preclinical characterization of mgd013, a pd-1 x lag-3 bispecific … · 2019. 9. 24. · pd-1- and...

Preclinical Characterization of MGD013, a PD-1 x LAG-3 Bispecific DART® Molecule

Abstract Results

Introduction

Presented at the Society for Immunotherapy of Cancer (SITC) 32nd Annual Meeting, November 8-12, 2017, National Harbor, Maryland © 2017 MacroGenics, Inc. All rights reserved.

Ross La Motte-Mohs, Kalpana Shah, Jennifer G. Brown, Doug Smith, Sergey Gorlatov, Valentina Ciccarone, James K. Tamura, Hua Li, Jill R. Rillema, Monica Licea, Christine Shoemaker, Leilei He, Farha Vasanwala, Jessica Hill, Arin Whiddon, Massimiliano Pascuccio, Shereen Saini, Francine Z. Chen, Anushka De Costa, Ann Easton,

Peter Lung, Jonathan Li, Kurt Stahl, Jeffrey Nordstrom, Scott Koenig, Ezio Bonvini, Syd Johnson and Paul A. Moore MacroGenics, Inc., Rockville, MD and South San Francisco, CA, USA

Conclusions

http://ir.macrogenics.com/events.cfm

NCT03219268

MGD013 Binds PD-1 and LAG-3 and Blocks Ligand Interactions

Structure of MGD013

Expression of Checkpoint(s) Across All TMAs* Overlapping Expression**

Tumor MicroArray (TMA) Spot Count for Particular Indication

LAG-3* Total

PD-1+ Total

LAG-3+PD-1+ Total

LAG-3+ PD-1+

PD-1+ LAG-3+

Lung Squamous Cell Carcinoma 18/36 50%

15/36 42%

10/36 28%

10/15 67%

10/18 56%

Lung Adenocarcinoma 19/33 58%

18/33 55%

15/33 46%

15/18 83%

15/19 79%

Triple Negative Breast Cancer 16/29 55%

12/29 41%

10/29 35%

10/12 83%

10/16 63%

*Spot Counts indicate the number of TMA positive for LAG-3, PD-1, or LAG-3 + PD-1 expression divided by the total TMAs**Spot Counts indicate the number of TMA with overlapping checkpoint expression of PD-1 and LAG-3

10-3 10-2 10-1 100 101 102

2000

4000

6000

8000

10000

Binding to PD-1+ NS0 Cells

Concentration (nM, LOG)

MFI

(APC

)

10-3 10-2 10-1 100 101 1021000

2000

3000

4000Binding to LAG-3+ NS0 Cells

Concentration (nM, LOG)

MFI

(APC

)

PD-1 and LAG-3 are Expressed on TILs

10-3 10-2 10-1 100 101 1020

5000

10000

15000

20000

PD-L1 Binding Blockade

Concentration (nM, LOG)

MFI

10-4 10-3 10-2 10-1 100 101 1020

1000

2000

3000

LAG-3 Binding Blockade

Concentration (nM, LOG)

MFI

MGD013 Enhances Antigen-driven T-cell Cytokine Function In Vitro

MGD013 Co-engages PD-1 & LAG-3

� MGA012 + MG anti-LAG-3 combination comparable to benchmark antibody combination

� MGD013 enhanced IFN-γ secretion beyond that observed with antibody combinations

MGD013 Disrupts PD1- & LAG-3- Mediated T-cell Inhibitory Signaling

MGD013 demonstrates a dose dependent blockade of the PD-1/PD-L1 axis comparable to anti-PD-1 mAbs as evaluated in PD-1 reporter models obtained from DiscoverX’s PathHunter® Enzyme Fragment Complementation Assay to inhibit SHP-2 activation (A) or Promega’s PD-1/PD-L1 Blockade Biosassay to release NF-AT blockade (B). Similarly, MGD013 demonstrates a dose dependent blockade of the LAG-3/MHC-class II axis comparable to a replica of BMS’s 25F7 [anti-LAG-3 mAb] evaluated in Promega’s LAG-3/MHC-class II Blockade Bioassay to release NF-AT blockade (C).

