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Cancer Therapy: Preclinical
TTI-621 (SIRPaFc): A CD47-Blocking InnateImmune Checkpoint
Inhibitor with BroadAntitumor Activity and Minimal
ErythrocyteBindingPenka S. Petrova1, Natasja Nielsen Viller1, Mark
Wong1, Xinli Pang1, Gloria H.Y. Lin1,Karen Dodge1,Vien Chai1, Hui
Chen1,Vivian Lee1,Violetta House1, Noel T.Vigo1, Debbie
Jin1,Tapfuma Mutukura1, Marilyse Charbonneau1, Tran Truong1,
Stephane Viau1,Lisa D. Johnson1, Emma Linderoth1, Eric L. Sievers1,
Saman Maleki Vareki2,3,Rene Figueredo2,3, Macarena Pampillo2, James
Koropatnick2,3, Suzanne Trudel4,Nathan Mbong4, Liqing Jin4, Jean
C.Y.Wang4,5, and Robert A. Uger1
Abstract
Purpose: The ubiquitously expressed transmembrane glyco-protein
CD47 delivers an anti-phagocytic (do not eat) signal bybinding
signal-regulatory protein a (SIRPa) on macrophages.CD47 is
overexpressed in cancer cells and its expression is asso-ciated
with poor clinical outcomes. TTI-621 (SIRPaFc) is a fullyhuman
recombinant fusion protein that blocks the CD47–SIRPaaxis by
binding to human CD47 and enhancing phagocytosis ofmalignant cells.
Blockade of this inhibitory axis using TTI-621 hasemerged as a
promising therapeutic strategy to promote tumorcell
eradication.
Experimental Design: The ability of TTI-621 to promote
mac-rophage-mediated phagocytosis of human tumor cells wasassessed
using both confocal microscopy and flow cytometry. Invivo antitumor
efficacy was evaluated in xenograft and syngeneicmodels and the
role of the Fc region in antitumor activity wasevaluated using
SIRPaFc constructs with different Fc tails.
Results: TTI-621 enhanced macrophage-mediated phagocy-tosis of
both hematologic and solid tumor cells, while sparingnormal cells.
In vivo, TTI-621 effectively controlled the growthof aggressive AML
and B lymphoma xenografts and wasefficacious in a syngeneic B
lymphoma model. The IgG1 Fctail of TTI-621 plays a critical role in
its antitumor activity,presumably by engaging activating Fcg
receptors on macro-phages. Finally, TTI-621 exhibits minimal
binding to humanerythrocytes, thereby differentiating it from CD47
blockingantibodies.
Conclusions: These data indicate that TTI-621 is activeacross a
broad range of human tumors. These results furtherestablish CD47 as
a critical regulator of innate immune sur-veillance and form the
basis for clinical development of TTI-621 in multiple oncology
indications. Clin Cancer Res; 1–12.�2016 AACR.
IntroductionThe phagocytic activity of macrophages is regulated
by both
activating ("eat") and inhibitory ("do not eat") signals. CD47,
awidely expressed transmembrane glycoprotein, serves as a
criticalinhibitory signal, suppressing phagocytosis by binding to
signal-
regulatory protein alpha (SIRPa) on the surface of
macrophages.Engagement by CD47 triggers tyrosine phosphorylation of
thecytoplasmic tail of SIRPa, leading to recruitment of the
Srchomology-2 domain containing protein tyrosine phosphatasesSHP-1
and SHP-2 and prevention of myosin-IIA accumulation atthe
phagocytic synapse (1). CD47 is believed to regulate thenatural
clearanceof senescent erythrocytes andplatelets by
splenicmacrophages (2, 3). In addition, the CD47–SIRPa
interactionmay represent an importantmechanismbywhichmalignant
cellsescape immune-mediated clearance.
CD47 has been shown to be overexpressed in numeroushematologic
malignancies, including acute myeloid leukemia(AML), acute
lymphoblastic leukemia (ALL), chronic lymphocyt-ic leukemia
(CLL),multiplemyeloma,myelodysplastic syndrome(MDS), and inmultiple
types of non-Hodgkin lymphoma (NHL),including diffuse large B-cell
lymphoma (DLBCL), mantle celllymphoma, and marginal cell lymphoma
(4–10). Similarly, ele-vated CD47 expression has been demonstrated
on solid tumors,including bladder, brain, breast, colon,
esophageal, gastric, kid-ney, leiomyosarcoma, liver, lung,melanoma,
ovarian, pancreatic,and prostate tumors (11–15). CD47 has been
found to be an
1Trillium Therapeutics Inc., Mississauga, Ontario, Canada.
2London RegionalCancer Program, London Health Sciences Centre,
Lawson Heath ResearchInstitute, London, Ontario, Canada.
3Department of Oncology, University ofWestern Ontario, London,
Ontario, Canada. 4Princess Margaret Cancer Center,University
HealthNetwork (UHN), Toronto,Ontario, Canada. 5Division
ofMedicalOncology and Hematology, Department of Medicine, UHN, and
Department ofMedicine, University of Toronto, Toronto, Ontario,
Canada.
Note: Supplementary data for this article are available at
Clinical CancerResearch Online
(http://clincancerres.aacrjournals.org/).
Corresponding Author: Robert A. Uger, Trillium Therapeutics
Inc., Mississauga,Ontario, Canada. Phone: 416-595-0627; Fax:
416-595-5835; E-mail:[email protected]
doi: 10.1158/1078-0432.CCR-16-1700
�2016 American Association for Cancer Research.
