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Development of an Antibody-drug Conjugate Targeting TIM-1 for the Treatment of Ovarian and Renal Cell Carcinoma
Lawrence J. Thomas, Laura Vitale, Thomas O’Neill, Ree Dolnick, Paul K. Wallace, Hans Minderman, Lauren E. Gergel, Eric M. Forsberg, James M. Boyer,
James R. Storey, Russell A. Hammond, Jenifer Widger, Karuna Sundarapandiyan, Andrea Crocker, Henry C. Marsh, Jr. and Tibor Keler
Celldex Therapeutics, Inc., Hampton, NJ 08827 and Needham, MA 02494; Flow and Image Cytometry Facility, Roswell Park Cancer Institute, Buffalo, NY 14263
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TIM-1 BACKGROUND
IHC ANALYSIS OF TIM-1 EXPRESSION
Ovarian Carcinoma
• TIM-1 (T cell Ig and mucin domain-containing protein-1) is a type I
transmembrane protein with an Ig V-set (IgV) domain and a mucin
domain with O-linked glycosylation.
Ig Domain
Mucin Domain
Cyto-Tail
[29-107]
[129-290]
[311-359]
IN VIVO ANTI-TUMOR ACTIVITY OF CR014 IgG1 vcMMAE AND CR014 IgG4 vcMMAE
CR014 mAb BINDING AND INTERNALIZATION
A
Normal Ovary
IGROV-1 (Ovarian)
Caki-1 (Renal)
A549 (Lung)
IGROV-1 Tumor Model
Caki-1 Tumor Model
A549 Tumor Model
A
C
IgG1-FITC
CR014-FITC
E
Tm
B
• TIM-1 is also known as kidney injury molecule-1 (KIM-1) and hepatitis
A virus cellular receptor-1 (HAVCR-1).
Normal Kidney
Renal Cell Carcinoma
• TIM-1 is expressed by activated T cells and B cells and thought to
function as a co-stimulatory molecule.
B
D
• TIM-1 deficient mice do not exhibit significant phenotypes or defects
in immune system development.
• TIM-1 is overexpressed in renal cell carcinoma and ovarian clear cell
carcinoma and the shed ectodomain is a predictive biomarker of
tumor progression.
ANTI-TIM-1 mAb, CR014, ADC
Clinical samples were stained with biotin-conjugated CR014 IgG4 antibody followed
by streptavidin–conjugated HRP and counterstained with hematoxylin and eosin.
TIM-1 expression was observed in 74% (42/57) of renal and 69% (11/16) of ovarian
carcinoma specimens.
CHARACTERIZATION OF IgG1 AND IgG4
VERSIONS OF ANTI-TIM-1
Panel A: The indicated tumor cells were incubated with 5mg/mL antibody for 20 minutes at
room temperature. Binding was detected with a PE-labeled goat anti-human IgG (Fc specific)
antibody and analyzed on a BD FACSCanto II flow cytometer. CR011 is a anti-GPNMB
antibody acting as a negative control. Panel B: Internalization of isotype control (left panel) or
CR014 IgG1 (right panel) was measured after incubation (40 min, 37oC) of IGROV-1 cells with
FITC-labeled antibodies. The cells were counterstained with anti-Class I (red) and DAPI (blue)
before analysis by confocal microscopy.
IN VITRO CYTOTOXICITY OF CR014 IgG1 vcMMAE
A
IGROV-1 (TIM-1+, GPNMB-)
Caki-1 (TIM-1+, GPNMB-)
ADC Mechanism
• CR014 was expressed as human IgG4
and IgG1 isotype.
• CR014 was conjugated to
monomethylauristatin-E (MMAE) using
Seattle Genetics’ Antibody Drug Conjugate
technology.
• We have developed CR014, a fully human monoclonal IgG4 and IgG1 antibody specific for
extracellular domain of TIM-1.
B
0.8
CR014-IgG1
0.8
0.7
CR014-IgG4
CR014 (IgG1)-vcMMAE
CR014 (IgG4)-vcMMAE
0.6
CR011
Mean OD
1
• This antibody was shown to bind purified recombinant TIM-1-Fc protein and TIM-1 expressed on a
variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma) and IGROV-1
(human ovarian adenocarcinoma) and A549 (lung carcinoma).