10-4 10-3 10-2 10-1 100 1010

20

40

60

80

100

Concentration (nM, LOG)

%N

oTr

eatm

entC

trl

MGA012Negative Control

MGD013

PD-L1-induced SHP-2 Activation

10-3 10-2 10-1 100 101 1020

10000

20000

30000

40000

PD-1 Jurkat + PD-L1 CHO

Concentration (nM, LOG)

RU

LLu

min

esce

nce

25F7*Nivolumab*MGD013

10-2 100 102100000

120000

140000

160000

180000LAG-3 Jurkat + Raji (SED Stimulated)

Concentration (nM, LOG)

RU

LLu

min

esce

nce MGA012

25F7*

MGD013

A Inhibition of SHP-2 Activation PD-1 Signaling LAG-3 SignalingB C“Brakes on” “Brakes released”

SED (50ng/ml)

Raji

CD4lo TCR

HLA-DR

Raji

JurkatReporter Line

NFAT luc2NFAT luc2

CD4lo TCR

HLA-DR

LAG-3

MGD013or

αLAG-3mAb

MGD013 Enhances Immune Responses in TME Models Compares favorably to PD-1 + LAG-3 mAb combination

MGD013, MGA012 (PD-1 mAb), MG’s LAG-3 mAb were evaluated and compared against the combination of nivolumab* + 25F7* for their ability to capture immune activation under the mimicry of the tumor microenvironment (TME). HT-29 colorectal cells were cultured with fibroblasts and PBMCs to recapitulate a stromal microenviroment (left panel) or with endothelial cells and PBMCs to recapitulate a vascular microenvironment (right panel). Immune profiling of checkpoint targets including adhesion molecules, cytotoxic granules, and cytokines were measured (DiscoverX).

0 50 100 150 200 250 300 350 400 450

Control IgG

25F7*

MG anti-LAG-3

Nivolumab*

MGA012

Nivolumab* + 25F7*

MGA012 + MG anti-LAG-3

MGD013

Relative IFN-γ Induction(% of 25 nM MGA012, mean± SEM)

0.006 nM0.024 nM0.09 nM0.39 nM1.56 nM6.25 nM25 nM

***

IFN-γ release by 25 nM MGA012 = 3276 ± 744 pg/mL

Ratio-paired t-test(25 nM group):

* p < 0.0262** p < 0.0022

NS = not significant

NS+PD-1 LAG-3

orPD-1

LAG-3

MGA012 MG anti-LAG-3 MGD013 Nivo+25F7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

Log

Rati

o

CD10

6/VC

AM1

CD87

/uPA

R

CEAC

AM5/

CD66

e

Colla

gen

l

Colla

gen

lll

CXCL

10/I

P10

Kera

tin

20

MM

P9 PAll

PBM

C Cy

toto

xici

ty

Gra

nzym

e B

IFN

γ

IL10

IL17

A

IL2

IL6

SRB

TNFα

sVEG

F

TIM

P2 tPA

uPA

CCL2

/MCP

1

CD10

6/VC

AM1

CD40

CD69

uPAR

CEAC

AM5/

CD66

e

Colla

gen

I V

CXCL

10/I

P10

CXCL

9/M

IG

Kera

tin

20

PBM

C Cy

toto

xici

ty

Gra

nzym

e B

IFN

γ

IL10

IL17

A

IL2

IL6

SRB

TNFα

Granzyme BIFN IL2 IL6IL17A

sVEGF

tPA Granzyme B

IFN

IL2IL6

IL10 IL17ATNF

StroHT29 VascHT29

Nivolumab* (anti-PD-1)MGD013

25F7 (anti-LAG-3)*

Maximal Binding of antigenBackground

* Replicas of nivolumab and 25F7 mAbs produced at MacroGenics based on published sequences

� MGD013 was engineered as a tetravalent bispecific DART molecule in a human hinge-stabilized IgG4 backbone.

� MGD013 is capable of simultaneously binding PD-1 and LAG-3.

� MGD013 blocks PD-1/PD-L1/PD-L2 and LAG-3/MHC-Class II interactions and resultant inhibitory signal with potency comparable to MGA012 (anti-PD-1), and replicas of nivolumab or 25F7 (anti-LAG-3).

� MGD013 enhances T-cell responses compared to individual mAbs or combination mAb blockade.

� MGD013 was well-tolerated and demonstrates favorable pharmacokinetics in cynomolgus monkeys.

Clinical testing of MGD013 in several cancer indications is ongoing [NCT03219268].

� PD-1 and LAG-3 are two coinhibitory molecules that deliver negative signals upon interaction with ligands expressed on tumor cells and/or antigen presenting cells (PD-L1, PD-L2, or MHC-II).

Background: Monoclonal antibodies (mAbs) that target the immune checkpoints, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1), have shown enhanced clinical antitumor activity when given in combination, triggering interest in determining whether additional checkpoint inhibitor combinations may afford enhanced clinical benefit. Lymphocyte-activation gene 3 (LAG-3) is another immune checkpoint expressed on activated T cells and tumor infiltrating lymphocytes (TILs). Recognizing the therapeutic potential of dual checkpoint blockade, we have engineered MGD013, a IgG4κ bispecific DART molecule, to bind PD-1 and LAG-3 concomitantly or independently and disrupt these nonredundant inhibitory pathways to further restore exhausted T-cell function.