ClinicalCancerResearch
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adverse prognostic factor where high CD47 expression
correlateswith more aggressive disease and poorer clinical
outcomes. Forexample, overall survival was significantly lower for
DLBCL ormantle cell lymphoma patients who had elevated CD47
expres-sion, and higher CD47 expression on tumor cells was
associatedwith significantly poorer event-free survival in patients
with CLL(7). Similar trends have been reported in other
hematologicmalignancies (5, 6, 8) and solid tumors (11, 13, 16,
17). Inaddition, there is evidence to suggest that increased CD47
expres-sion is associated with the transition from low-risk to
high-riskMDS and subsequent transformation to AML (10). These
findingsare consistent with tumor cells exploiting the suppressive
CD47–SIRPa axis to evade macrophage-mediated destruction.
BlockingCD47 has thus emerged as a promising therapeutic strategy
andseveral studies have shown that interrupting the
CD47–SIRPasignaling pathway using anti-CD47 mAbs promotes
antitumoractivity against human cancers both in vitro and in vivo
(6–9, 18).However, the expression of CD47 on erythrocytes raises
concernsabout the potential for anti-CD47 mAbs to cause
hemolyticanemia, as seen in preclinical studies (19). Furthermore,
eryth-rocyte CD47 constitutes a massive antigen sink that may limit
theability of CD47-targeting agents to reach tumor cells.
Finally,CD47-targeting agents bound to erythrocytes may cause
interfer-ence with blood typing tests.
TTI-621 (SIRPaFc) is a novel innate immune checkpoint inhib-itor
that binds humanCD47 and prevents it fromdelivering a "donot eat"
signal to macrophages. It is designed to function as adecoy
receptor, binding CD47 on the surface of tumor cells andblocking
its anti-phagocytic "do not eat" signal, thereby
allowingmacrophages to phagocytose malignant cells. Moreover, the
IgG1Fc region of SIRPaFc can interact with human Fcg receptors
onmacrophages to further enhance phagocytosis, tumor
antigenpresentation, and effective antitumor activity. Here, we
fullycharacterize this novel agent and demonstrate that
TTI-621strongly binds to a wide range of human tumors and
inducespotent phagocytosis of human tumor cells in vitro and in
vivowhilesparing most normal cells. Although the decoy receptor
binds
circulating platelets and leukocytes, TTI-621 shows only
minimalbinding to human erythrocytes, thereby mitigating concerns
ofanemia associated with anti-CD47 mAbs. These results
furtherestablish CD47 as a critical regulator of innate immune
surveil-lance and form the basis for clinical development of
TTI-621 inmultiple oncology indications.
Materials and MethodsSIRPaFc proteins
TTI-621 consists of the N-terminal V domain of human
SIRPa(GenBankAAH26692) fused to the human IgG1 Fc region
(hinge-CH2-CH3, UniProtKB/Swiss-Prot, P01857). Variant proteinswere
generated in which the identical human SIRPa domain waslinked to a
human IgG4 Fc region (hinge-CH2-CH3, UniProtKB/Swiss-Prot, P01861)
or an IgG4 Fc region which was mutated toremove residual Fc
interactions (20). Both IgG4-based fusionproteins contained a
hinge-stabilizing mutation that prevents theformation of intrachain
disulfide bonds (21). Two mouse surro-gate SIRPaFcs were
constructed, one using the N-terminal Vdomain from NOD mouse SIRPa
(22) and the second using amutated (CV1) N-terminal V domain of
human SIRPa (23). Inboth mouse surrogates, the SIRPa domains were
linked to amouse IgG2a Fc (hinge-CH2-CH3,
UniProtKB/Swiss-Prot,P01863). All constructs were generated by
overlapping PCR usingstandard molecular biology techniques and
expressed in stablytransfected CHO-S cells (Invitrogen). Proteins
were purified fromculture supernatant using protein A and
hydrophobic interactionchromatography, concentrated, and residual
endotoxin removed.Control human IgG1 and mouse IgG2a Fc proteins
lacking theSIRPa domain were also generated and similarly purified.
Allproteins displayed >99% purity by HPLC and
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Viably frozen primary tumor cells from the peripheral blood
orbone marrow of patients with B-cell ALL, T-cell ALL, MDS, andAML
were obtained from the University Health Network (UHN)BioBank
(Toronto, Canada) according to the proceduresapproved by the
Research Ethics Board of UHN.
Human macrophages were prepared from heparinized wholeblood
obtained from normal healthy human donors (Biolog-ical Specialty
Corporation); informed consent was obtainedfrom all donors.
Peripheral blood mononuclear cells (PBMC)were isolated over
Ficoll-Paque Plus density gradient (GEHealthcare) and CD14þ
monocytes were isolated from PBMCsby positive selection using CD14
antibody-coated MicroBeadseparation (Miltenyi Biotec). Monocytes
were differentiatedinto macrophages by culturing for at least 10
days in X-Vivo-15 media (Lonza) supplemented with M-CSF
(PeproTech).One day prior to phagocytosis assays, the
monocyte-derivedmacrophages were primed with IFNg (PeproTech) to
generateM1 macrophages or with IL4 (Peprotech) to generate
M2macrophages. Unless otherwise specified, all phagocytosisassays
were carried out using M1 macrophages. When required,macrophages
were harvested using Enzyme-Free Cell Dissoci-ation Buffer
(ThermoFisher).
Tumor cell bindingCell lines or primary patient sampleswere
added induplicate to
96-well plates and incubated with titrated amounts of
biotiny-lated TTI-621 or biotinylated isotype-matched control IgG
Fc,together with Near-IR LIVE/DEAD Fixable Dead Cell Stain
(Invi-trogen) for 30 minutes on ice. Cells were washed, stained
withphycoerythrin (PE)-conjugated streptavidin
(eBioscience),washed, and resuspended in Stabilizing Fixative
(BDBiosciences).Flow cytometry was performed on a FACSVerse flow
cytometer(BD Biosciences). Data were analyzed using FlowJo
software(Treestar Inc.). Half-maximal effective concentration
(EC50)values were calculated using a sigmoidal dose–response curve
inGraphPad Prism software.
