Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
MGD019, a PD-1 x CTLA-4 Tetravalent Bispecific DART® Protein, Provides Optimal Dual Checkpoint Blockade
Alexey Berezhnoy, Kurt Stahl, Kalpana Shah, Daorong Liu, Jessica Saul, Jonathan Li*, Douglas Smith*, Jill Rillema*, Sergey Gorlatov, Valentina Ciccarone, Ralph Alderson, Hua Li, James Tamura, Jennifer Brown, Bradley Sumrow, Jon Wigginton, Ezio Bonvini, Gundo Diedrich, Paul A. Moore
MacroGenics Inc., Rockville, MD and *Brisbane, CA, USA
©2019 MacroGenics, Inc. All rights reserved.Presented at the 5th International Cancer lmmunotherapy Conference, September 25–28, 2019, Paris, France
MGD019 Supports Homeostatic T-cell Proliferation and Memory T-cell Expansion in Cynomolgus Monkeys
0 100 200 300 4000
200
400
600
800
1000T-cell Proliferation in Peripheral Blood
Time (hours)
CD4+ K
i67+ C
ells
% C
hang
e fr
om B
asel
ine
Vehicle PD-1 x CTLA-4 DART (MGD019)PD-1 mAb (parental)
Group 1 Vehicle Group 2 (10 mg/kg) Group 3 (40 mg/kg) Group 4 (100 mg/kg)0
5
10
15
20
25Activated CD8 Cells
CD
25+ C
D8
Cel
ls (%
)
0
10
20
30
40
50CD8 T-cell Proliferation
Ki6
7+ CD
8 C
ells
(%)
0
20
40
60
80
ICOS Expression onCD4 T Cells
ICO
S+ CD
4 C
ells
(%)
A. B.
C.
D.
0.00
0.05
0.10
Spleen Weights
Mea
n Sp
leen
to
Brai
n W
eigh
tRa
tio
at T
erm
inal
Nec
rops
y
PD-1 x CTLA-4 DART(MGD019)
Vehicle PD-1 mAb(parental)
0
20
40
60
80 Memory
CD
28+C
D95
CD
4 C
ells
(%)
Group 1 Vehicle Group 2 (10 mg/kg)Group 3 (40 mg/kg) Group 4 (100 mg/kg)
Vehicle 100 mg/kg
CD28
CD95
A. Cynomolgus monkeys were infused IV Q1W for 3 weeks with 75 mg/kg MGD019 (3M/3F) and, in a separate study, 100 mg/kg of parental anti-PD-1 mAb (2M/2F). Ki67 expression was quantified by flow cytometry. B. Spleen weights at terminal necropsy were calculated as fraction of brain weight. C. Cynomolgus monkeys were injected weekly with the indicated amounts of MGD019. Shortly after the 4th injection, splenocytes of necropsied animals were analyzed for expression of CD25, Ki-67, and ICOS. D. Cynomolgus monkeys were injected weekly with indicated amounts of MGD019. Shortly after the 4th injection, T cells of necropsied animals were analyzed for expression of CD28 and CD95 by flow cytometry.
Phase I StudyA Phase 1, First-in-Human, Open-Label, Dose Escalation and Cohort Expansion Study of MGD019: Key Study ObjectivesPrimary Objective: ■■ Characterize safety, including tolerability, dose limiting toxicities (DLTs), and maximum tolerated dose (MTD) or maximum administered dose (MAD) if no MTD is defined, of MGD019 given intravenously to patients with advanced solid tumors
Secondary Objectives:■■ Characterize PK and immunogenicity■■ Describe preliminary evidence of antitumor activity using conventional RECIST v1.1 and immune-related RECIST
Exploratory Objectives:■■ Explore relationship between PK, pharmacodynamics, and antitumor activity; immune-regulatory effects of MGD019, including measures of immune cell activation/exhaustion in peripheral blood and/or tumor biopsy specimens; relationships among PD-1, PD-L1, and CTLA-4 expression on tumor cells and immune cell infiltrates within biopsy specimens (including CD4+ and CD8+ T cells) and antitumor activity; relationship between gene profiles and antitumor activity; relationship between tumor mutational burden and antitumor activity
Study Design
Cervical CancerN = 16
Soft Tissue SarcomaN = 16
Dose Escalation
CPI-Naïve RCCN = 16
CPI-Naïve NSCLCN = 20
Post-PD-1 NSCLCN = 20
Post-PD-1 SCCHNN = 16
Cohort Expansion
MTD/MADAll-comers Advanced Solid TumorsUp to 7 Planned Cohorts
