pe t o es t e e pe t t t o t t e s pt t e teg t o t e et g e te p t e o o g s o e se te tt e s g t e e s st e e stop e e s e p eg s oo e e Lee ott ppe e o o t

e o og ge

ese te t t e e sso t o o e ese eet g t t

g o

e te e o tos s o poptot e s o t o s op ge t o espo se

te st s o te e pe t t o es

o es o eo o to

• Pleiotropic negative regulator of immune responses, promoting tissue homeostasis

• Reduces immune cell activation by clearing apoptotic cells• Dampens signals at the interphase of innate and adaptive immunity to reduce

T-cell effector functions• MERTK deficiency associated with antitumor responses characterized by

increased inflammatory cytokine production

o t s• Aberrantly expressed in multiple cancer cell types, including solid tumors and

hematologic malignancies• Intrinsic driver of tumor growth and survival via PI3K, MAPK, and STAT

pathways

Lymphocyte Isolation RNA-Seq (SMART-Seq® 2)

CD45+ Sort

*

5 11 16 21 25Days Post-Implant

0

500

1000

1500

2000

2500

Tum

or V

olum

e (m

m3 )

IsotypeSRF2

*64% TGI; p < 0.0293

GAS6

Apoptotic Cell

Alters MacrophageResponses to Stimuli

MERTK

Promotes Efferocytosisand Reduces Inflammatory

Responses

TNFIL-12IL-6

o s o s

o e t e s o et o tt o e

• Genetic disruption of MERTK leads to alterations in RPE cells and vision loss.• Inhibition of MERTK using a small molecule inhibitor leads to changes in

structure of the RPE and photoreceptors in treated mice.• The known permeability restriction of large molecules to pass the blood-retinal

barrier presents the possibility that the RPE would be spared during MERTK antibody treatment.

e est o Will a selective MERTK antibody lead to RPE toxicity?

o t o Perform a repeat-dose cynomolgus monkey study to evaluate RPE toxicity.

o o g s o e o o og t es g• 2 MERTK antibodies with distinct epitope binding properties• 30 mg/kg, 100 mg/kg, and vehicle control arms (N = 3 per group)• Antibody injected IV on Days 1, 8, 15, 22, 29; study termination Day 30-32• Endpoints: ophthalmic examinations (predose, Day 14, Day 30);

electroretinograms (ERG) (predose, Day 16, Day 28)• Evaluation of postmortem retinal histology

o et o o og t gs• No abnormal in-life observations with MERTK antibody administration

» Normal ophthalmic examinations and ERG measurements• Overall pathology findings:

» Normal retinal histology in vehicle-treated animals » Microscopic findings observed for both antibodies, but generally occurred at a greater incidence and severity with SRF1 (higher affinity) than with SRF2 (lower affinity)

• A panel of fully human MERTK blocking antibodies was developed.

• In vitro, MERTK blocking antibodies: » Inhibited GAS6 ligand-driven AKT phosphorylation and CD20 upregulation in Kasumi-2 cells

» Inhibited primary human and murine macrophage efferocytosis of apoptotic Jurkat cells

• In vivo, MERTK blocking antibodies: » Showed efficacy in syngeneic murine tumor models as monotherapies and in combination with anti-PD-L1

» Led to gene expression changes indicative of immune cell activation and monocyte infiltration

e e e es Burstyn-Cohen Neuron 2012 Cook JCI 2013 Del Amo Prog Retin Eye Res 2017 Dransfield Cell Death Dis 2015 Duncan IOVS 2003

Lemke Nat Rev Immunol 2008 Liu BMC Bioinformatics 2016 Mukherjee Lancet 2017 Sayama Tox Path 2018 Vollrath PLOS Genetics 2015

• A multidose study in cynomolgus monkeys revealed that MERTK therapeutic antibodies disrupted the integrity of the RPE.

• Because of observed treatment-related retinal disruption, further development of therapeutic MERTK antibodies was not pursued.

• As several therapeutics that block MERTK function are currently in preclinical development, a thorough evaluation of retinal toxicity is warranted.