Erythrocyte bindingErythrocytes were isolated from
sodium-heparinized whole
blood from healthy human donors (Biological Specialty
Cor-poration) by centrifugation followed by several washes withPBS.
The resulting packed erythrocytes were diluted in PBS andadded in
duplicate to 96-well plates. Binding was performed byincubating
erythrocytes with titrated amounts of TTI-621, anti-CD47 mAbs
[BRIC126 (Serotec), 2D3 (eBioscience), CC2C6(BioLegend), B6H12
(in-house), 5F9 (in-house)]. Cells werewashed and subsequently
stained with biotin-conjugated anti-human IgG Fc PAN (Hybridoma
Reagent Laboratory), followedby detection with PE-conjugated
streptavidin (eBioscience).Flow cytometry was performed on a
FACSVerse flow cytometer(BD Biosciences).
Hemagglutination assaysTitrated amounts of TTI-621 or anti-CD47
mAbs (up to
3 mmol/L) were added to wells containing erythrocytesdiluted in
PBS, and the plates were incubated overnight at37�C in 5% CO2. The
extent of hemagglutination was assessedby scoring each well on a
scale of 1 to 6, with 1 representing theabsence of hemagglutination
and 6 representing completehemagglutination.
Phagocytosis assaysConfocal-based phagocytosis assay. Tumor
cells were labeled withCellTrace CFSE (Life Technologies) and added
to primedmacrophages in 24-well plates at a 1:5 effector:target
ratio.Macrophages and tumor cells were cocultured for 2 hours
at37�C in 5% CO2 in the presence of TTI-621 or control Fcprotein
and subsequently stained with Alexa Fluor 555–con-jugated Wheat
Germ Agglutinin (Invitrogen). Phagocytosis wasassessed by confocal
microscopy on a Quorum Wave FX-X1Spinning Disc Confocal System and
images were analyzedusing Velocity software (PerkinElmer). A
phagocytosis indexwas calculated as: (number of tumor cells inside
macrophages/number of macrophages) � 100; counting at least 200
macro-phages per sample. All tumor cells counted were confirmed
tobe internalized using z-stack images. Statistical significance
wascalculated by unpaired t test versus isotype control
usingGraphPad Prism software.
Flow cytometry–based phagocytosis assay. Tumor cells were
labeledwith Violet Proliferation Dye 450 (BD Biosciences) and added
toprimedmacrophages in 96-well plates at a 1:5 effector:target
ratio.Macrophages and tumor cells were cocultured for 2 hours at
37�Cin 5% CO2 in the presence of TTI-621 or control Fc protein
andsubsequently stained with Near-IR LIVE/DEAD Fixable Dead
CellStain (Invitrogen), APC-conjugated anti-human CD14
(61D3,eBioscience), and PE-conjugated anti-human CD11b
(ICRF44,eBioscience), washed and resuspended in Stabilizing
Fixative (BDBiosciences). Cells were acquired on a FACSVerse flow
cytometer,and data were analyzed using FlowJo software (Treestar
Inc.).Macrophages were identified as live, single, CD14þCD11bþ
cells.Doublets were excluded by SSC-W and SSC-H
discrimination.Percent phagocytosis was assessed as the percent of
macrophagesthat were VPD450þ. The gating strategy and
representative dotplots are shown in Supplementary Fig. S1.
Statistical significancewas calculated by unpaired t test versus
isotype control usingGraphPad Prism software.
AML xenograftsAML xenografts were performed in 10-week-old
female
NOD.SCID mice bred and maintained in the Barrier Unit atthe UHN
Animal Facility (Toronto, Canada). One day prior totransplantation,
mice were sublethally irradiated (275 cGy)and pretreated with
anti-CD122 antibody (0.2 mg/mouse) todeplete residual host NK
cells. On the day of transplantation,viably frozen mononuclear
cells collected from AML patients90543 and 90191 were thawed,
counted, and transplantedintrafemorally into the preconditioned
mice at a dose of 5 �106 cells/mouse in a total volume of 30 mL.
Twenty-one daysafter engraftment, mice were dosed with TTI-621 (8
mg/kg) orequimolar amount of control human IgG1 Fc (5.4 mg/kg)
at0.3 mL/mouse, 3 times/week for 4 weeks. Upon euthanization,bone
marrow from injected and noninjected bones wascollected and stained
with mouse anti-human antibodiesincluding CD47-FITC, CD33-PE,
CD19-PC5, CD45-APC,CD34-APCCy7, CD38-PECy7. After staining, washed
cells wererun on an LSRII flow cytometer (BD Biosciences).
Events(10,000–20,000) were collected for each sample. Collecteddata
were analyzed by FlowJo software to assess AML engraft-ment levels
in the injected femur, noninjected bones, and inthe spleen as
determined by the percentage of humanCD45þCD33þ cells.
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B-cell lymphoma xenograftsLymphoma xenografts were performed in
6- to 7-week-old
female NOD.Cg-PrkdcscidHrhr/NCrHsd (SHrN) mice, a hairlessSCID
strain, obtained from Harlan Laboratories (Montreal,Canada) and
maintained at the Victoria Research LaboratoriesVivarium (London
Health Sciences Centre). Raji and Namalwacells (1 � 106 per
injection) were injected subcutaneously intoeach flank of SHrNmice
in a volume of 0.1 mL PBS (i.e., 2 tumorinjection sites per mouse).
Toledo cells (1 � 107 per injection)were injected in 50% Matrigel
(ECM gel, Sigma-Aldrich) in avolumeof 0.1mL into the leftflank for
SHrNmice.Micewere keptunder isoflurane-mediated anesthesia during
the injections. Threedays after Raji andNawalma tumor cell
implantation and 10 daysafter Toledo tumor cell implantation,
animals received either 10mg/kg of mouse SIRPaFc (NOD SIRPa) or
6.75 mg/kg controlIgG2a Fc, daily 5 times per week, for 3 weeks by
intraperitonealinjection. Tumor volumes were estimated twice weekly
by stan-dard calipermeasurements of length andwidth then calculated
asfollow: p/6 � (longest diameter) � (shortest diameter)2.