Methods: Proprietary PD-1 and LAG-3 mAbs were generated and selected based on binding characteristics, biophysical properties, the ability to block their respective receptor/ligand axes and to synergize in T-cell stimulation assays. Humanized sequences were incorporated into a tetravalent bispecific DART format and benchmarked against combinations of replicas of the approved PD-1 mAb (nivolumab) and BMS-986016 anti-LAG-3 mAb (25F7), which is currently under clinical evaluation. MGD013 biological activity was evaluated in various primary cell-based immune assays. Safety was assessed in cynomolgus monkey toxicology studies performed at MPI (Mattawan, MI) under Institutional Animal Care and Use Committee-approved protocols.

Results: MGD013 bound with high affinity to human and cynomolgus monkey PD-1- and LAG-3-expressing cells and blocked PD-1/PD-L1, PD-1/PD-L2 and LAG-3/HLA (MHC-II) interactions, with resultant signaling blockade. Functional characterization revealed enhanced cytokine secretion in response to antigen stimulation that was greater than that of the combination of individual equimolar amounts of PD-1 and LAG-3 mAbs. MGD013 was well tolerated in a repeated-dose (Q1Wx4) cynomolgus monkey toxicology study. Except for the occurrence of watery feces in a few animals, no MGD013-related adverse findings were noted, including hematological or clinical chemistry changes, serum cytokine levels or gross and microscopic histological findings, establishing a no-observed-adverse-effect level (NOAEL) of 100 mg/kg.

Conclusion: MGD013 is a bispecific DART molecule capable of simultaneously blocking the PD-1 and LAG-3 pathways, resulting in enhanced T-cell activation compared to single or combination mAb blockade. MGD013 has demonstrated a favorable preclinical safety and toxicological profile and is currently initiating clinical testing [NCT03219268].

Rationale

General StructuremAb1 VL

mAb1 VH

mAb2 VH

mAb2 VL

mAb1 VHmAb2 VL

mAb1 VL mAb2 VH

Fc

Fc

� High affinity binding to PD-1 and LAG-3 that compares favorably to nivolumab* (anti-PD-1) or 25F7* (anti-LAG-3)

Trip

le N

egat

ive

Brea

st C

ance

r

H&E LAG-3PD-1

T-cell Clonal ExpansionCytokine SecretionEffector Function

Tumor-directed Migration

Enhancement of T-cell response following SEB stimulation

* Indicates replicas of nivolumab and/or 25F7 (anti-LAG-3)

Well Tolerated in Cynomolgus Monkeys

Pilot Toxicology Study Design

�1-hour IV infusion at 100 (1 male) or 150mg/kg (2 males) �QW x 2

GLP Toxicology Study Design

�1-hour IV infusion at 10, 40 or 100 mg/kg (5 animals/sex/group) �QW x 4; 10-week recovery

Observations

�Well tolerated. Drug-related changes were limted to watery feces at ≥ 40 mg/kg with no impact to body weight. �Nonadverse increase in incidence of mononuclear cell infiltrates. �No cytokine release

Conclusion �NOAEL = 100 mg/kg; MTD > 150 mg/kg

� Combination mAb blockade of PD-1 and LAG-3 in animal models resulted in enhanced antitumor immunity than either mAb alone and is actively being tested clinically.

� MGD013 is a checkpoint inhibitor DART molecule currently under clinical evaluation that has been designed to restore T-cell effector function and enhance antitumor activity by simultaneously targeting PD-1 andLAG-3.

A

Binding of MGD013 to NS0-PD-1+ (A) and NS0-LAG-3+ (B) engineered cells was assessed by FACS analysis. Inhibition of soluble PD-L1 binding to NS0-PD-1+ cells (C) or soluble LAG-3 binding to class II+ Daudi cells (D), respectively, was assessed by FACS analysis. Similar data were obtained for soluble PD-L2 binding to NS0 -PD-1+ cells (data not shown).

B

C D

Indicated molecules were evaluated using enzyme fragment complementation assay employing PathHunter® U2OS PD-1/LAG-3 dimerization cell line (DiscoverX).

10-4 10-2 100 1020.0

1.0

2.0

3.0

4.0

5.0

Concentration (nM, LOG)

Rel

ativ

eD

imer

izat

ion

Act

ivity

Nivolumab*25F7*

MGD013Nivolumab* + 25F7*

Light

MGD013 (PD1 x LAG3) hinge stabilized IgG4 tetravalent, bispecific DART molecule