A n t i - T I G I T ( 1 0 A 7 . m I g G 2 a . D L E ) A n …€¢ Our data describe a novel Fc...
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0 2 4 6 8 10 0.0 0.5 1.0 1.5 2.0 CD4 + non-Tregs Days post-injection Fold-change CD4 + non-Tregs Fc R Co-Engagement by Anti-TIGIT Monoclonal Antibodies Enhances T cell Functionality and Antitumor Immune Responses Dhan Chand 1 , Jeremy D. Waight 1 , Elena Paltrinieri 1 , Sylvia Dietrich 1 , Mark Bushell 1 , Mathew Costa 1 , Randi Gombos 1 , Nicholas S. Wilson 1 , Jennifer S. Buell 1 , Robert B. Stein 1 , Alexander Duncan 1 , David A. Savitsky 1 1 Agenus Inc. or subsidiary thereof (current or former employee), Lexington, MA Presented at the American Association for Cancer Research Annual Meeting Atlanta, GA, USA • March 29– April 3, 2019 #2390 TIGIT: A Key Regulator of the Immune Synapse CT26: Fc-dependent anti-tumor activity Human: Fc-dependent T cell priming (in vitro) Anti-TIGIT Requires Intact FcүR Interactions for Anti-Tumor Activity and Improved T cell Stimulation Fc:FcүR Co-Engagement Enhances T cell Responsiveness References 1 Waight et al., Cancer Cell. 2018; 33(6):1033-1047; 2 Zhang et al., Nat Immunol. 2018; 19(7):723-732 • Our data describe a novel FcR-dependent mechanism of action that enhances the therapeutic activity of anti-TIGIT antibodies in preclinical studies • Murine FcRIV and human FcRIIIA are critical mediators of anti-TIGIT function • Tumor control is dependent on enhancing CD8 T cell and NK cell function 2 but not Treg depletion • Enhanced FcRIIIa co-engagement via Fc engineering further enhances T cell responsiveness • This novel mechanism extends to antibodies targeting CTLA-4 and could inform the optimal design for a new class of Fc-engineered antibodies for cancer immunotherapy 1 . Conclusions Anti-TIGIT Antagonist Activity Depends on Fc үRIV Engagement to Mediate Optimal Immune Modulation In Vivo The Importance of Fc-FcүR Co-Engagement for Improved T Cell Responses Extends to Antibodies Targeting CTLA-4 …but not PD-1 -3 -2 -1 0 1 2 0 2 4 6 8 10 12 Antibody g/ml [log] IL-2 (fold-change) hIgG1 hIgG1.N27A hIgG1.DLE hIgG1.DLE hIgG1.N297A hIgG1 Isotype control Anti-CTLA-4 Blockade of FcүRs Anti-CTLA-4 Hypothesis: Optimizing Fc-FcR co-engagement enhances the activity of anti-TIGIT and anti-CTLA-4 antagonist antibodies 1 . TIGIT and CTLA-4 antibodies with increased binding affinities to activating Fc receptors FcRIV (CD16-2, mouse) or Fc RIIIA (CD16a, human) augment T cell priming by improving the quality of the immune synapse between a T cell and an antigen presenting cell (APC). 0.1 1 10 0 50000 100000 150000 Fc RI Antibody [ g/mL] MFI (APC) 0.1 1 10 0 2000 4000 6000 Fc RIIB Antibody [ g/mL] MFI (APC) 0.1 1 10 0 20000 40000 60000 80000 Fc RIII Antibody [ g/mL] MFI (APC) 0.1 1 10 0 20000 40000 60000 Fc RIV Antibody [ g/mL] MFI (APC) 10 15 20 25 30 0 1000 2000 3000 Days post implantation Tumor volume (mm 3 ) Isotype Control (mIgG2a) anti-CTLA-4 (mIgG2a.DLE) anti-TIGIT (mIgG2a) anti-TIGIT (mIgG2a.DLE) Enhanced FcүR Co-Engagement by Anti-TIGIT Antibodies Promotes Better Tumor Control 0 9 12 14 18 22 26 28 0 1000 2000 3000 Isotype Control (mIgG2a) Days post implantation Tumor volume (mm 3 ) 0 9 12 14 