ADC Bifunctional Binding
Naked mAb Binding
1.2
Mean OD
• CR014 binds to mucin domain of TIM-1
and cross reacts with non-human primate
TIM-1.
SUMMARY AND FUTURE DIRECTIONS
• TIM-1 expression is upregulated in several human cancers, most notably in renal cell and ovarian
carcinomas, but has very restricted expression in healthy tissues thus representing a promising
target for antibody mediated therapy.
• Fully human mAb CR014 was produced
by immunization of human Ig expressing
mice (XenoMouse®) with the extracellular
domain of human TIM-1.
• Kd of CR014 for human TIM-1 is 2.71 x
10-9 M.
Panel A: Groups of NCr Nude mice were implanted with 2x106 IGROV-1 cells s.c.. On day 0, mice were divided into four groups with equivalently sized tumors. CR014 IgG1 vcMMAE was
administered i.p. on days 0, 4, 8 and 12 (arrows). SEMs are indicated. Panel B: Mice were treated as in A and dosed with indicated amounts of CR014 IgG1 vcMMAE on days 0, 4, 8 and
12 (arrows). Kaplan-Meier survival curves from pooled data of 6 independent experiments. Panel C. Female Nude (NCr) mice received 5.6x105 Caki-1 cells s.c. Mice were divided into four
groups with equivalently sized tumors on day 0 and treated with the indicated amounts of CR014 IgG4 vcMMAE, or saline (n=8) i.p. on days 0, 4, 8, and 12 (arrows). Panel D: Comparison
of CR014 IgG1 vcMMAE versus CR014 IgG4 vcMMAE in the renal Caki-1 model (not significantly different from each other, p<0.2566). Panel E: Groups of NCr Nude mice were implanted
with 1x106 A549 cells s.c.. Mice were dosed with CR014 IgG1 vcMMAE (n= 12) or saline (n=12), i.p. on days 0, 4, 8 and 12 (arrows). SEMs are indicated.
0.6
0.4
A549 (TIM-1+, GPNMB-)
CR011-vcMMAE
SK-MEL-2 (TIM-1-, GPNMB+)
0.5
0.4
• The ADCs were shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1 expressing cell lines, but not on TIM-1
negative cell lines.
0.3
0.2
0.2
0
0.001
• CR014 IgG1 vcMMAE and CR014 IgG4 vcMMAE showed significant anti-tumor activity in Caki-1, IGROV-1 and A549 xenograft models, .
0.1
0.01
0.1
Concentration, mg/mL
1
10
0
0.001
• Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by
cells in vitro.
• Antibody-drug conjugates (ADC) were produced with CR014 covalently linked to a potent cytotoxin, monomethyl auristatin E (MMAE).
0.01
0.1
Concentration, mg/mL
1
• Similar ADC activity was observed with CR014 IgG1 or IgG4.
10
Panel A: Size Exclusion Chromatography performed on a TSKgel G3000. Panel B:
Plates were coated with TIM-1-Fc and blocked with BSA/PBS, followed by an
incubation with the mAb or ADC’s. The naked mAb were detected with an HRP
labeled goat anti-human IgG F(ab’)2 specific reagent. Bifunctional binding of the
ADC’s was detected with a mouse anti-MMAE antibody, followed by an HRP labeled
goat anti-mouse IgG antibody.
• We have selected the CR014 IgG1 for manufacturing and IND-enabling studies in order to advance CR014 IgG1 vcMMAE (CDX-014)
towards clinical studies in renal cell carcinomas and potentially other TIM-1 expressing tumors.
The indicated tumor cells were plated overnight (5,000/well) and allowed to adhere. The next day,
dilutions of ADC’s were added and the plates were incubated for an additional 96 hours. Viability
was assessed with Cell-Titer Glo kit from Promega. CR011 is an anti-GPNMB antibody.
AKNOWLEDGEMENTS
The authors would like to thank V. Pollack, J. MacDougall, W. LaRochelle, K. Borrelli, J. Christopher, C. Pilsmaker, S. Round, A. Sandland,
R. Weaver and J. Hunter for contributions to this work.

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