Tumorvolumes were monitored until they reached the maximum vol-ume
of approximately 1,500 mm3 or maximum permissiblemarkers of
discomfort in the mice were reached (i.e., mousediscomfort or body
weight loss reached maximum allowablelevels), at which time the
mice were sacrificed. All studies wereconducted according to the
animal care guidelines described bythe Canadian Council on Animal
Care (CCAC) andmonitored byThe Western University Animal Use
Subcommittee. Statisticalsignificance was calculated by two-way
ANOVA using GraphPadPrism software.
Syngeneic B-cell lymphoma modelFemale BALB/c mice (6–8 weeks
old) were purchased from
Charles River Laboratories and housed in the University of
Tor-onto animal facility. A20 cells (2 � 106) were injected
subcuta-neously into the right hind flank of 8-week-old BALB/c
femalemice in a volume of 0.1 mL. When the tumors were
palpable(approximately 60 mm3), they were randomized and
injectedintratumorally with 200 mg (10 mg/kg) of a mouse
SIRPaFcsurrogate (CV1 SIRPa) fusion protein in a 50 mL volume of
PBS.Control groups were injected with vehicle alone in a
50-mLvolume. Animals were dosed twice weekly for a total of five
doses.Tumors were monitored three times a week and tumor volumewas
calculated as 1/2 length � width2. Tumor volumes weremonitored
until one or more tumor dimensions reaches themaximum permissible
measure (15 mm), or when maximum
permissible markers of discomfort were observed, at which
timethe mice were sacrificed. All animal procedures were approved
bythe animal care committee of the University of Toronto
inaccordance with the CCAC. Statistical significance was
calculatedby two-way ANOVA using GraphPad Prism software.
ResultsStructure of TTI-621
TTI-621 (SIRPaFc) was generated by directly linking thesequences
encoding the N-terminal CD47 binding domain ofhuman SIRPa with the
Fc domain of human IgG1 (Fig. 1). TheSIRPa region
interactswithCD47,while the Fc region binds to Fcgreceptors.
TTI-621 is secreted by a genetically engineered Chinesehamster
ovary (CHO) cell line as a 77-kDa disulfide-linked, N-glycosylated
homodimer consisting of two identical 345 aminoacid chains.
TTI-621binds toCD47and enhancesmacrophagephagocytosisof tumor
cells in vitro
The binding of TTI-621 toCD47 onmalignant human cells
wasassessed by flow cytometry. TTI-621was found to bind strongly
toa panel of 19 tumor cell lines derived from patients representing
awide range of both hematologic and solid tumors (Supplemen-tary
Table S1). TTI-621 also exhibited strong binding to primarytumor
samples obtained from the blood of patients with B-cellacute
lymphoblastic leukemia (B-ALL), T-ALL, and AML, andbone marrow
samples from patients with MDS, with an averagebinding EC50 value
of 197 � 182 nmol/L (Supplementary TableS2). CD47 is widely
expressed on normal cells, and TTI-621 alsodemonstrated binding to
human CD4þ T cells, CD8þ T cells, Bcells, platelets, natural killer
(NK) cells, granulocytes, monocytes,and NK T cells from the
peripheral blood of healthy donors(Supplementary Table S3).
The ability of TTI-621 to promote
macrophage-mediatedphagocytosis of human tumor cells was assessed
using bothconfocal microscopy and flow cytometry.
Monocyte-derivedmacrophages were cocultured with tumor cells for
two hours,and in cultures left untreated or treatedwith a control
Fc fragment,macrophages exhibited a low level of phagocytosis,
consistentwith CD47-mediated suppression. In contrast, blockade of
CD47on the target cells using TTI-621 significantly increased
macro-phage phagocytosis of tumor cells (Fig. 2A). Compared with
acontrol Fc protein, TTI-621 promoted macrophage phagocytosisof 77%
(23/30) of tumor cell lines established from patients with
Figure 1.
Structure of TTI-621. TTI-621 consists ofthe N-terminal domain
of human SIRPa(shown in red) linked to a human IgG1Fc region (shown
in blue). The hingeand inter-chain disulfide bonds areshown as
black lines.
Petrova et al.
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Figure 2.
TTI-621 promotes macrophage-mediated phagocytosis of human tumor
cells in vitro. A, Representative scanning confocal microscopy
images after macrophageswere cocultured with a primary AML patient
sample for 2 hours in the presence of 10 mmol/L TTI-621 or control
IgG1 Fc protein. Tumor cells and macrophages arestained green and
red, respectively. B, Macrophage-mediated phagocytosis of
established human tumor cell lines from patients with B-cell
malignancies(n ¼ 17), myeloid malignancies (n ¼ 7), T-cell
malignancies (n ¼ 6), skin cancers (n ¼ 7), and other solid cancers
(n ¼ 5) in the presence of 1 mmol/L TTI-621(black bars) or control
IgG1 Fc protein (white bars). Phagocytosis was quantified by
determining a phagocytosis index (number of engulfed tumor cells
per 100macrophages) using confocal microscopy or measuring
percentage phagocytosis by flow cytometry, as described in the
Materials and Methods section.C, Macrophage-mediated phagocytosis
of primary human tumor samples from patients with hematologic
malignancies (n ¼ 33) in the presence of 1 mmol/LTTI-621 (black
circles) or control IgG1 Fc protein (white circles). D,
Representative titration of TTI-621 (black circles) on a primary
AML patient sample. Control Fcprotein (white circle) was tested at
1 mmol/L. E, Macrophage-mediated phagocytosis of primary AML tumor
sample or normal monocytes was assessed byconfocal microscopy in
the presence of 1 mmol/L TTI-621 or control IgG1 Fc. Statistical
significance was assessed by unpaired t test versus Fc control (� ,
P < 0.05;�� , P < 0.01; ��� , P < 0.001; NS, not
significant).