Entry CriteriaKey Inclusion Criteria■■ Dose escalation: Patients with histologically proven, unresectable, locally advanced or metastatic solid tumors for whom no approved therapy with demonstrated clinical benefit is available or patients who are intolerant to standard therapy■■ Cohort expansion phase: Disease-specific prior therapy requirements to be specified■■ ECOG performance status of 0–1■■ Life expectancy ≥12 weeks■■ Measurable disease as per RECIST 1.1 for the purpose of response assessment must either (a) not reside in a field that has been subjected to prior radiotherapy or (b) have demonstrated clear evidence of radiographic progression since the completion of prior radiotherapy and prior to study enrollment■■ All patients must have an identified formalin-fixed, paraffin embedded tumor specimen for immunohistochemical evaluation of pharmacodynamic markers of interest■■ Acceptable laboratory parameters and adequate organ reserve
Key Exclusion Criteria■■ In patients who have previously received an immune checkpoint inhibitor, toxicities related to the CPI must have resolved to ≤ Grade 1 or baseline. Patients with well controlled immune endocrinopathies secondary to prior checkpoint therapy are eligible■■ Patients with symptomatic CNS metastases. Patients with history of CNS metastasis must have been treated, must be asymptomatic, and must not have concurrent treatment for the CNS disease, progression of CNS metastases on MRI or CT for at least 14 days after last day of prior therapy for the CNS metastases, or concurrent leptomeningeal disease or cord compression■■ Patients who experienced the following Grade 3 CPI-related AEs are ineligible: ocular AE, changes in liver function tests that met the criteria for Hy’s law, neurologic toxicity, colitis, renal toxicity, pneumonitis■■ Patients with prior therapy with a combination of monoclonal antibodies against PD-1/PD-L1 and CTLA-4 will be excluded in Cohort Expansion■■ Patients with any history of known or suspected autoimmune disease with certain exceptions■■ History of prior allogeneic bone marrow, stem cell, or solid organ transplantation■■ History of trauma, major surgical procedure, systemic antineoplastic therapy, or investigational therapy within 4 weeks and treatment with radiation therapy within 2 weeks prior to initiation of study drug administration
Conclusions■■T cells co-expressing PD-1 and CTLA-4 are more prevalent in tumors compared to healthy tissues■■MGD019 binds to and blocks its targets with increased activity on dual PD-1/CTLA-4-expressing cells■■MGD019 does not deplete peripheral blood Tregs in vitro■■ In cynomolgus monkeys, MGD019 demonstrates IgG4-like PK and an acceptable safety profile similar to that observed with PD-1 blockade alone, while demonstrating biological effects of CTLA-4 antagonism
References
1. Selby M. et al. Preclinical Development of Ipilimumab and Nivolumab Combination Immunotherapy: Mouse Tumor Models, In Vitro Functional Studies, and Cynomolgus Macaque Toxicology. PLoS One. 2016 Sep 9;11(9):e0161779.
Abstract■■Combinatorial blockade of PD-1 and CTLA-4 has shown clinical benefit beyond that observed with individual mAbs, albeit with increased toxicity. Co-expression of the checkpoint receptors creates an opportunity for selective, enhanced blockade of dual PD-1/CTLA-4 expressing cells through use of a bispecific inhibitor targeting both pathways simultaneously■■Consistent with previous reports, multiplex in-situ hybridization (ISH) demonstrated enrichment of PD-1/CTLA-4-dual positive cells in neoplastic lesions relative to normal tissues, where distinct populations expressed CTLA-4 or PD-1. PD-1 x CTLA-4 DART proteins were designed as either bivalent or tetravalent bispecific engagers with a human IgG4 backbone; in vitro characterization revealed that both 1x1 and 2x2 bispecific PD-1 x CTLA-4 formats block each checkpoint molecule, with enhanced blockade of CTLA-4 over that achieved with anti-CTLA-4 mAb on dual expressing cells. As expected, 1x1 format molecule was less efficient in blocking PD-1 in cell models lacking CTLA-4 expression, while 2x2 molecule demonstrated full PD-1 blockade. Likewise, the 2x2 format molecule effectively blocks CTLA-4 ligand interactions on PD-1negCTLA-4pos cells, while