Normal Cynomolgus Monkey Retinal H istology

B

Vacuolation of the Photoreceptor O uter Segments

Cell Displacement in the RPE (Foamy Cells) Single-Cell Necrosis in the O uter Nuclear Layer

ARPE: Retinal Pigmented EpitheliumO S: Photoreceptor O uter SegmentsIS: Photoreceptor Inner SegmentsO NL: O uter Nuclear LayerO PL: O uter Plexiform LayerINL: Inner Nuclear LayerIPL: Inner Plexiform LayerGCL: Ganglion Cell Layer

O S

IS

O NL

O PL

INL

RPE

SRF1(100 mg/kg)

SRF2(100 mg/kg)

SRF1(100 mg/kg)Vehicle

SRF2(100 mg/kg)

SRF1(100 mg/kg)

SRF2(100 mg/kg)

H istology of A) w ild-type and B) Mertk-/ - murine retina

Burstyn-Cohen, Neuron 2012

o o e o e o o e o e ( o ot e p

o o e o e ( t L o t o

st t e es e o espo ses + o L p o tes

Le t Gene expression changes in CD45+ tumor infiltrating lymphocytes are displayed by volcano plot where log2 fold-change between SRF2 treatment vs isotype control is shown on the x-axis and p-value is represented on the y-axis. g t Top 51 genes upregulated by SRF2 are highlighted by heatmap and hierarchically clustered; gene expression is row-normalized.

o o o g t o es

p ess o s s o t o e te o s

Transcriptional profiles from ImmGen consortium identify gene signatures specifically enriched in mouse immune cell subsets. Each subset-specific signature was analyzed for SRF2-mediated bias in CD45+ transcriptional profiles from the MC38 model. The proportion of genes biased for each signature was rank-ordered and analyzed for immune cell subset enrichment.

o L p o te e e g MC38 tumors (n = 5/group) harvested on Day 10 post-dosing were minced with a razor blade and digested with collagenase type 1 and DNase. CD45+ cells were FACS-sorted into 96-well plates and prepared for RNA sequencing using standard SMART-Seq® 2 protocols.

e t e t o otes toto o oe o e t

Based on analyses of genes induced by SRF2, transcript counts for relevant cytotoxic genes were extracted and plotted for each replicate sample.

es o o o te e t e sso te g t es

ImmGen Cell Type % of Cell-Specific Genes Biased

Monocyte Colony Forming Unit 83Dendritic Cell, Liver 83Classical Monocytes, MHCII- 83Classical Monocytes, MHCII+ 83Monocyte 81Dendritic Cell, Lung 81Dendritic Cell, Small Intestine 81Neutrophils, Synovial Fluid 81Peritoneal Neutrophils, Thioglycolate 81CD4 T cell 52Spleen Naïve CD4 4+ 8- TCR+ 25- 62Lhi 44lo 51Mesenteric LN Naïve CD4 4+ 8- TCR+ 25- 62Lhi 44lo 51Pancreatic LN CD4, BDC Islet-Reactive TCR Tg 51Subcutaneous LN Memory-Phenotype CD8 4- 8+ TCR+ 25- 44hi 122hi 51Peyer Patches Naïve CD8 4- 8+ TCR+ 25- 62Lhi 44lo 51Thymic TCRγ-δ, All DN 51Thymic Vg1.1+ Vd6.3+, Immature 51

0

1000

2000

3000

4000

Nor

mal

ized

Tra

nscr

ipt C

ount

Nor

mal

ized

Tra

nscr

ipt C

ount

Nor

mal

ized

Tra

nscr

ipt C

ount

0

50

100

150

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Isotype SRF2 Isotype SRF2 Isotype SRF2Isotype SRF2 Isotype SRF2 Isotype SRF2

GZMB KLRA7PRF1 TNFRSF9KLRB1 XCL1

SRF2 Induces Cytotoxic Genes SRF2 Induces NK Markers SRF2 Induces T-Cell and Myeloid Markers of Activation

Scml2Axin2Slc22a18Cdh1DgkeClec10aGmprPemtWnt9aDbtTrim25DlatMid2Scmh1H19Tspan32Gpr107FerSlfn4Hddc2Dazap2Clec2gNarfCdc45Mcts1MntBrat1Btbd17Gna12Klf6Scpep1SdhdCdh4RtcaBcl6bComtCcnd2Cox5aNgfrCkmt1Itgb2Xpo6Pih1d2ArvcfAlox12Cav2Rem1Tbx4Gnai3Drp2Tfe3