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C
60402000
500
1,000
1,500
2,000
Burkitt Lymphoma(Raji)
Day post-engraftment
Dosing window
Dosing window
Rituximab
Control FcSIRPαFc
Rituximab
Control FcSIRPαFc
******
60402000
500
1,000
1,500
2,000
Burkitt Lymphoma(Namalwa)
Day post-engraftment
Dosing window
Rituximab
Control FcSIRPαFc***
***
D
E
A
B
% A
ML
Engr
aftm
ent
% A
ML
Engr
aftm
ent
% A
ML
Engr
aftm
ent
% A
ML
Engr
aftm
ent
% A
ML
Engr
aftm
ent
% A
ML
Engr
aftm
ent
Control FcTTi-621Control FcTTi-6210
25
50
75
100
0
25
50
75
100
P = 0.003
P = 0.008
P = 3×10–8 P = 0.003 P = 0.02
Non-injected bone marrow
Non-injected bone marrow
Injected bone marrow
Injected bone marrow
Control FcTTI-621
Control FcTTi-621Control FcTTi-621 Control FcTTI-621
012345678
Spleen
Spleen
P = 0.0004
4030201000
500
1,000
1,500
2,000
0
500
1,000
1,500
2,000
B Lymphoma(A20)
Diffuse large B-cell lymphoma(Toledo)
Day post-engraftment
40 60200Day post-engraftment
Tum
or v
olum
e (m
m3 )
Tum
or v
olum
e (m
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Tum
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olum
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Tum
or v
olum
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VehicleDosing window ***
F
Figure 3.
TTI-621 and its mouse surrogate are efficacious in vivo. A and
B, NOD.SCID mice were preconditioned with sublethal irradiation and
anti-CD122 antibody(to deplete residual NK cells) and then
transplanted with AML cells from patient #0905443 (A) or patient
#090191 (B) by intrafemoral injection. Treatment with TTI-621 (8
mg/kg i.p. 3�/week for 4 weeks) or control IgG1 Fc protein was
initiated 21 days post-transplantation. The percent AML engraftment
(% cells expressinghuman CD45 and CD33 markers) was assessed by
flow cytometry. Each symbol represents one mouse, bars indicate
mean values. P values were determinedby t test versus Fc control
protein. Data shown are representative of 9 separate AML patient
xenografts. C–E, SHrN mice (n ¼ 5 per group) receivedsubcutaneously
implanted Raji (C), Namalwa (D), or Toledo (E) cells. Three days
after implantation (Namalwa and Raji) or 10 days after implantation
(Toledo), micewere dosed intraperitoneally with either a mouse
surrogate SIRPaFc (10 mg/kg), control mouse IgG2a Fc (6.67 mg/kg),
or rituximab (8 mg/kg) five timesa week for 3 weeks (indicated by
the arrow heads). Tumor volumes were estimated by caliper
measurement from both flanks and the means for thosemeasurements
were calculated in mm3. (Continued on the following page.)
Petrova et al.
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hematologicmalignancies and 67%(8/12) of human solid cancercell
lines (Fig. 2B). A marked prophagocytic effect of TTI-621 wasalso
observed on primary samples from patients with AML,
MDS,multiplemyeloma, B-ALL, and T-ALL (Fig. 2C). TTI-621
enhancedmacrophage-mediated killing of 97% (32/33) of primary
bloodcancer samples tested. Drug activity was further characterized
bytitrating TTI-621 on selected human tumor cell lines (n¼ 13)
andprimary tumor samples (n¼4) (representative data in Fig. 2D).
Assummarized in Supplementary Table S4, TTI-621 treatmentresulted
in a saturable, dose-dependent phagocytic response withan average
EC50 of 10 � 14 nmol/L.
We then assessed the effect of TTI-621 on macrophage-medi-ated
phagocytosis of normal cells in vitro. As shown in Fig. 2E, TTI-621
potently increased phagocytosis of primary AML tumor cells,while
sparing normal peripheral blood monocytes, indicatingthat
TTI-621-enhanced phagocytosis is tumor cell-specific.
Collectively, these in vitro data demonstrate that
TTI-621induces potent, tumor-specific macrophage phagocytosis
acrossa broad range of hematologic and solid tumors. In fact, we
havenot observed a tumor type that is refractory to TTI-621
treatment,consistent with prior data demonstrating that the CD47
immunecheckpoint is widely used by malignant cells to escape
immunesurveillance (5, 24).
TTI-621 and mouse surrogate SIRPaFc have potent
antitumoractivity in vivo
To determine whether the potent effects of TTI-621 in
vitrotranslated into in vivo antitumor activity, we employed an
AMLxenograft model using primary patient samples. Engrafted
micewere treated with TTI-621 or an Fc fragment control three
times/week for 4 weeks. Although control-treated animals
exhibitedsignificant engraftment, particularly in the injected
bonemarrow,TTI-621 treatment significantly reduced the tumor burden
inbonemarrow and spleen (Fig. 3A and B). In fact, tumor cells
wereundetectable in most animals following TTI-621 therapy.