the 1x1 design is significantly less efficient. The tetravalent bispecific molecule was designated as MGD019 and selected for clinical development■■ In primary cell assays in which both PD-1 and CTLA-4 contribute to inhibit T-cell activation, MGD019 enhances antigen-driven in vitro T-cell activation to a level comparable to the combinatorial PD-1 plus CTLA-4 blockade. Tumor microenvironment models that recapitulate vascular or stromal compartments confirmed MGD019 induces in vitro immune response profiles comparable to those observed with replicas of ipilimumab plus nivolumab. Unlike ipilimumab, however, MGD019 does not reduce the number of peripheral blood Tregs ex vivo■■MGD019 was well tolerated in cynomolgus monkeys, with no mortality or significant adverse findings up to 100 mg/kg QWx4. T-cell proliferation in the periphery and expansion in lymphoid organs was observed, with increases in ICOS+ CD4 cells and memory T cells, findings attributable to the CTLA-4 blocking arm, since the anti-PD-1 mAb precursor was devoid of these activities ■■ In summary, MGD019 offers the convenience of a single molecule administration for dual checkpoint blockade. In addition to providing full blockade on cells expressing PD-1 or CTLA-4 individually, MGD019 exploits dual target avidity resulting in preferential engagement and enhanced blockade on cells that express both checkpoint molecules, a feature that could provide additional benefits given the preeminent co-expression of PD-1 and CTLA-4 by TILs. These data indicate support clinical testing of MGD019 in cancer patients
Introduction
T Cells Co-expressing PD-1 and CTLA-4 Are Prevalent Among TILs Compared to Healthy Tissues
Ovarian CancerNormal Tonsil
Expression of PD-1 (red) and CTLA-4 (blue) mRNA in tissue samples of normal human tonsils (left) or ovarian cancer (right) visualized by RNAscope™.
Rationale and Format Selection for Dual PD-1 and CTLA-4 Targeting
2x2
1x1
10-6 10-4 10-2 100 102 10-6 10-4 10-2 100 102 10-6 10-4 10-2 100 1020
500
1000
1500PD-1+ Cells
Bind
ing
(ahF
c, M
FI)
0
2000
4000
PD-1+ CTLA-4+ Cells
Control IgG PD-1 x CTLA-4 2x2PD-1 x CTLA-4 1 x1
0
50
100
150
200PD-1 Blockade
Liga
nd B
indi
ng (M
FI)
0
100
200
300
400CTLA-4 blockade
0
500
1000
1500
2000CTLA-4 Blockade on
PD-1+ CTLA-4+
0
200
400
600
800
1000CTLA-4+ Cells
0
50000
100000
150000
200000
250000PD-1 Reporter
T-ce
ll Si
gnal
ing
(RLU
)
6000
8000
10000
12000
CTLA-4 Reporter
0
500
1000
1500
2000
2500PD-1+ CTLA-4+ Reporter
Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL)
10-6 10-4 10-2 100 102 10-6 10-4 10-2 100 102 10-6 10-4 10-2 100 102
Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL)
10-6 10-4 10-2 100 102 10-6 10-4 10-2 100 102 10-6 10-4 10-2 100 102 104
Concentration (µg/mL) Concentration (µg/mL) Concentration (µg/mL)
A. B. C.
D. E. F.
G. H. I.
2x2 and 1x1 DART molecules were evaluated for binding to (A, B, C), ligand blockade (D, E, F) and restoration of T-cell signaling (G, H, I) in PD-1 only, CTLA-4 only or PD-1+CTLA-4 models by flow cytometry and reporter gene activation.
PD-1+ CTLA-4 Combo:PD-1xCTLA-4 2x2:
TumorHealthy TissuesCirculation
++++ +++++++ +
PD-1xCTLA-4 1x1: +++ -
T CellPD-1
CTLA-4T Cell
CTLA-4T Cell
PD-1PD-1
Rationale for PD-1xCTLA-4 Dual Checkpoint Targeting Strategy
Results
MGD019, a Tetravalent Bispecific Fc DART Molecule
MGD019 Molecular Structure
2x2 Fc DART
CTLA-4
PD-1
CTLA-4
PD-1
IgG4
10-8 10-6 10-4 10-2 100 1020.0
1.0
2.0
3.0
4.0
5.0
PD-1/CTLA-4 Dimerization
Concentration (nM)
Rela
tive
Dim
eriz
atio
n A
ctiv
ity
Control IgGCTLA-4 mAb (parental)PD-1 mAb (parental) PD-1 + CTLA-4 mAbs combo
PD-1 x CTLA-4 Fc DART (MGD019)
0.0001 0.01 1 100 0.0001 0.01 1 1000
1000
2000
3000
4000
Binding to PD-1+ Cells
Concentration (nM)
MFI
0
2000
4000
6000
8000
Binding to CTLA-4+ Cells
Concentration (nM)
MFI
0.0001 0.01 1 1000
10000
20000
30000
Binding toPD-1+/CTLA-4+ Primary T Cells
Concentration (µg/mL)
MFI
A.
