margetuximab mediates greater fc-dependent anti-tumor ......©2019 macrogenics, inc. all rights...
TRANSCRIPT
©2019 MacroGenics, Inc. All rights reserved.
Margetuximab Mediates Greater Fc-dependent Anti-tumor Activities than Trastuzumab or Pertuzumab In VitroLiqin Liu1, Yinhua Yang1, Robert Burns1, Jonathan Li2, Haiquan Li1, Sergey Gorlatov1, Paul Moore1, Jeffrey Nordstrom1
MacroGenics, Inc., Rockville, MD and Brisbane, CA
Presented at the American Association for Cancer Research Annual Meeting 2019, March 29–April 3, 2019, Atlanta, GA
http://ir.macrogenics.com/events.cfm
1538
AbstractIntroduction: Margetuximab (M) is an investigational Fc-engineered anti-HER2 monoclonal antibody (mAb) with potential for greater immune-mediated anti-tumor activity than trastuzumab (T). M and T bind the same subdomain IV epitope, but M binds with higher affinity to activating Fc receptor, CD16A (FcgRIIIA), and lower affinity to inhibitory Fc receptor, CD32B (FcgRIIB). Pertuzumab (P) binds to a subdomain II epitope and has the same wild type Fc domain as T.Purpose: We compared in vitro properties of M, T and P and combinations of M+P and T+P to evaluate Fc domain contributions to anti-HER2 mAb mediated activities. HER2 binding: By surface plasmon resonance, M, T and P bound to the HER2 extracellular domain with high affinity. Binding affinities of M and T were unchanged if P was pre-bound, indicating lack of interaction. M, T and P exhibited comparable binding to HER2-expressing cell lines.Inhibition of HER2 ECD shedding: M and T mediated comparable inhibition of HER2 ECD shedding, whereas P was ineffective.Inhibition of proliferation: Anti-proliferative activities of M and T toward HER23+ cells (N87, SKBR3 or BT474) in the absence or presence of ligands (EGF or HRG1b) were similar, whereas P exhibited weaker activity. In the absence of ligands, M+P or T+P were comparably as active as M or T alone. In presence of ligands, M+P and T+P were generally more active than M or T alone. ADCC: Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with N87 and SKBR3 target cells (HER23+, expressing a reporter gene to measure viability) and NK effector cells from donors with differing CD16A158 genotypes. M was 7- to 84-fold more potent than T, as well as 69- to 744-fold more potent than P. Greater differences generally were seen with effector NK cells bearing the more common CD16A158 FF and VF genotypes than with the less common, high affinity CD16A158 VV genotype. While addition of P enhanced mean potency of T by 2- to 3-fold, no such effect was seen with M; however, M or M+P was 7- to 25-fold more potent than T+P. Results with JIMT-1 cells (HER22+) were similar, albeit with lower differential than with HER23+ target cells. NK cell activation: NK cells were monitored by flow cytometry following incubation of peripheral blood mononuclear cells with HER2+ target cells and mAbs. M induced greater expression of markers of activation (CD137), cytolytic capability (granzyme B, perforin) and proliferation (Ki67) than T. An anti-HER2 mAb with an inactivated Fc domain was ineffective, indicating that induced changes in NK cells were Fc-dependent.Conclusion: M and T bind HER2 with high affinity and exhibit anti-proliferative activity that is enhanced by addition of P if ligands (EGF or HRG1b) are present. Relative to T, M mediates superior ADCC with effector cells of all CD16A genotypes and promotes greater NK cell activation and expansion. The M+P combination maintains superior ADCC compared to the T+P combination.