The presence of CD47 on nontumor tissue has the potential
tobindSIRPaFc and remove it fromcirculation, potentially
resultingin a significant antigen sink effect. As TTI-621 does not
bind tomouse CD47 (data not shown), TTI-621 treatment of
xenograftrecipient does notmodel this antigen sink effect. To
overcome thislimitation, mouse surrogate fusion proteins (mSIRPaFc)
wereconstructed using the mouse IgG2a Fc region, allowing for
fulleffector function, analogous to the human IgG1 Fc region in
TTI-621. Treatment of mice with mSIRPaFc may thus more closelymimic
the anticipated pharmacokinetic and Fc effector activityprofile of
TTI-621 in human subjects. The in vivo efficacy ofmSIRPaFc was
assessed in three aggressive B-cell lymphomaxenograft models:
Namalwa and Raji (Burkitt lymphomas) andToledo (DLBCL). Hairless
NOD.SCID (SHrN) mice wereimplanted subcutaneously with tumor cells
and treated withmSIRPaFc five times/week for 3 weeks starting
either 3 days afterengraftment (Namalwa and Raji) or 10 days after
engraftment
(Toledo). mSIRPaFc treatment markedly reduced the growth ofRaji
tumors (Fig. 3C) and completely ablated Namalwa andToledo tumors
(Fig. 3D and E); in the latter two models, mostmice remained
tumor-free 60 days after inoculation. Moreover,mSIRPaFc was
superior to rituximab therapy in both Namalwaand Toledo
xenografts.
To overcome the limitations inherent with xenograft models,we
also assessed whether mSIRPaFc could reduce tumor burdenin an
immunocompetent syngeneic system. BALB/c mice weresubcutaneously
inoculated with A20 B-cell lymphoma cells, andmSIRPaFc was
administered by intratumoral injection twiceweekly starting 7 days
postengraftment. As shown in Fig. 3F,mSIRPaFc treatment
significantly reduced the growth of A20tumors, confirming that CD47
blockade with mSIRPaFc is alsoefficacious in animals with an intact
immune system.
Collectively, these in vivo data suggest that blockade of
theCD47–SIRPa axis using SIRPaFc has broad applicability across
avariety of malignancies.
Blockade of CD47 using SIRPaFc requires an IgG1 Fc tail
formaximum potency
Engagement of Fcg receptors (FcgR) on macrophages bySIRPaFc may
deliver a prophagocytic signal that could augmentthe effect of CD47
blockade. TTI-621 possesses an IgG1 Fc tail,allowing for binding to
the high-affinity receptor FcgRI (CD64) aswell as to the
low-affinity receptors FcgRII (CD32) and FcgRIII(CD16). To
determine whether the IgG1 Fc tail is required formaximum potency,
we compared the in vitro activity of TTI-621with a variant SIRPaFc
inwhich the IgG1 Fc region of TTI-621wasreplaced with an IgG4 Fc
tail. IgG4 Fc regions bind well to CD64but have weaker interactions
than IgG1 with CD32 and CD16(25). We compared the prophagocytic
activity of both SIRPaFcsusing classically activated (M1) and
alternatively activated (M2)macrophages. We have previously shown
that M1 macrophagesare CD32hi CD64hiin vitro, whereas M2
macrophages are CD32hi
CD64lo (26). TTI-621 enhanced phagocytosis by both macro-phage
subsets equally well. In contrast, SIRPaFc with an IgG4 tailinduced
significantly less phagocytosis by M2 macrophages (Fig.4A). These
data suggest that an IgG1 tail is necessary for
SIRPaFc'senhancement of phagocytosis by both M1 and M2
macrophages.
We next compared the in vivo activity of TTI-621 and the
variantIgG4-containing SIRPaFc in the AML xenograft model. We
alsotested a SIRPaFc with amutated IgG4 Fc region that is
completelydevoid of Fc effector functions. As shown in Fig. 4B,
treatmentwith all three SIRPaFc constructs reduced tumors to
undetectablelevels in the spleen. In the injected femur and
noninjected bonemarrow, TTI-621 treatment completely ablated tumor
growth inall but one mouse. SIRPaFc with an IgG4 tail reduced
tumorburden in the noninjected bone marrow, but not in the
injectedfemur compared with controls, whereas the mutated IgG4
fusionprotein was unable to control tumor burden in either
bonemarrow compartment (Fig. 4B).
(Continued.) Micewere sacrificedwhen tumor volumes exceeded
1,500mm3 orwhen therewas extensive ulceration. Mean tumor volumes
are recorded only for timepoints in which �1 mouse per group was
sacrificed. Data shown are representative of 4 (Raji), 2 (Namalwa),
and 2 (Toledo) independent experiments.Mice were terminated at a
tumor volume of 1,500 mm3. F, 2 � 106 A20 cells were implanted
subcutaneously into the right flank of Balb/c mice on day 0.Mice
were randomized (n¼ 9–10mice per treatment arm) when themean tumor
size was palpable at which time the tumorswere approximately 60mm3
in volume.A mouse surrogate SIRPaFc (10 mg/kg) or vehicle was given
bi-weekly by intratumoral administration. Mice were sacrificed when
at least one tumordimension exceeded 15 mm. Mean tumor volumes �
SEM were recorded only for time points in which �2 mice per group
were sacrificed. Data shown arerepresentative of two independent
experiments. Statistical significance was assessed by two-way ANOVA
(��� , P < 0.001).
TTI-621 Is a Novel Antitumor Immune Checkpoint Inhibitor
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IgG1 (
TTI-6
21)
IgG4
IgG1 (
TTI-6
21)
IgG4
0
50
% M
ax P
hago
cyto
sis
100
150 M1 Mϕ M2 Mϕ**
SIRPa
Fc(Ig
G4-m
ut)
SIRPa
Fc (Ig
G4)
SIRPa
Fc (Ig
G1) (T
TI-62
1)
IgG4-m
ut Fc
contr
ol
IgG1 F
c con
trol
0
0
Blockade of the CD47–SIRPa interaction: No Yes Yes
20
40
60
80
25
50
75
100
Injected femurP < 0.001
P < 0.01 P < 0.001
% A
ML
Engr
aftm
ent
% A
ML
Engr
aftm
ent
% A
ML
Engr
aftm
ent
SIRPa
Fc (Ig
G4-m
ut)
SIRPa
Fc (Ig
G4)
SIRPa
Fc (Ig
G1) (T
TI-62
1)
IgG4-m
ut Fc
contr
ol
IgG1 F
c con
trol
0
25
50
75
100P < 0.001
P < 0.001
Non-injected BM
P < 0.05
SIRPa
Fc (Ig
G4-m
ut)
SIRPa
Fc (Ig
G4)
SIRPa
Fc (Ig
G1) (T
TI-62
1)
IgG4-m
ut Fc
contr
ol
IgG1 F
c con
trol
012345678
Spleen
P < 0.01
P < 0.01
P < 0.001
A
B
C
Nodr
ug
mIgG
1
Contr
ol Fc
2D
3
B6H1
2
TTI-6
21
% P
hago
cyto
sis
Figure 4.