Isotype SRF2

** **

*

0 5 10 150

500

1000

1500

2000

Days on Study

IsotypeSRF2Anti-PD-L1SRF2 + anti-PD-L1

0 5 10 150

300

600

900

1200

Days Post-Implant

IsotypeSRF1vSRF2v

5 11 16 21 25Days Post-Implant

Tum

or V

olum

e (m

m3 )

Tum

or V

olum

e (m

m3 )

0

500

1000

1500

2000

2500

*47%TGI; p = 0.0292

Tum

or V

olum

e (m

m3 )

*37% TGI; p = 0.02**48% TGI; p = 0.0002

IsotypeSRF2

*64% TGI; p < 0.0293

0.5 1.0 2.0

5E-04

5E-03

5E-02

5E-01

Fold Change SRF2 vs Isotype

Log(

t−te

st p

−e

s so

tpe

Gzmb

Xcl1

Tnfrsf9Prf1

Gpr155

Mgat2

Klra7

Tef

Eed

Crlf2

R3hcc1

Dennd6a

Mettl17

Slfn3

Zfp346

Adamts10

Pank4

Pced1a

Cep295Mllt11

0.2 0.5 1.0 2.0 5.0

Monocyte Precursor

Fold Change SRF2 vs Isotype

Log

(t−te

st p

−val

ue)

81%

0.2 0.5 1.0 2.0 5.0

Fold change SRF2 vs isotype

0.010.02

0.050.100.20

0.501.00

0.010.02

0.050.100.20

0.501.00

Mature Splenic B Cell

51%

10410310210110010-1-25

Concentration (ng/mL)

% o

f Con

trol

25

50

75

100

SRF1SRF2

125

0

UNC1062+ GAS6

MER590+ GAS6

GAS6

Control

105104103102

GAS6No GAS6SRF Ab

1041030

50

0

100

150

200

pAKT S473IgG CD20

MER590

SRF1

SRF2

Control

104103102

t o g t o

o g t o es t op ge e o tos s

t o es to o o te e e op ges Monocyte-derived macrophages were stained with SRF1, SRF2, or a commercially available anti-MERTK monoclonal antibody (MER590, BioLegend) and analyzed by flow cytometry.

o g t o es t e osp o t o s e s Kasumi-2 B cell ALL cells were serum starved overnight and incubated for 1 h in the presence of anti-MERTK antibodies. Cells were stimulated with 20 μg/mL GAS6 for 30 min, fixed, and stained for pAKT S473.

o g t o es t e p eg t o s e s Kasumi-2 cells were screened for cell surface markers regulated by GAS6 stimulation (LEGENDScreen, BioLegend). CD20 was identified as a GAS6 responsive receptor that was inhibited by the MERTK small molecule inhibitor UNC1062 (AK Scientific), the anti-MERTK antibody MER590 (BioLegend), SRF1, and SRF2. Cells were incubated for 1 h with MERTK antibodies or small molecule inhibitors and cultured with 20 μg/mL GAS6 or PBS for 3 days. Cells were stained for CD20 expression and analyzed by flow cytometry.

e o tos s ss e e o e e ts poptot e et e g to op ges

t op ge e o tos s

Human monocyte-derived macrophages (left graph) or murine bone marrow-derived macrophages (right graph) were pre-incubated with antibodies or controls for 1 h. Apoptotic Jurkat cells were then added and incubated overnight. Cells were fixed and analyzed to determine the number of Jurkat cells remaining vs negative control (isotype) and positive control (UNC1062).

Human monocyte-derived macrophages were incubated with controls, small molecules, or antibodies for 1 h. Apoptotic Jurkat cells were then added and incubated overnight. Apoptotic Jurkat cells were almost completely efferocytosed in the isotype control well. UNC1062 inhibited efferocytosis but not apoptotic cell tethering to macrophages, while the anti-MERTK antibody prevented both tethering and efferocytosis. Images were captured by brightfield microscopy.