Binding Properties
Margetuximab Has Increased Affinity for Both Allotypes of CD16A (Activating FcgR) and Decreased Affinity for CD32B (Inhibitory FcgR)
Antigen Antibody ka (1/M•s) kd (1/s) KD (nM) M/T Affinity Ratio (1/KD)
CD16A 158V1 MT
4.7 x 105
4.3 x 10527 x 10-3
105 x 10-357
244 4.3
CD16A 158F1 MT
5.8 x 105
2.8 x 10561 x 10-3
155 x 10-3105554 5.3
CD32B2 MT
NDND
NDND
8719 0.2
ka = association rate constant, kd = dissociation rate constant, KD = equilibrium dissociation constant, M = molar, s = second, ND = not determined1Binding of monomeric of human CD16A ECD to antibody captured on immobilized HER2-His2Binding of dimeric human CD32B ECD/aglycosyl Fc fusion to antibody captured on immobilized HER2-His
Comparable Binding to HER2 by Margetuximab (M) and Trastuzumab (T)■■Margetuximab or trastuzumab binding to HER2 is unaffected by prior pertuzumab (P) binding
Antigen Antibody ka (1/M•s) kd (1/s) KD (nM)
HER21
M 3.2 x 105 10.0 x 10-4 3.1M (+ P)2 3.5 x 105 10.0 x 10-4 2.9
T 3.7 x 105 9.5 x 10-4 2.6T (+ P)2 4.2 x 105 11.0 x 10-4 2.6
P 2.5 x 105 1.6 x 10-4 0.6ka = association rate constant, kd = dissociation rate constant, KD = equilibrium dissociation constant, M = molar, s = second1Binding of antibodies to human HER2 (ECD)-His protein captured on anti-penta-His surface2Binding of M or T measured after P was preloaded
Comparable Binding to SKBR-3 (HER23+) Cells by Margetuximab (M), Trastuzumab (T) and Pertuzumab (P)■■ Margetuximab or trastuzumab binding to HER2+ cells is unaffected by pertuzumab
10-410-310-210-1100 101 102 103 104 10-410-310-210-1100 101 102 103 1040
10000
20000
30000SKBR-3 Cells
Concentration (ng/mL) Concentration (ng/mL)
MFI
MTPControl
0
10000
20000
30000
MFI
SKBR-3 Cells ± PM
TM (+ P)
T (+ P)
Binding of Alexa-Fluor-488-labeled M, T or P. Binding of Alexa-Fluor-488-labeled M or T in the absence or presence of unlabeled P.
Fc-independent Activities
Comparable Inhibition of HER2 Extracellular Domain (ECD) Shedding by Margetuximab and Trastuzumab
100 101 102 103 1040
20
40
60
80
100
120SKBR-3
Concentration (ng/mL)
HER
2 EC
D in
Gro
wth
Med
ium
(% n
o tr
eatm
ent)
M
T
P
M (Fc-null)
Neg Ctrl
■■M, T and Fc-null (aglycosylated) variant of M block HER2 ECD shedding■■Pertuzumab (P) does not block HER2 ECD shedding
Anti-proliferative Activities of Margetuximab and Trastuzumab Are Improved by Combination with Pertuzumab
Neg Ctrl M T P M+P T+P
No Added Ligand
20
40
60
80
100
BT474
Concentration (µg/mL)
% M
ediu
m C
ontr
ol
Emax (M+P)
Emax (M)
50
60
70
80
90
100
SKBR-3
Concentration (µg/mL)
% M
ediu
m C
ontr
ol
Emax (M+P)
Emax (M)
10-3 10-2 10-1 100 101 10210-3 10-2 10-1 100 101 102
EGF (Ligand for EGFR)
20
40
60
80
100
BT474
Emax (M)
Emax (M+P)
70
80
90
100SKBR-3
Emax (M)
Emax (M+P)
Concentration (µg/mL)10-3 10-2 10-1 100 101 102
Concentration (µg/mL)10-3 10-2 10-1 100 101 102
% M
ediu
m C
ontr
ol
% M
ediu
m C
ontr
ol
Heregulin 1b (Ligand for HER3/4)
70
80
90
100
BT474
% M
ediu
m C
ontr
ol
70
80
90
100
SKBR3
% M
ediu
m C
ontr
ol
Emax (M)
Emax (M+P)
Concentration (µg/mL)10-3 10-2 10-1 100 101 102
Emax (M)
Emax (M+P)
Concentration (µg/mL)10-3 10-2 10-1 100 101 102
Dashed lines indicate the maximal level of inhibition of proliferation (Emax) by M alone or M+P combined.