SIRPaFc with an IgG1 Fc tail has potent antitumor efficacy. A,M1
and M2 monocyte-derived macrophages were generated by priming for
24 hours with IFNg or IL4,respectively. Macrophage phagocytosis of
a DLBCL cell line (Toledo) was assessed by flow cytometry (%
phagocytosis) in the presence of SIRPaFc with anIgG1 Fc tail
(TTI-621) or an IgG4 Fc tail (both at 1 mmol/L concentration). Data
shown represent n ¼ 5 donors. B, NOD.SCID mice were preconditioned
withsublethal irradiation and anti-CD122 antibody (to deplete
residual NK cells) and then transplanted with AML cells from
patient #090191 by intrafemoral injection.Treatment with SIRPaFc (8
mg/kg i.p. 3�/week for 4 weeks) or control IgG1 Fc protein was
initiated 21 days post-transplantation. The percent AML
engraftment(% cells expressing human CD45 and CD33 markers) was
assessed by flow cytometry. Each symbol represents one mouse, bars
indicate mean values.P values were determined by one-way ANOVA.
Data shown are representative of two independent experiments. C,
Monocyte-derived macrophages weregenerated as described and primed
for 24 hourswith IFNg . Macrophagephagocytosis of aDLBCL cell line
(Toledo)was assessed by flowcytometry (%phagocytosis)in the
presence of TTI-621, anti-CD47 mAbs B6H12 or 2D3, or
isotype-matched controls (all at 1 mmol/L).
Petrova et al.
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-
The contribution of the Fc region raises the question of
whetherTTI-621 activity requires neutralization of the CD47 "do not
eat"signal, orwhether it simplyopsonizesCD47-expressing cells for
Fcreceptor–mediated destruction, similar to antibodies that
triggerclassical antibody-dependent cellular phagocytosis (ADCP).
Toaddress this, we compared the in vitro activity of TTI-621 to
twoisotype-matched (mouse IgG1) anti-CD47 antibodies: cloneB6H12,
which blocks the CD47–SIRPa interaction and thenon-neutralizing
clone 2D3. As shown in Fig. 4C, 2D3 inducesa low level of
phagocytosis, attributable to opsonization of CD47without blockade
of the CD47–SIRPa interaction. B6H12 ismoreeffective than 2D3 at
inducing the phagocytosis of a human Blymphoma target, indicating
that efficient phagocytosis in thissystem requires blockade of
theCD47–SIRPa interaction. TTI-621exhibits even greater activity
than B6H12, which presumablyreflects the combined effect of CD47
blockade andmore effectiveFc receptor engagement by the TTI-621
human IgG1 Fc region.
Collectively, these data show that SIRPaFc with an IgG1
tail(TTI-621) is significantly more potent at promoting
phagocytosisin vitro and controlling tumor burden in vivo, and that
both CD47blockade and Fc-mediated effector functions contribute to
themechanism of action of TTI-621.
TTI-621 induces anemia in non-human primates but bindsminimally
to human erythrocytes
A significant concern with CD47-blocking agents is related tothe
high expression of CD47 on human erythrocytes and the
potential for such agents to cause anemia, as seen in
preclinicalstudies (19). To assess the risk of anemia andother
adverse events,primate repeat-dose toxicology studies of
TTI-621were conductedin non-human primates. Cynomolgus monkeys were
selected asrelevant species based on the high CD47 sequence
homology(97.6% identity to human CD47) and cross-reactivity
studies
The principal dose-limiting toxicity observed in
cynomolgusmonkeys was anemia, which occurred at repeat doses of 3
mg/kgor greater. In addition to anemia, other cytopenias,
includingthrombocytopenia, lymphopenia, neutropenia, and
monocyto-penia were observed, although these were reversible and
withoutclinical sequelae (see Supplementary Fig. S2 for
representativehematology values). The bone marrow exhibited
evidence ofregenerative responses, notably erythropoiesis. No
effects wereobserved on neurologic, cardiovascular, or other
systems.
Despite the strong binding of TTI-621 to monkey erythrocytes,we
observed only minimal binding to human erythrocytes (Fig.5A), which
may be due to species-specific differences in themobility of CD47
in erythrocyte membranes (data not shown).Importantly, the low
binding profile of TTI-621 to human ery-throcytes is in contrast to
the strong binding demonstrated by fivedifferent anti-CD47 antibody
clones (Fig. 5A). Minimal bindingof TTI-621 was observed on
erythrocytes from all 43 healthydonors tested regardless of gender,
ABO blood group, or rhesusantigen status (Fig. 5B). Consistent with
these binding data, TTI-621 did not induce hemagglutination of
human erythrocytes invitro (Fig. 5C). The lack of significant
binding of TTI-621 tohuman
A
Hem
aggl
utin
atio
n sc
ore
(mea
n ±
SE)
0.1 1 10Concentration (nmol/L)
100
2D3B6H12BRIC1265F9TTI-621
1,000 10,0000.0101234567
B
TTI-6
21
BRIC
126
2D3
CC2C
6
B6H1
25F
9
hIgG1
Fc
mIgG
1
mIgG
2b0
500
1000
1500
100,000
200,000
300,000
CD47 mAbs Controls
Mea
n flu
ores
cenc
e in
tens
ity
C
TTI-621 5F9B6H12CC2C62D3BRIC126
Fluorescence intensity
Figure 5.
