Embed Size (px)
Citation preview
GITRLipid raft
Isotype
INCAGN1876
GITR
FcγR
Downstreamsignaling Higher order clustering
and robust downstreamsignaling
INCAGN1876, a Unique GITR Agonist Antibody That Facilitates GITR Oligomerization
Abstract
Glucocorticoid-induced TNFR family related protein (GITR, CD357 or TNFRSF18) is a member of the tumor necrosisfactor receptor superfamily (TNFRSF). Like other T cell co-stimulatory TNFR family members, GITR utilizes multipleoligomerization states to regulate the initiation of downstream signaling during T cell activation by antigen presentingcells (APCs). The formation of receptor superclusters, comprised of two or more trimeric molecules, has been definedfor multiple TNFRs as a means of regulating downstream signal amplification. For co-stimulatory TNFRs, like GITR,CD137 and OX40, signaling outcomes in T cells are primarily mediated via the NFkB pathway that promotes cellsurvival and effector cell activities in response to suboptimal T cell receptor (TCR) stimulation. It has beenhypothesized that the manipulation of the oligomeric states of co-stimulatory TNFRs using antibodies may havetherapeutic utility in enhancing the activity of tumor-reactive T cells, either as single agents or in combination withother immunomodulatory or immune education strategies.
Here we describe a structure-based analysis of two functionally distinct classes of anti-human GITR antibodies thatstabilize unique conformational states of the receptor. INCAGN1876, a human IgG1 monoclonal anti-GITR antibody,was found to engage a conformational epitope located within a β-turn of the extracellular domain of GITR. Thisantibody binding site modified the equilibrium of GITR monomer, dimer and trimers to promote receptoroligomerization, resulting in downstream NFkB signaling. Notably, this mode of INCAGN1876 receptor engagementenabled it to effectively activate the GITR pathway in recently primed T cells. By contrast, a second reference anti-GITR antibody required concomitant TCR co-engagement in order to modulate the GITR pathway. High contentconfocal analysis was used to evaluate the kinetics of GITR clustering by both classes of anti-GITR antibody,confirming our T cell functional analysis. The ability of INCAGN1876 to engage and effectively activate GITR onrecently primed T cells may enable them to overcome suppressive features of the tumor microenvironment. Notably,INCAGN1876 was shown to promote T cell co-stimulation both as a single agent and in combination with otherantibodies targeting PD-1, CTLA-4 and OX40. Finally, we compared the pharmacologic activity of INCAGN1876 to Fcvariants of this antibody with diminished binding to the inhibitory Fcγ receptor (FcγR), CD32B. The superiority of anIgG1 antibody in these assays was consistent with the potential to achieve optimal GITR clustering by FcγRs, whilemaintaining the potential for FcγR-mediated effector cell activity directed toward intratumoral GITRhigh regulatoryT cells. INCAGN1876 is currently under evaluation in Phase 1/2 studies in subjects with advanced metastatic solidtumors (NCT02697591).
3643Ana Gonzalez,1 Mariana Manrique,1 Lukasz Swiech,1 Thomas Horn,1 Jeremy Waight,1 Yuqi Liu,1 Shiwen Lin,1 Dennis Underwood,1 Ekaterina Breous,1,2 Olivier Léger,2 Volker Seibert,1,2 Taha Merghoub,3 Roberta Zappasodi,3 Gerd Ritter,4 David Schaer,3 Kevin Heller,5Kimberli Brill,5 Peggy Scherle,5 Gregory Hollis,5 Reid Huber,5 Marc van Dijk,1,2 Jennifer Buell,1 Robert Stein,1 and Nicholas S. Wilson1
1Agenus Inc., Lexington, MA; 2Agenus Switzerland Inc., Basel, Switzerland; 3Memorial Sloan Kettering Cancer Center, New York, NY; 4The Ludwig Institute for Cancer Research, New York, NY; 5Incyte Corporation, Wilmington, DE
Presented at theAmerican Association for Cancer Research Annual Meeting 2017Washington, DC, USA • April 1–5, 2017
• INCAGN1876 preferentially binds to dimers, trimers, and higher order GITR complexes, as compared with reference antibody
• INCAGN1876 promotes GITR forward signaling in recently activated T cells in the presence and absence of concomitant TCR stimulation
• INCAGN1876 efficiently promotes the formation of GITR clusters on the surface of cells that correlates with GITR forward signaling
• INCAGN1876 functions optimally on an IgG1 Fc backbone, and combines with anti-OX40, anti-CTLA-4, or anti-PD-1 antibodies to enhance T cell cytokine production
• INCAGN1876 has at least 3 potential mechanisms of action:– Promote tumor-specific T cell priming in the context of APCs alone or in combination with other
immunomodulatory antibodies– Enhance recently activated tumor-specific T cell function in the absence of APCs– Selectively depletes intratumoral populations of activated regulatory T cells
Summary
Scan code to download acopy of this poster or visit:
http://bit.ly/2nHofHI
References1. Schaer DA, et al. Curr Opin Immunol. 2012;24:217–224.2. Bulliard Y, et al. J Exp Med. 2013;210:1685–1693.3. Wajart H, et al. Cell Death Differ. 2015;22:1727–1741.4. White AL, et al. Cell. 2015;27:138–148.5. Benjamini Y, et al. Biometrika. 2006;93:491–507.