18 22 26 28 0 1000 2000 3000 Anti-CTLA-4 (mIgG2a.DLE) Days post implantation Tumor volume (mm 3 ) 0 9 12 14 18 22 26 28 0 1000 2000 3000 Anti-TIGIT (10A7.mIgG2a) Days post implantation Tumor volume (mm 3 ) 0 9 12 14 18 22 26 28 0 1000 2000 3000 Anti-TIGIT (10A7.mIgG2a.DLE) Days post implantation Tumor volume (mm 3 ) 0 2 4 6 8 10 0 5 10 15 20 25 CD8 + T cells Days post-injection Fold-change CD8 + T cells 0 2 4 6 8 10 0.0 0.3 0.6 0.9 1.2 FoxP3 + Tregs Days post-injection Fold-change FoxP3 + Tregs Anti-TIGIT Has a Novel Mechanism of Immune Modulation that Does Not Include Treg Depletion 1 2 3 4 5 6 0 5 10 15 IL-2 (fold change) *** *** 1. Isotype Control (IgG1.DLE) 2 Anti-TIGIT (hIgG1) 3 Anti-TIGIT (hIgG1.DLE) 4 Isotype Control (IgG1.DLE) 5 Anti-TIGIT (hIgG1) 6 Anti-TIGIT (hIgG1.DLE) + Anti-PD-1 + Isotype IgG4 1 2 3 4 5 6 0 2 4 6 8 IL-2 (fold change) + Anti-LAG-3 + Isotype IgG4 1. Isotype Control (IgG1.DLE) 2. Anti-TIGIT (hIgG1) 3. Anti-TIGIT (hIgG1.DLE) 4. Isotype Control (IgG1.DLE) 5. Anti-TIGIT (hIgG1) 6. Anti-TIGIT (hIgG1.DLE) * *** Enhanced FcүRIIIA Engagement by Anti-TIGIT Further Improves T Cell Responsiveness In Vitro (Human) 0.001 0.01 0.1 1 10 100 0 50000 100000 150000 200000 Antibody [ng/ml] Relative Light Units (RLU) hIgG1 hIgG1.DLE hIgG1.N297A hIgG1 hIgG1.DLE IgG1.N297A Anti-TIGIT Isotype Control A B C A B C D Figure 5. A. IL-2 production (day 4) by human PBMCs stimulated with a suboptimal concentration of SEA peptide together with increasing concentrations of anti-TIGIT clone 347_02_A10 (hIgG1 ⚫), Fc-enhanced anti- TIGIT (hIgG1.DLE ⚫) or isotype control (hIgG1.DLE ). B. Jurkat cells genetically engineered to express FcRIIIA (V/V) upstream of a NFAT-dependent luciferase reporter were co-cultured with TIGIT-expressing CHO cells and increasing doses of anti-TIGIT or isotype control antibodies. Signaling was assessed based upon luciferase expression shown as relative light units (RLU). C. IL-2 production (day 4) by human PBMCs stimulated with a suboptimal concentration of SEA peptide together with 10 μg/ml of anti-TIGIT (hIgG1), Fc- enhanced anti-TIGIT (hIgG1.DLE) or isotype control (hIgG1.DLE) alone or in combination with 10 μg/ml anti- PD-1 (Nivolumab) or D. anti-LAG-3 (Clone 25F7) A B C Figure 3. A. BALB/c mice (n=10-12/group) with established CT26 tumors (~50mm 3 ) were administered i.p. with 200 μg of anti-TIGIT clone 10A7 (mIgG2a ◼), Fc-enhanced anti-TIGIT clone 10A7 (mIgG2a-DLE ◼), Fc-enhanced anti-CTLA-4 clone 9D9 (mIgG2a.DLE ◼) or isotype control ( ) on days 9, 13 and 16. Data representative of 3 independent experiments. B. Growth curves showing individual tumor volumes over time of treated CT26-bearing BALB/c mice. C. Binding profiles of IgG Fc variants of a non-TIGIT control antibody to CHO-cells stably expressing FcRI, FcRIIB, FcRIII, or Fc RIV. Binding was assessed by flow cytometry and reported as mean fluorescence intensity (MFI). A B C D A B C Figure 4. A. Experimental model. Balb/c mice with established CT26 tumors (50-80mm 3 ) were administered i.p with a single dose of 200 μg of anti-TIGIT clone 10A7 (mIgG2a ◼), Fc-enhanced anti- TIGIT clone 10A7 (mIgG2a-DLE ◼), isotype control (mIgG2a ⚫), Fc-enhanced isotype control (mIgG2a.DLE), or