An FSC-SSC gate was drawn on a no-Jurkat control well to determine the background number of macrophages in the gate. A macrophage plus apoptotic Jurkat well treated with isotype control represents maximal efferocytosis. A macrophage plus apoptotic Jurkat well treated with UNC1062 represents minimal efferocytosis.

e o tos s ss t g t teg

Apoptotic Jurkat cells remaining: 44000 50 µM

10 µM UNC1062

50 µMApoptotic Jurkat cells remaining: 29000

Anti-MERTK Antibody

Apoptotic Jurkat cells remaining: 1800 50 µM

Isotype Control

Jurkat71.3

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

10 µg/mL SRF1

N = 39414

Jurkat74.2

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

10 µM UNC1062

Jurkat cells

N = 46842

Jurkat12.3

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

Isotype Control

N = 2040

Jurkat6.05

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

No Jurkat Control

N = 502

Macrophages

SSC

-A

FSC-A

SRF1

100 101 102 10310010-1 101 102 103 104-20

0

20

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Concentration (ng/mL)

% In

hibi

tion

of E

ffero

cyto

sis

-20

0

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Concentration (ng/mL)

% In

hibi

tion

of E

ffero

cyto

sis

SRF2SRF2

Macrophage MERTK binds via its ligand GAS6 to exposed phosphatidylserine on the surface of apoptotic cells, leading to efferocytosis. MERTK deficiency increases production of proinflammatory cytokines after macrophage activation.

558

• MERTK, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is a promising therapeutic target expressed on macrophages that can regulate innate antitumor immune responses.

• MERTK regulates efferocytosis by macrophages and governs how these cells respond to apoptotic debris.

• Reduced tumor growth in Mertk-deficient mice is accompanied by increased inflammatory cytokine production and immune cell activation.

• MERTK is also expressed in retinal pigmented epithelium (RPE) cells of the eye where it mediates phagocytosis of photoreceptor outer segment fragments.

• Mutations that disrupt MERTK expression or kinase activity lead to marked retinal degeneration and blindness in mice, rats, and humans.

• Permeation of large molecules to the retina is restricted by the blood-retinal barrier, which may restrict therapeutic IgG molecules from accessing RPE cells.

• In order to inhibit MERTK in macrophages while sparing RPE cells, a diverse panel of high-affinity antibodies to MERTK was developed and studied in vitro and in vivo.

• To investigate effects on RPE biology with MERTK antibodies, a multidose, 4-week cynomolgus monkey study with several in-life and postmortem ophthalmologic endpoints was designed.

• o Determine if MERTK antibodies that modulate tumor immunity in murine syngeneic models will cause disruption of the RPE in cynomolgus monkeys.

Antibody Human Affinity (M)

Human Avidity (M)

Murine Avidity (M)

Human Efferocytosis IC50 (ng/mL)

Murine Efferocytosis IC50 (ng/mL)

SRF1 8.7E-11 3.3E-10 Not Active 37.3 Not ActiveSRF2 4.4E-09 5.1E-10 3.6E-09 605.2 121

Model # Cells Strain Dosing Start Dose Days DosedCT26 2.5E5 Balb/C 2 days post-inoculation Isotype / SRF2 (50 mg/kg IP) 3, 7, 9, 13, 17

MC38 (mono) 1E6 C57BL/6 2 days post-inoculation Isotype / SRF1v / SRF2v (20 mg/kg IP) 2, 5, 9, 12MC38 (combo) 1E5 C57BL/6 Tumor size 70 mm3 Isotype / SRF2 (20 mg/kg IP) ± anti-PD-L1 (10 mg/kg IP) 0, 3, 7, 10

N = 10/group. Tumors were measured 3x/week. Data shown are mean ± SEM. SRF2v (SRF2 variant) has enhanced murine cross-reactivity.

• Fully human IgG4• No human or murine AXL/TYRO3 binding• Block GAS6 binding• Selective against a > 4500 human membrane protein panel • SRF1 has picomolar affinity to MERTK; SRF2 is murine cross-reactive

Analysis• Cells fixed with paraformaldehyde

• Number of Jurkat cells remaining in culture

measured

Efferocytosis• Antibodies and controls

added to macrophages• Apoptotic Jurkat cells layered on and incubated overnight

Apoptosis induction• Jurkat cells exposed to UV

light for 5 min followed by 2-3 h of culture

Macrophage differentiation• Human monocytes

differentiated into macrophages with 4-7 days of M-CSF