Author DisclosuresAna Gonzalez, Ekaterina Breous, Mariana Manrique, Lukasz Swiech, Thomas Horn, Jeremy Waight, Yuqi Liu, Shiwen Lin, OlivierLéger, Dennis Underwood, Volker Seibert, Marc van Dijk, Jennifer Buell, Robert Stein, Nicholas S Wilson: Agenus Inc.: Employment/consultancy and Stock Ownership. Kevin Heller, Kimberli Brill, Reid Huber, Peggy Scherle, Gregory Hollis: IncyteCorporation: Employment and Stock Ownership. Taha Merghoub, David Schaer, Roberta Zappasodi, Gerd Ritter: Nothing to disclose.
AcknowledgementsLayout and printing support was provided by Evidence Scientific Solutions, Philadelphia, PA, funded by Incyte Corporation.
The GITR Pathway Promotes T Cell Co-stimulation
INCAGN1876 Promotes GITR Signaling in Recently Activated T Cells, Including in the Absence of Concomitant TCR Stimulation
INCAGN1876 Demonstrates Increased Activity as an IgG1 Fc and Cooperates With Other Immunomodulatory Antibodies
INCAGN1876 Mediates GITR Clustering on the Surface of Cells That Correlates With T Cell Activation
INCAGN1876 Promotes GITR Signaling in Recently Activated T Cells, Including in the Absence of Concomitant TCR Stimulation
INCAGN1876 Favors Binding to Higher Order GITR Complexes
Fcγ Receptors Facilitate GITR Clustering by INCAGN1876
A. GITR-expressing T cells (Jurkat) were treated with or without bis(sulfosuccinimidyl)suberate (BS3), lysed, and immunoprecipitated using GITR antibodies (either INCAGN1876 or 1580 [a reference GITR antibody]) versus pull-down with an isotype control antibody. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and blotted for detection of GITR.
B. Western blot signal was normalized within a lane and within the cell lysate fraction. Significant P values were calculated with the 2-stage step-up method of Benjamini, Krieger, and Yekutieli.5 Each column represents n = 3 individual experiments and error bars represent standard deviation.5
A. Confocal microscopy images of stably transfected GITR-GFP Jurkat cells co-cultured with GITR ligand (GITR-L), plate-bound INCAGN1876, 1580, or isotype control antibody. Arrows point to clusters.
B. Average GITR clusters per cell, as quantified from the image shown in A.
C. Jurkat cells expressing GITR-GFP (green) stained with a cholera toxin subunit B (CT-B), plus an anti-CT-B antibody to stain lipid rafts (red) and co-cultured with plate bound INCAGN1876 or an isotype control antibody.
A. Schematic representing experimental conditions.B. Human PBMCs stimulated with INCAGN1876, 1580, or isotype control antibodies alone.C. Activated primary human PBMCs (48 hours with an anti-CD3 antibody) re-stimulated with
INCAGN1876,1580, or isotype control antibody.D. Activated primary human PBMCs re-stimulated with GITR antibodies together with TCR
stimulation (anti-CD3 antibody).
Hypothesis: INCAGN1876 binds to GITR expressed on the surface of recently activated T cells and facilitates GITR clustering via Fcγ receptor (FcγR) interaction mediating higher order receptor clustering and downstream signaling (modified from ref. 3).
A B
A. INCAGN1876-mediated primary T cell response: Staphylococcal enterotoxin A (SEA) peptide plus INCAGN1876 expressed in different Fc variants(IgG1, IgG2, IgG2 h2b fixed hinge [containing a mutation that stabilizes the hinge region]4).