anti-GITR clone DTA-1 (100 μg, mIgG2a ⚫). Tumors were analyzed on days 1, 3, 5 and 10 post treatment by flow cytometry for changes in T cell frequency. B. Frequency of intratumoral FoxP3 + Tregs. C. CD4 + non-Tregs and D. CD8 + T cells by flow cytometry pre- (t=0 hr), and post-antibody injection. N=3 mice per group per timepoint. Data representative of 3 independent experiments. Figure 2. A. Experimental model (tumor-free system). C57BL/6 mice were administered i.p. with 150 μg of SEB together with a 200 μg dose of anti-TIGIT clone 10A7 (mIgG2a), or isotype control (mIgG2a) in combination with 200 μg FcRIV blocking antibody (clone 9E9). T cells in the peripheral blood or spleen (not shown) were evaluated by flow cytometry on days 3, 6 and 10. B. CD4 + V8 + T effector cells and C. CD8 + V8 + T effector cells were evaluated on day 3 post-stimulation by flow cytometry. N=4 mice/group, and data are representative of two independent experiments. A B C Figure 1. A. BALB/c mice with established CT26 tumors (~50mm 3 ) were administered twice weekly intraperitoneally (i.p.) for two weeks with 200 μg of anti-TIGIT clone 10A7 (mIgG2a ◼ or mIgG2a-N297Q ◼) or mIgG2a isotype control (⚫). B. IL-2 production (day 4) by human PBMCs stimulated with 100 ng/ml of SEA peptide and 10 μg/ml of anti-TIGIT clone 10A7 (hIgG1 ◼ or hIgG1-N297A ◼) or corresponding isotype controls ( ⚫ and ◼, respectively). C. IL-2 production (day 4) by PBMCs stimulated with SEA peptide (100 ng/ml) and anti-TIGIT hIgG1 (10 μg/ml) with (◼) or without (◼) pre-blockade of FcRIIIA. Fc RIIIA + (V/V) Jurkat NFAT Reporter 0.01 0.1 1 10 100 1000 0 200 400 600 800 1000 Antibody [ g/ml] IL-2 [pg/ml] hIgG1.DLE hIgG1 hIgG1.DLE Anti-TIGIT Isotype Control Isotype (msIgG2a) anti-TIGIT (mIgG2a) 0 10 20 30 40 50 CD4 + V 8 Effector T cells (Day 3) % CD4 + V 8 Effector T cells Control anti-FcyRIV ** Isotype (msIgG2a) anti-TIGIT (mIgG2a) 0 10 20 30 40 CD8 + V 8 Effector T cells (Day 3) % CD8 + V 8 Effector T cells Control anti-FcyRIV ** Single co-injection 1. Staphylococcus enterotoxin B (SEB) antigen (150 μg, i.p.) 2. Anti-TIGIT or isotype control antibody (200 μg, i.p.) 3. Anti-FcRIV antibody (200 μg, i.p) Human PBMCs Author Disclosures Dhan Chand, Jeremy D. Waight, Elena Paltrinieri, Sylvia Dietrich, Mark Bushell, Mathew Costa, Randi Gombos, Jennifer S. Buell, Robert B. Stein, Alexander Duncan, David A. Savitsky: Agenus Inc.: Employment and Stock Ownership. Nicholas S. Wilson: Agenus Inc.: Former Employment and Stock Ownership. Anti-TIGIT Fc Isotype Fc mutations Characteristics Mouse mIgG2a - - mIgG2a.N297Q N297Q Reduced FcR binding (“Fc silent”) mIgG2a.DLE S241D.A332L.I334E > FcR binding (“Fc enhanced”) Human IgG1 - IgG1.N297A N297A Reduced FcR binding (“Fc silent”) IgG1.DLE S239D.A330L.I332E > FcRIIIA binding (“Fc enhanced”) FcүR Binding Characteristics of Antibody Fc Variants Used Figure 6. A. IL-2 production (day 4) by human PBMCs stimulated with a suboptimal concentration of SEA peptide together with anti-CTLA-4 variants or corresponding hIgG1 isotype controls. B. IL-2 production (day 4) by PBMCs following blockade of the indicated FcRs with FcR-specific antibodies (10 μg/ml) prior to co- incubation with SEA peptide and