C. D.
B.MGD019 Co-engages PD-1 and CTLA-4
Binding of MGD019 or control molecules to Jurkat/PD-1 (A) or Jurkat/CTLA-4 (B) cells measured by flow cytometry. C. Binding of MGD019 or control molecules to in vitro activated primary human T cells by flow cytometry. D. Co-engagement of PD-1 and CTLA-4 on the cell surface by MGD019 measured by DiscoverX™ enzyme complementation assay.
MGD019 Does Not Affect the Circulating Treg Population
104
105
103
0
-103 0 103 104 105
FoxP3
CD4
104
105
103
0
-103 0 103 104 105
FoxP3
CD4
Control mAb Ipilimumab Replica
104
105
103
0
-103 0 103 104 105
FoxP3
CD4
104
105
103
0
-103 0 103 104 105
FoxP3
CD4
Ipilimumab IgG4 MGD0195
10
15Donor A
FoxP
3+ Cel
ls(%
of C
D4+ T
Cel
ls)
FoxP
3+ Cel
ls(%
of C
D4+ T
Cel
ls)
10
Donor B
Peripheral blood mononuclear cells from healthy donors were treated in vitro with 1 µg of test articles under suboptimal CD3 stimulation. Fraction of FoxP3+ cells was measured by intracellular flow cytometry at 48 hours.
hIgG Control Ipilimumab replica (IgG4)Ipilimumab replica (IgG1) MGD019 (IgG4)
Enhanced Blockade of CTLA-4 by MGD019 on Dual Expressing Cells
0
5000
10000
15000
20000PD-1 Blockade on PD-1+ CTLA-4- Cells
mAbs or DARTs (nM)PD
-L1
Bind
ing
(MFI
)0
1000
2000
3000
4000CTLA-4 Blockade on CTLA-4+ PD-1- Cells
mAbs or DARTs (nM)
B7-1
Bin
ding
(MFI
)
0
200
400
600
800
1000
1200PD-1 Blockade on PD-1+ CTLA-4+ Cells
PD-L
1 Bi
ndin
g (M
FI)
0
200
400
600
800
1000
1200
1400CTLA-4 Blockade on PD-1+ CTLA-4+ Cells
B7-1
Bin
ding
(MFI
)
10-3 10-2 10-1 100 101 10310210-8 10-6 10-4 10-2 100 104102
mAbs or DARTs (nM) mAbs or DARTs (nM)10-4 10-3 10-2 10-1 100 102101
10-6 10-4 10-2 102100
10-4 10-3 10-2 10-1 100 102101
Control IgG CTLA-4 mAb (parental)PD-1 mAb (parental) PD-1 + CTLA-4 mAbs combo PD-1 x CTLA-4 DART (MGD019)
Anchoring via PD-1 Contributes to anti-CTLA-4-mediated Blockade of B7 Binding
0
200
400
600
800CTLA-4 Blockade on PD-1+ CTLA-4+ Cells
Concentration (µg/mL)
B7-1
Bin
ding
(MFI
)
Control IgGCTLA-4 mAbPD-1 + CTLA-4 mAbs combo
PD-1 x CTLA-4 DARTPD-1 x CTLA-4 DART + PD-1 mAb
PD-1 or CTLA-4 binding to their respective soluble ligands on Jurkat cells engineered to express PD-1 and/or CTLA-4 was measured by flow cytometry.
MGD019 Enhances T-cell ActivationA. B.
0 0.0001 0.003 0.06 1.25 25 5000
500
1000
1500
2000
2500
SEB Concentration (ng/mL)
IL-2
pg/
mL
Activation of Primary T Cells
0
2000
4000
6000
8000
10000
Dual PD-1/CTLA-4 Reporter Assay
mAbs or DARTs (nM)
Lum
inec
ence
10-2 10-1 100 101 102 103
Control IgGCTLA-4 mAb (parental)PD-1 mAb (parental) PD-1 + CTLA-4 mAbs combo
PD-1 x CTLA-4 DART (MGD019)
A. PBMC were activated by SEB in the presence of 10 µg/mL of the indicated molecules. IL-2 secretion was measured at 96h by ELISA. B. Expression of IL-2 reporter cassette in Jurkat cells co-expressing PD-1 and CTLA-4 (Promega) co-incubated with APC in the presence of the indicated molecules.