TCR stimulation of PBMCs stimulated with INCAGN1876 alone or in combination with:B. Anti-OX40 antibody (plus anti-CD3).
C. Anti-CTLA-4 antibody (plus anti-CD3).
D. Anti-PD-1 antibody (SEA peptide).
Paradigm: GITR signaling in the context of TCR activation enhances effector T cell activation, cytokine production, and survival (modified from ref. 1).
A
C
A. GITR signaling (NFκB activation) (relative light units [RLU]) mediated by Fc crosslinked INCAGN1876, 1580, or an isotype control antibody in the absence of TCR (anti-CD3 antibody) stimulation, as measured using a reporter assay.
B. NFκB activation by cross-linked INCAGN1876, 1580, or an isotype control antibody with concomitant TCR stimulation.
B
C D
A
B
A
A
MHC-I TCR CD28 CD86 GITR GITR-L
Naïve T cell
GITR-L
EFFECTOR PHASE
SECONDARY PHASE
INITIAL PRIMING
24–72 hours
Activated APC
GITR expression upregulated
No GITRstimulus
GITRengagement
• Reduced T cell expansion/survival• Decreased cytokine production
• Enhanced effector T cell expansion/survival• Increased proinflammatory cytokine production
APC, antigen-presenting cell.
GITR Immunoprecipitate From T Cells Quantitation of GITR Immunoprecipitate
NFκB Activation Without TCR Stimulation
NFκB Activation With TCR Stimulation
Assay Conditions Naïve T Cells (without TCR)
Re-stimulation (without TCR)
Re-stimulation (with TCR)
Z-stacks
Confocal Microscopy
SEA Assay
INCAGN18761580
6000
0
RLU
3000
1000
0.1
1500
500
1 10 100
Antibody concentration (µg/mL)
INCAGN18761580Isotype
B
Isotype
Aver
age
rece
ptor
clu
ster
s pe
r cel
l
25
0
15
1 2 10 17
20
10
5
3 4 5 6 7 1698 11 12 13 14 15
GITR-L
INCAGN1876
1580
25
0
15
20
10
5
25
0
15
20
10
5
25
0
15
20
10
5
B DC
800
0
IL-2
(pg/
mL) 600
400
200
4000
0
IL-2
(pg/
mL) 3000
2000
1000
INCAGN1876INCAGN1876 lgG2INCAGN1876 lgG2 fixed hinge
10
8
0
IL-2
fold
indu
ctio
n(lg
Gva
riant
/isot
ype
cont
rol r
atio
)
6
4
2
6 4
Antibody concentration (µg/mL)
10 6 4 10 6 4
Isotype
INCAGN1876Anti-OX40 antibodyINCAGN1876 + anti-OX40 antibodies
0
Donor 1
Donor 4
Donor 2
Donor 3
50 100
IL-2 fold induction
150
INCAGN1876Anti-CTLA-4 antibodyINCAGN1876 + anti-CTLA-4 antibodies
0
Donor 1
Donor 4
Donor 2
Donor 3
40 60
IL-2 fold induction
8020
INCAGN1876Anti-PD-1 antibodyINCAGN1876 + anti-PD-1 antibodies
0
Anti-PD-1 #1
Anti-PD-1 #2
Anti-PD-1 #1
10 30
IL-2 fold induction
4020
0 100 300 500200 400
IL-2 secretion
Naïve T cell
+/- TCR-stimulation
Activated T cell
160
11080
60
50
40
30
-+ + + +
- -Input
INC
AGN
1876
1580
1580 IgG
IgG
Monomer
Dimer
Trimer
Higher order
BS3 – + + + – – + –
INC
AGN
1876
INCAGN1876
Confocal Stacks 3DTop
Bottom
Top
Bottom
Top
Bottom
Top
Bottom
Bottom Top
Bottom Top
Bottom Top
Bottom Top
Isotype
GITR-L
INCAGN1876
1580
Trim
er
Tetr
amer
Pent
amer
Hex
amer
Mon
omer
Dim
er
15
0
GIT
R o
ligom
ers
bind
ing
effic
ienc
y
10
50.009
0.86
0.08
0.007
0.002 0.003
Anti-OX40 Combination
Anti-CTLA-4 Combination
Anti-PD-1 Combination
800
0
IL-2
(pg/
mL) 600
400
200
6000
0
RLU
4000
2000
0.1 1 10 100
INCAGN18761580Isotype
Antibody concentration (µg/mL)
Anti-PD-1 #2