anti-CTLA-4 hIgG1 antibody (10 μg/ml). C. IL-2 production (day 4) by human PBMCs stimulated with SEA peptide and 10 μg/ml of anti-PD-1 antibodies or isotype control. Binding to mouse Fcү Receptors 0 5 10 15 20 25 30 35 0 1000 2000 3000 Days post implantation Tumor volume (mm 3 ) Isotype mIgG2a mIgG2a mIgG2a.N297Q Anti-TIGIT mIgG2a mIgG2a.DLE mIgG2a mIgG2a mIgG2a.DLE Anti-TIGIT Anti-GITR Isotype Control 1 2 3 4 Prevention of CD226 co-stimulation TIGIT-induced suppression of T and NK cells Enhance Treg suppressive activity Induce immunosuppressive DCs ( IL-10, TGF-) *** 1 2 3 0 2 4 6 8 10 IL-2 (fold-change) 1. Isotype control 2. Anti-TIGIT hIgG1 3. Anti-TIGIT hIgG1 + Fc RIIIA blockade *** 1 2 3 4 0 2 4 6 8 IL-2 (fold-change) 1. hIgG1 2. hIgG1-N297A 3. hIgG1 4. hIgG1-N297A Isotype control Anti-TIGIT *** *** *** ** mIgG2a.DLE mIgG2a.N297A mIgG2a
Transcript of A n t i - T I G I T ( 1 0 A 7 . m I g G 2 a . D L E ) A n …€¢ Our data describe a novel Fc...
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FcR Co-Engagement by Anti-TIGIT Monoclonal Antibodies Enhances T cell Functionality and Antitumor Immune Responses
Dhan Chand1, Jeremy D. Waight1, Elena Paltrinieri1, Sylvia Dietrich1, Mark Bushell1, Mathew Costa1, Randi Gombos1, Nicholas S. Wilson1, Jennifer S. Buell1, Robert B. Stein1, Alexander Duncan1, David A. Savitsky1
1Agenus Inc. or subsidiary thereof (current or former employee), Lexington, MA
Presented at the
American Association for CancerResearch Annual MeetingAtlanta, GA, USA • March 29– April 3, 2019
#2390
TIGIT: A Key Regulator of the Immune Synapse
CT26: Fc-dependent anti-tumor activity
Human: Fc-dependent T cell priming (in vitro)
Anti-TIGIT Requires Intact FcүR Interactions for Anti-Tumor
Activity and Improved T cell Stimulation
Fc:FcүR Co-Engagement Enhances T cell Responsiveness
References1Waight et al., Cancer Cell. 2018; 33(6):1033-1047; 2 Zhang et al., Nat Immunol. 2018; 19(7):723-732
• Our data describe a novel FcR-dependent mechanism of action that enhances the therapeutic
activity of anti-TIGIT antibodies in preclinical studies
• Murine FcRIV and human FcRIIIA are critical mediators of anti-TIGIT function
• Tumor control is dependent on enhancing CD8 T cell and NK cell function2 but not Treg depletion
• Enhanced FcRIIIa co-engagement via Fc engineering further enhances T cell responsiveness
• This novel mechanism extends to antibodies targeting CTLA-4 and could inform the optimal
design for a new class of Fc-engineered antibodies for cancer immunotherapy1.
Conclusions
Anti-TIGIT Antagonist Activity Depends on FcүRIV Engagement
to Mediate Optimal Immune Modulation In Vivo
The Importance of Fc-FcүR Co-Engagement for Improved T Cell
Responses Extends to Antibodies Targeting CTLA-4
…but not PD-1
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Blockade of FcүRsAnti-CTLA-4
Hypothesis: Optimizing Fc-FcR co-engagement enhances the activity of anti-TIGIT and anti-CTLA-4
antagonist antibodies1. TIGIT and CTLA-4 antibodies with increased binding affinities to activating Fc
receptors FcRIV (CD16-2, mouse) or FcRIIIA (CD16a, human) augment T cell priming by improving
the quality of the immune synapse between a T cell and an antigen presenting cell (APC).