Characterization of MGD019-mediated Enhancement of T-cell Activation
Tumor Microenvironment Models Transcriptional Changes inAllo-activated T Cells
MGD01946 genes
upregulated
Ipilimumab + nivolumab36 genes
upregulated
-0.5-0.4-0.3-0.2-0.10.00.10.20.30.40.50.60.7
Log
Rati
o
Ipilimumab* + nivolumab* (20 nM) Ipilimumab + nivolumab (6.7 nM)Ipilimumab + nivolumab (2.2 nM) Ipilimumab + nivolumab (0.74 nM)
-0.5-0.4-0.3-0.2-0.10.00.10.20.30.40.50.60.7
CCL2
/MCP
1CD
106/
VCAM
1CD
40CD
69uP
ARCE
ACAM
5/CD
66e
Colla
gen
IVCX
CL10
/IP1
0CX
CL9/
MIG
Kera
tin 2
0PB
MC
Cyto
toxi
city
Gra
nzym
e B
IL10
IL17
AIL
2IL
6SR
B
Log
Rati
o
MGD019 (20 nM) MGD019 (6.7 nM)MGD019 (2.2 nM) MGD019 (0.74 nM)
CD10
6/VC
AM1
CD87
/uPA
RCE
ACAM
5/CD
66e
Colla
gen
ICo
llage
n III
CXCL
10/I
P10
Kera
tin 2
0M
MP9
PAII
PBM
C Cy
toto
xici
tyG
ranz
yme
BIF
Nγ
IFN
γ
IL10
IL17
AIL
2IL
6SR
BTN
Fα
TNFα
sVEG
FTI
MP2 tPA
uPA
StroHT29 VascHT29
Ipilimumab + Nivolumab
MGD019
23 genes 23 genes
IL-10 IL-213 genes
IL-10
Transcriptional changes in MLR assay: T cells were re-isolated from 72h MLR assay after treatment with 1 µg/mL each ipilimumab and nivolumab or MGD019 at 1 µg/mL, and RNAseq was performed using Illumina HiSeq platform. Gene alignment, filtering for 750 immune- related genes and differential expression analysis were done using Strand NGS software. Genes were clustered based on upregulation in both donors.
DiscoverX StroHT29 and VascHT29 TME models: HT-29 colorectal cells were cultured with stromal cells and PBMCs to recapitulate a tumor microenviroment (DiscoverX). Protein expression was measured following treatment with mAbs or MGD019. *Replicas of nivolumab and ipilimumab were constructed on the basis of published sequences.
MGD019 GLP Toxicology Study
FindingPD-1xCTLA-4 (MGD019) aPD-1
(parental)
10 mg/kg 40 mg/kg 100 mg/kg ≥100 mg/kgAdverse clinical signs - - - -Body weight loss - - - -Increased spleen weight + ++ ++ -Lymphoid hyperplasia in spleen - + ++ -GI tract inflammation - - - -Circulating cytokines - - - -T-cell proliferation (Ki67+) + ++ ++ +/-
■■MGD019 was well-tolerated compared to prior reported PD-1+CTLA-4 mAb combination1
MGD019 Pharmacokinetics and Receptor Occupancy
0 7 14 21 28 35 42 49 56 63 70 77 84 91100
101
102
103
104
105
106
107
Study Day
MG
D01
9 (n
g/m
L)
MGD019 Serum Concentration
100 mg/kg/dose40 mg/kg/dose10 mg/kg/dose0 mg/kg/dose
0 7 14 21 28 35 42 49 56 63 70 77 84 910
10
20
30
40
50
60
70
80
90
100
110
120
Study Day
Bind
ing
to C
D4+ /
PD-1
+ Cel
ls (%
)
Receptor Occupancy
Cynomolgus monkeys (3F/3M) were infused with 10, 40 or 100 mg/kg/dose MGD019 at Day 1, 8, 15, and 22. Serum concentration was measured by ELISA (right) and receptor occupancy was measured by flow cytometry (left).
Abstract B027