of its close association with pros-tate cancer1-2, disease progression3-8, and overall disease outcome9.
Here, we expand on previous reports10 to further characterize the in vitro activity, pharmacokinetics, and in vivo efficacy of ES414 in mouse xenograft models.
ADAPTIR Therapeutics
Antibody
αPSMA x αCD3 ADAPTIR Molecule
ES414
ααPSMA x αCD3 scFv-scFv
ADAPTIR molecules are bispecific antibody-like therapeutics containing two sets of binding domains linked to immunoglob-ulin Fc domains to extend the half-life of the molecule in vivo. In order to avoid interactions with other components of the im-mune system that could lead to CD3 clustering and non-spe-cific T cell activation, the Fc region of αPSMA x αCD3 ADAPTIR molecule ES414, has been engineered to minimize complement fixation and interaction with Fcg receptors. The anti-PSMA binding domain is a humanized single chain variable fragment (scFv) derived from a murine antibody that binds human and non-human primate (NHP) PSMA. The anti-CD3 binding do-main is a humanized scFv adapted from a murine antibody that binds human and NHP CD3. This platform differs from other bispecific antibody formats like scFv-scFv11 that have also been shown to be active at redirected T cell cytotoxicity.
Mechanism of Action
Multiple bispecific antibody formats have been shown to redi-rect T cell cytotoxicity by physically cross-linking the T cell re-ceptor complex to a cell surface antigen on target cells. This drives formation of a cytotoxic synapse and triggers tumor cell lysis in a target antigen-dependent fashion.12 The intercellular linkage also stimulates T cell activation and proliferation.13
In Vitro Cytotoxicity
C o n c e n t r a t io n ( p M )
% s
pe
cif
ic l
ys
is
0
5
1 0
1 5
2 0
2 5
1 1 0 1 0 0 1 0 0 0
E S 4 1 4 + M D A -P C a -2 b
s c F v -s c F v + M D A - P C a - 2 b
C o n c e n tr a t io n ( p M )
% s
pe
cif
ic l
ys
is
0
1 0
2 0
3 0
4 0
5 0
0 .1 0 .3 1 3 .2 1 0 3 1 .6 1 0 0
E S 4 1 4 + C 4 -2 B ( P S M A + )
E S 4 1 4 + D U -1 4 5 ( P S M A -)
Protein EC50
ES414 2.7 ± 0.6 pMscFv-scFv* 99 ± 10 pM
Cell Line EC50 (ES414)C4-2B 1.1 ± 0.1 pM
DU-145 n/a
A. MDA-PCa-2b (PSMA+) target cells were loaded with 51Cr and incu-
bated with anti-PSMA x anti-CD3 ADAPTIR molecule (ES414) or an-
ti-PSMA x anti-CD3 scFv-scFv along with human T cells for 4 hours.
B. C4-2B (PSMA+) target cells or DU-145 (PSMA-) target cells were
loaded with 51Cr and incubated with either anti-PSMA x anti-CD3
ADAPTIR protein (ES414) along with human T cells for 4 hours. The
percentage of target cell lysis was measured by specific 51Cr release
into the supernatant as compared to total target cell lysis with NP-40.
In Vitro T-cell Proliferation
P ro life r a t io n o f C D 4 + T c e l ls
C o n c e n t r a t io n ( p M )
Pe
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ell
s0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0
0
1 0
2 0
3 0
4 0
5 0
P ro life r a t io n o f C D 8 + T c e l ls
C o n ce n tra t io n (p M )
Pe
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idin
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s
0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 00
2 0
4 0
6 0
8 0
1 0 0P B M C + E S 4 1 4
P B M C + E S 4 1 4 + C 4 -2 B
P B M C + s c F v - s c F v
P B M C + s c F v - s c F v + C 4 -2 B
P B M C + C 4 -2 B
P B M C o n ly
Human PBMC were labeled with CFSE and incubated for 4 days at 37°C with either ADAPTIR molecule alone or with ADAPTIR protein and C4-2B
(PSMA+) target cells rendered non-proliferative by Mitomycin C treatment. The ratio of target cells to T cells was 1:3. Data is plotted as the
percent of T cells that proliferated, as assessed by loss of CFSE fluorescence.
In Vitro Cytokine ReleaseIL -2
[n M ]
pg
/ml
0 .0 0 0 5 0 .0 0 5 0 .0 5 0 .5 5 5 0 5 0 00
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
T N F -
[n M ]
pg
/ml
0 .0 0 0 5 0 .0 0 5 0 .0 5 0 .5 5 5 0 5 0 00
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0IF N -
[n M ]
pg
/ml
0 .0 0 0 5 0 .0 0 5 0 .0 5 0 .5 5 5 0 5 0 00
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
E S 4 1 4E S 4 1 4 + C 4 -2 Bs c F v -s c F vs c F v -s c F v + C 4 -2 BP B M C o n lyC 4 -2 B o n ly
IL -1 7
[n M ]
pg
/ml
0 .0 0 0 5 0 .0 0 5 0 .0 5 0 .5 5 5 0 5 0 00
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
Purified human T cells were incubated for 4 hours at 37°C with bispecific proteins alone or with bispecific proteins and C4-2B (PSMA+) target
cells rendered non-proliferative by Mitomycin C treatment. The ratio of target cells to T cells was 1:3. Cytokine levels were determined by
Milliplex MAP analysis.