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Enhanced FcүR Co-Engagement by Anti-TIGIT Antibodies
Promotes Better Tumor Control
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Anti-TIGIT Has a Novel Mechanism of Immune Modulation that
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Enhanced FcүRIIIA Engagement by Anti-TIGIT Further
Improves T Cell Responsiveness In Vitro (Human)
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Figure 5. A. IL-2 production (day 4) by human PBMCs stimulated with a suboptimal concentration of SEA
peptide together with increasing concentrations of anti-TIGIT clone 347_02_A10 (hIgG1 ⚫), Fc-enhanced anti-
TIGIT (hIgG1.DLE ⚫) or isotype control (hIgG1.DLE ). B. Jurkat cells genetically engineered to express
FcRIIIA (V/V) upstream of a NFAT-dependent luciferase reporter were co-cultured with TIGIT-expressing CHO
cells and increasing doses of anti-TIGIT or isotype control antibodies. Signaling was assessed based upon
luciferase expression shown as relative light units (RLU). C. IL-2 production (day 4) by human PBMCs
stimulated with a suboptimal concentration of SEA peptide together with 10 µg/ml of anti-TIGIT (hIgG1), Fc-
enhanced anti-TIGIT (hIgG1.DLE) or isotype control (hIgG1.DLE) alone or in combination with 10 µg/ml anti-
PD-1 (Nivolumab) or D. anti-LAG-3 (Clone 25F7)
A
B C
Figure 3. A. BALB/c mice (n=10-12/group) with established CT26 tumors (~50mm3) were administered i.p.
with 200 µg of anti-TIGIT clone 10A7 (mIgG2a ◼), Fc-enhanced anti-TIGIT clone 10A7 (mIgG2a-DLE ◼),
Fc-enhanced anti-CTLA-4 clone 9D9 (mIgG2a.DLE ◼) or isotype control () on days 9, 13 and 16. Data
representative of 3 independent experiments. B. Growth curves showing individual tumor volumes over
time of treated CT26-bearing BALB/c mice. C. Binding profiles of IgG Fc variants of a non-TIGIT control
antibody to CHO-cells stably expressing FcRI, FcRIIB, FcRIII, or FcRIV. Binding was assessed by flow
cytometry and reported as mean fluorescence intensity (MFI).
A
B C D
A
B C
Figure 4. A. Experimental model. Balb/c mice with established CT26 tumors (50-80mm3) were
administered i.p with a single dose of 200 µg of anti-TIGIT clone 10A7 (mIgG2a ◼), Fc-enhanced anti-
TIGIT clone 10A7 (mIgG2a-DLE ◼), isotype control (mIgG2a ⚫), Fc-enhanced isotype control
(mIgG2a.DLE), or anti-GITR clone DTA-1 (100 µg, mIgG2a ⚫). Tumors were analyzed on days 1, 3, 5 and
10 post treatment by flow cytometry for changes in T cell frequency. B. Frequency of intratumoral FoxP3+
Tregs. C. CD4+ non-Tregs and D. CD8+ T cells by flow cytometry pre- (t=0 hr), and post-antibody injection.
N=3 mice per group per timepoint. Data representative of 3 independent experiments.
Figure 2. A. Experimental model (tumor-free system). C57BL/6 mice were administered i.p. with 150 µg of
SEB together with a 200 µg dose of anti-TIGIT clone 10A7 (mIgG2a), or isotype control (mIgG2a) in
combination with 200 µg FcRIV blocking antibody (clone 9E9). T cells in the peripheral blood or spleen
(not shown) were evaluated by flow cytometry on days 3, 6 and 10. B. CD4+ V8+ T effector cells and C.
CD8+ V8+ T effector cells were evaluated on day 3 post-stimulation by flow cytometry. N=4 mice/group,
and data are representative of two independent experiments.
A
B C
Figure 1. A. BALB/c mice with established CT26 tumors (~50mm3) were administered twice weekly
intraperitoneally (i.p.) for two weeks with 200 µg of anti-TIGIT clone 10A7 (mIgG2a ◼ or mIgG2a-N297Q
◼) or mIgG2a isotype control (⚫). B. IL-2 production (day 4) by human PBMCs stimulated with 100 ng/ml
of SEA peptide and 10 µg/ml of anti-TIGIT clone 10A7 (hIgG1 ◼ or hIgG1-N297A ◼) or corresponding
isotype controls (⚫ and ◼, respectively). C. IL-2 production (day 4) by PBMCs stimulated with SEA peptide
(100 ng/ml) and anti-TIGIT hIgG1 (10 µg/ml) with (◼) or without (◼) pre-blockade of FcRIIIA.