Fc-dependent Activities
Margetuximab (M) Mediates ADCC with Greater Potency than Trastuzumab (T) or Pertuzumab (P)ADCC of SKBR-3/GF Cells by NK Cells of Different CD16A158 Genotypes (E:T = 2:1)■■SKBR-3/GF cells constitutively express luciferase, which allows specific measurement of target cell viability
0
20
40
60
80
100
CD16A158 FF
CD16A158 VF
CD16A158 VV
Concentration (ng/mL)
Targ
et C
ell V
iabi
lity
(% o
f Con
trol
)
0
20
40
60
80
100
Targ
et C
ell V
iabi
lity
(% o
f Con
trol
)
0
20
40
60
80
100
Targ
et C
ell V
iabi
lity
(% o
f Con
trol
)
MTPM+PT+PNeg Ctrl
10-310-4 10-2 10-1 100 101 102
Concentration (ng/mL)10-310-4 10-2 10-1 100 101 102
Concentration (ng/mL)10-310-4 10-2 10-1 100 101 102
ADCC EC50 Values Obtained with NK Cells of Different CD16A Genotypes■■For each NK cell donor:
– M is always more potent (lower EC50) than T, which is always more potent than P – Combination of M + P is always more potent than combination of T + P
■■Similar results obtained with N87/GF and JIMT-1/GF target cells
M T P M+P T+P0.0001
0.001
0.01
0.1
1
ng/m
L
CD16A158 FFCD16A158 VFCD16A158 VV
Black bar = mean EC50 value.
Margetuximab (M) Induces Greater Activation and Proliferation of NK Cells than Trastuzumab (T)
Induction of CD137
M (Fc-null)
T
M
0 2 4 6 80246
10
20
30
40CD137
Day
% o
f NK
Cells
MTM (Fc-null)
PBMCs and HER2(+) N87 cells incubated with anti-HER2 Abs
0.07 32 2.7 3.1 4.4 11.4
9.62.92.01.8
0.8 1.1 3.1 6.50.3
15
105
104
103
0
103 104 1050
105
104
103
105
104
103
0
103 104 1050
105
104
103
0
103 104 1050
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
105
104
103
0
103 1040
CD56CD13
7
Day=1 Day=2 Day=3 Day=4 Day=7Day=0
105
104
103
0
103 104 1050
105
104
103
0
103 104 1050
Induction of Ki67
0
20
40
60
80Ki67
0 2 4 6 8Day
% o
f NK
Cells
MTM (Fc-null)
3.73 24.57.09 51.3 68.9 67.8
3.04 15.2 27.3 39.2 40.0
3.92 3.77 2.40 3.00 8.03
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
105
104
103
-103
0
-103 103 104 1050
CD56
Ki67
Day=1 Day=2 Day=3 Day=4 Day=7Day=0
M (Fc-null)
T
M
Induction of Granzyme B
0
20
40
60
80Granzyme B
0 2 4 6 8Day
% o
f NK
Cells
MTM (Fc-null)
48.9023.40 56.30 58.00 75.10
36.30 40.40 34.10 48.10
35.4021.80 24.00 19.00
73.30
44.10
8.430
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
105
104
103
-102
102
0
-103 103 104 1050
CD56
GB
Day=1 Day=2 Day=3 Day=4 Day=7Day=0
M (Fc-null)
T
M
Induction of Perforin
42.20.04 49.7 23.8 67.7
25.5 7.58 30.7 37.432.2
0.97 4.41 19.2 11.720.5
M (Fc-null)
T
M60.7
Perforin
Day
% o
f NK
Cells
MTM (Fc-null)
0
20
40
60
80
0 2 4 6 8
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
105
104
103
0
-103 103 104 1050
CD56
PF
Day=1 Day=2 Day=3 Day=4 Day=7Day=0
Conclusions
Binding PropertiesCD16A (activating FcgR) M > TCD32B (inhibitory FcgR) M < THER2 (in absence or presence of P) M ≈ T
Fc-independent ActivitiesHER2 ECD shedding inhibition M ≈ T
Anti-proliferation (in absence or presence of EGF or heregulin)M ≈ T
M+P ≈ T+P
Fc-dependent Activities ADCC (with effector cells of all CD16A158 genotypes)M > T > P
M+P > T+PNK cell activation and expansion M > T
■■Margetuximab mediates ADCC in vitro with greater potency than trastuzumab and promotes greater NK cell activation and expansion ■■Combination of margetuximab + pertuzumab mediates ADCC in vitro with greater potency than combination of trastuzumab + pertuzumab