In Vivo Efficacy Model: C4-2B
C4-2B Tumor Volumes
Ave.
Tu
mo
r V
olu
me (
mm
3)
Treatment Day 0, 4, 8
Treatment Groups (n=15):
T cells + 3 μg ES414
T cells + 0.3 μg ES414
T cells + 1 μg ES414
T cells + Vehicle
Matrigel only (no tumor cells)
T cells + 0.1 μg ES414
Study Day
C4-2B Serum PSA
Day 54 Serum PSA (ng/mL)
T cells + 3 μg ES414
T cells + 0.3 μg ES414
T cells + 1 μg ES414
T cells + Vehicle
Matrigel only (no tumor cells)
T cells + 0.1 μg ES414
Ave.
Tu
mo
r V
olu
me (
mm
3)
Treatment Day 0, 4, 8
Treatment Groups (n=15):
T cells + 3 μg ES414
T cells + 0.3 μg ES414
T cells + 1 μg ES414
T cells + Vehicle
Matrigel only (no tumor cells)
T cells + 0.1 μg ES414
Study Day
2 million C4-2B cells were mixed with 1 million purified human T cells
in solubilized basement membrane matrix and injected subcutaneously
into the flank of male NOD/SCID mice. ES414 was administered by
IV injection on days 0, 4, and 8. Animals were monitored for tumor
growth and weight loss endpoint criteria. Tumor volumes are plotted
as the mean with the standard error. Averaged tumor volumes incor-
porate the last recorded tumor volume carried forward after end-
point was reached. All ES414 treatment groups showed a statistically
significant inhibition of time to tumor progression (TTP) p ≤ 0.01.
Log-rank test was used for comparing study groups and Kaplan-Meier
method was used to estimate median TTP.
Serum samples were collected from all animals on day 54 post tumor
challenge. PSA levels were determined using Meso Scale Discovery
(MSD) human serum PSA assay. All treatment groups showed a
statistically significant decrease in serum PSA, compared to vehicle
control (Comparisons with a vehicle control using non-parametric
Wilcoxon rank-sum test p ≤ 0.005).
In Vivo PharmacoKinetics in BALB/c mice
Mea
n C
onc.
(μg
/mL)
Time (hr)
ELISA Assay
Mea
n C
onc.
(μg
/mL)
Time (hr)
Activity Assay
Description ELISA Activity
Apparent terminal elimination half life 41.5 hr 38.03 hr
Time of maximum observed concentration 0.25 hr 0.25 hr
Maximum observed concentration, occurring at Tmax 114.3 µg / mL 531 µg / mL
Volume of distribution based on the terminal phase 238.7 mL / kg 79 mL / kg
Serum clearance 3.99 mL / hr / kg 1.44 mL / hr / kg
Area under the curve from the time of dosing extrapolated to infinity 2671 hr*µg / mL 7375 hr*µg / mL
ES414 protein (200 µg) was administered to three female BALB/c mice per group as a bolus intravenous injection. Serum was harvested
from terminal bleeds at indicated time points, and concentrations of protein were determined by both ELISA and a functional redirected T cell
cytotoxicity assay. Pharmacokinetic disposition parameters for proteins were estimated by non-compartmental analysis using WinNonlin™
Professional software (v5.3) and applying the precompiled model 201 for IV bolus administration and sparse sampling.
In Vivo Efficacy Model: MDA-PCa-2b
MDA-PCa-2b Tumor Volumes
Treatment Groups (n=12):
T cells + 30 μg ES414
T cells + 0.3 μg ES414
T cells + 3 μg ES414
T cells + Vehicle (true control)
Matrigel only (no tumor cells)
Vehicle (Tumor only, no T cells)
30 μg ES414 (no T-cells)
Ave.
Tu
mo
r V
olu
me (
mm
3)
Treatment Day 0, 4, 8 Study Day
MDA-PCa-2b Serum PSA
Day 56 Serum PSA (ng/mL)
T cells + 30 μg ES414
T cells + 0.3 μg ES414
T cells + 3 μg ES414
T cells + Vehicle
Matrigel only (no tumor cells)
Treatment Groups (n=12):
T cells + 30 μg ES414
T cells + 0.3 μg ES414
T cells + 3 μg ES414
T cells + Vehicle (true control)
Matrigel only (no tumor cells)
Vehicle (Tumor only, no T cells)
30 μg ES414 (no T-cells)
Ave.