FcRIIIA+ (V/V)
Jurkat NFAT Reporter
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Single co-injection
1. Staphylococcus enterotoxin B (SEB) antigen (150 µg, i.p.)
2. Anti-TIGIT or isotype control antibody (200 µg, i.p.)
3. Anti-FcRIV antibody (200 µg, i.p)
Human PBMCs
Author DisclosuresDhan Chand, Jeremy D. Waight, Elena Paltrinieri, Sylvia Dietrich, Mark Bushell, Mathew Costa, Randi Gombos, Jennifer S. Buell, Robert B. Stein, Alexander Duncan, David A. Savitsky: Agenus Inc.: Employment and Stock Ownership. Nicholas S. Wilson: Agenus Inc.: Former Employment and Stock Ownership.
Anti-TIGIT Fc Isotype Fc mutations Characteristics
Mouse
mIgG2a - -
mIgG2a.N297Q N297QReduced FcR binding
(“Fc silent”)
mIgG2a.DLE S241D.A332L.I334E> FcR binding
(“Fc enhanced”)
Human
IgG1 -
IgG1.N297A N297AReduced FcR binding
(“Fc silent”)
IgG1.DLE S239D.A330L.I332E> FcRIIIA binding
(“Fc enhanced”)
FcүR Binding Characteristics of Antibody Fc Variants Used
Figure 6. A. IL-2 production (day 4) by human PBMCs stimulated with a suboptimal concentration of SEA
peptide together with anti-CTLA-4 variants or corresponding hIgG1 isotype controls. B. IL-2 production (day 4)
by PBMCs following blockade of the indicated FcRs with FcR-specific antibodies (10 µg/ml) prior to co-
incubation with SEA peptide and anti-CTLA-4 hIgG1 antibody (10 µg/ml). C. IL-2 production (day 4) by human
PBMCs stimulated with SEA peptide and 10 µg/ml of anti-PD-1 antibodies or isotype control.
Binding to mouse Fcү Receptors0 5 1 0 1 5 2 0 2 5 3 0 3 5
0
1 0 0 0
2 0 0 0
3 0 0 0
D a y s p o s t im p la n ta t io n
Tu
mo
r v
olu
me
(m
m3
)
Is o ty p e m Ig G 2 a
m Ig G 2 a
m Ig G 2 a .N 2 9 7 Q
A n ti-T IG IT
0 2 4 6 8 1 0
0 .0
0 .3
0 .6
0 .9
1 .2
F o x P 3+
T re g s
D a y s p o s t- in je c tio n
Fo
ld-c
ha
ng
e
Fo
xP
3+
Tre
gs m Ig G 2 a
m Ig G 2 a .D L E
m Ig G 2 a
m Ig G 2 a
m Ig G 2 a .D L E
A n ti-T IG IT
A n t i-G IT R
Is o ty p e C o n tro l
1
2
3
4
Prevention of CD226 co-stimulation
TIGIT-induced suppression of
T and NK cells
Enhance Treg suppressive activity
Induce immunosuppressive DCs ( IL-10, TGF-)
* * *
1 2 3
0
2
4
6
8
1 0
IL-2
(fo
ld-c
ha
ng
e)
1 . Is o ty p e c o n tro l
2 . A n ti-T IG IT h Ig G 1
3 . A n ti-T IG IT h Ig G 1
+ F c R IIIA b lo c ka de
* * *
1 2 3 4
0
2
4
6
8
IL-2
(fo
ld-c
ha
ng
e)
1 . h Ig G 1
2 . h Ig G 1 -N 2 9 7 A
3 . h Ig G 1
4 . h Ig G 1 -N 2 9 7 A
Is o ty p e c o n tro l
A n ti-T IG IT
* * * * * *
* * *
* *
0 .1 1 1 0
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
F c R I
A n tib o d y [g /m L ]
MF
I (A
PC
)
m Ig G 2 a .D L E m Ig G 2 a .N 2 9 7 Am Ig G 2 a