Tu
mo
r V
olu
me (
mm
3)
Treatment Day 0, 4, 8 Study Day
2 million MDA-PCa-2b cells were mixed with 1 million purified human
T cells in solubilized basement membrane matrix and injected subcu-
taneously into the flank of male NOD/SCID mice. ES414 was adminis-
tered by IV injection on days 0, 4, and 8. Animals were monitored for
tumor growth and weight loss endpoint criteria. Tumor volumes are
plotted as the mean with the standard error. Averaged tumor vol-
umes incorporate the last recorded tumor volume carried forward after
endpoint was reached. All ES414 treatment groups in the presence of
T cells showed a statistically significant inhibition of tumor growth (p
≤ 0.001) was seen from day 39 onwards (Comparisons with T-cell
vehicle control using t-test).
Serum samples were collected from all animals on day 56 post tumor
challenge. PSA levels were determined using Meso Scale Discovery
(MSD) human serum PSA assay. All treatment groups showed a
statistically significant decrease in serum PSA, compared to negative
control (Comparisons with a control using non-parametric Wilcoxon
rank-sum test p ≤ 0.0004).
Summary
▪ ES414 redirected T cell cytotoxicity in a target-dependent fashion against PSMA(+) cell lines in vitro at low picomolar con-centrations. This activity occurred at a lower concentration than that seen with an alternative bispecific antibody platform (scFv-scFv).
▪ T cells were activated and proliferated in response to ES414 in the presence of PSMA(+) target cells; no T cell activation or proliferation was observed in the absence of target cells.
▪ ES414 induced less cytokine release upon target cell lysis than that observed with an alternative bispecific antibody platform (scFv-scFv).
▪ ES414 showed an extended serum half-life in mice (~40 hours), which is longer than that seen with alternative bispecific antibody fragment platforms11.
▪ Treatment with ES414 showed statistically significant inhibition of subcutaneous tumor outgrowth in the presence of human T cells in two independent mouse xenograft models of prostate cancer (C4-2B, MDA-PCa-2b). In both models, all treatment groups also showed a statistically significant decrease in serum PSA compared to negative controls.
Conclusions
These studies show that ES414, an αPSMA x αCD3 ADAPTIR molecule, redirects T cell cytotoxicity in vitro and in vivo in preclinical models and merits further investigation as a potential therapeutic for the treatment of CRPC.
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JW, Chung LWK, Isaacs WB. Prostate cancer: biology, genetics and the new therapeutics. Totowa, NJ: Humana Press. pp. 307–326.
(2) Wolf, P. (2011) “Chapter 4: Prostate Specific Membrane Antigen as Biomarker and Therapeutic Target for Prostate Cancer.”, in Spiess PE, Prostate Cancer – Diagnostic and Therapeutic Advances, InTech. pp 81-100.
(3) Su LS, et al. Alternatively Spliced Variants of Prostate-specific Membrane Antigen RNA: Ratio of Expression as a Potential Measurement of Progression. Cancer Research (1995) 44, 1441.
(4) Wright GL, et al. Expression of prostate-specific membrane antigen in normal, benign, and malignant prostate tissues. Urologic Oncology: Seminars and Original Investigations. (1995) 1, 18.
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(8) Burger, M.J. et al Expression analysis of delta-catenin and prostate-specific membrane-antigen: their potential as diagnostic markers for prostate cancer. Int J Cancer (2002) 100(2), 228.
(9) Ross JS et al, Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clin Cancer Res (2003) 9(17), 6357.
(10) Sewell, T et al, anti-PSMA x anti-CD3 bispecific antibody redirects T cell cytotoxicity in castrate-resistant prostate cancer models. 2012 EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, Dublin, Ireland.
(11) Friedrich, M et al. Regression of Human Prostate Cancer Xenografts in Mice by AMG212/BAY2010112, a Novel PSMA/CD3-Bispecific BiTE Antibody Cross-Reactive with Non-Human Primate Antigens. Mol. Cancer Ther. (2012), 11, 2664.
(12) Dreier T, et al. Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. Int. J. Cancer (2002) 100, 690.
(13) Wolf E, et al. BiTEs: bispecific antibody constructs with unique anti-tumor activity. Drug Discovery Today (2005) 10, 1237.
ADAPTIR™ is the new trademark for Emergent BioSolutions Inc.’s modular protein technology that was previously identified using the SCORPION™ (multi-specific protein therapeutic) and SMIP™ (mono-specific protein therapeutic) trademarks. ADAPTIR and any and all Emergent BioSolutions Inc. brand, product, service and feature names, logos and slogans are trademarks or registered trademarks of Emergent Biosolutions Inc. or its subsidiaries in the United States or other countries. All rights Reserved.
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