TTI-621 exhibits minimal binding to human erythrocytes. A, Human
erythrocytes were stained with saturating concentrations of TTI-621
or CD47-specificantibodies (clones BRIC126, 2D3, CC2C6, B6H12, or
5F9) and analyzed by flow cytometry. Representative histograms are
shown,with specific staining shown in blackand isotype control
staining in gray. B, Summary data showing the mean fluorescence
intensity for 43 erythrocyte donors. C, Hemagglutination assays
wereconducted with human erythrocytes and titrated amounts of
TTI-621- or CD47-specific antibodies. The extent of
hemagglutination was assessed by blindedscoring on a scale of 1 to
6, with 1 representing the absence of hemagglutination and 6
representing complete hemagglutination.
TTI-621 Is a Novel Antitumor Immune Checkpoint Inhibitor
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erythrocytes thus offers a significant advantage over
CD47-block-ing mAbs.
DiscussionApproved immune checkpoint inhibitors have extended
the
survival of multiple subgroups of cancer patients and thus
trans-formed modern oncology. Although the focus thus far has
beenon blockade of checkpoints that suppress T-cell responses
(e.g.,PD-1 or PD-L1), there is growing recognition that the
innateimmune system plays an important role in the initiation
andpropagation of enduring antitumor responses and CD47 hasrecently
emerged as a key checkpoint of innate immunity. Ourfindings
demonstrate that SIRPaFc (TTI-621) is an effective decoyreceptor
that enhances macrophage-mediated phagocytosis in abroad spectrum
of human hematologic and solid tumors, both invitro and in
xenograft models. More than 97% of primary patientsamples tested
were sensitive to the antitumor effects of TTI-621,suggesting that
this therapeutic approach will have broad appli-cability in human
cancer.
Importantly, blockade of CD47 by TTI-621 selectively
inducedphagocytosis of malignant cells over normal cells, providing
atherapeutic window for treatment of patients in the clinic.
Pref-erential macrophage phagocytosis of AML cells over normal
cordblood/bone marrow cells has also been reported for an
IgG4-based SIRPaFc fusion protein, even when nonmalignant cells
outnumbered the AML cells by a 2:1 ratio (27). In addition,
amouse anti-human CD47-neutralizing antibody did not
inducephagocytosis of normal peripheral blood B cells (7) or
normalhuman pancreatic ductal epithelial cells and pancreatic
stellatecells (15).
The specificity for tumor cells is thought to result from
theexpression of prophagocytic signals such as calreticulin
onmalig-nant cells but not on normal cells. Calreticulin is known
to triggermacrophage-mediated phagocytosis, and the phagocytosis
ofcancer cells induced by CD47 blockade can be completely
inhib-ited by antagonizing the interaction between calreticulin and
itsreceptor (28). It is hypothesized that tumor cells evade
phago-cytosis because the inhibitory CD47 pathway counterbalances
theprophagocytic calreticulin signal. Selectively targeting CD47
withTTI-621 promotes killing of tumor cells while sparing low
calre-ticulin-expressing normal cells. There are likely to be other
as yetunidentified prophagocytic signals on tumor cells that may
varydepending on the tissue type fromwhich the tumor is derived.
Thebroad efficacy of TTI-621 across tumor types suggests that
target-ing the CD47–SIRPa axis exploits the reliance of tumor cells
onCD47-mediated suppression of phagocytosis regardless of
theirspecific underlying prophagocytic signals.
The potent in vivo effects of TTI-621 were attenuated when
theIgG1 Fc tail of the fusion protein was substituted by an IgG4
tailwith reduced Fc-mediated effector function, or with an
inertmutated IgG4 tail, indicating that Fc effector function is
critical
Figure 6.
Proposed mechanism of action of TTI-621–mediated CD47 antitumor
activity. A, CD47 sends an inhibitory signal to macrophages by
binding to SIRPa.B, TTI-621 binds to CD47 on tumor cells and blocks
this interaction, (C) while engaging FcgR onmacrophages, (D)
leading tomacrophage-mediated phagocytosis oftumor cells. E,
Macrophages that have phagocytosed target cells can present tumor
peptides in the context of MHC to tumor-specific CD8þ T cells, (F)
activatingthe adaptive immune response and leading to destruction
of tumor cells by cytotoxic CD8þ T cells.
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-
for achieving maximum potency of SIRPaFc. These observa-tions
are consistent with a prior report demonstrating thatengineered
high affinity SIRPa monomers that bind stronglyto CD47 but lack an
Fc region are inactive on their own (29)and suggest that maximal
antitumor activity is obtainedthrough blockade of the CD47 "do not
eat" signal and simul-taneous delivery of a prophagocytic ("eat")
signal throughmacrophage FcgRs. In line with this, our data suggest
thatTTI-621 does not work simply by opsonization of CD47 andADCP,
but triggers phagocytosis through CD47 blockade aswell as
simultaneous activation through FcgRs.
Although CD47 has recently emerged as a promisingimmuno-oncology
target, concerns have been raised regardingthe potential for anemia
and an erythrocyte antigen sink, due tothe expression of high
levels of CD47 on human red blood cells(30, 31). In this regard,
TTI-621 exhibits an advantage over anti-CD47 antibodies, in that it
binds only minimally to human redblood cells. A similar observation
has recently been reported byan independent group (32). The minimal
binding of TTI-621 tohuman erythrocytes may be due to the
association of CD47with the erythrocyte spectrin cytoskeleton (30),
which results inreduced membrane mobility (33) and a consequent
failure tocluster CD47 effectively. Consistent with this theory, we
havepreviously shown strong binding of TTI-621 to human
ery-throcytes when CD47 is first preclustered using a
nonblockingCD47 antibody (34).
While it is acknowledged that TTI-621 binds to humanplatelets
and leukocytes (and thus may be associated with thedevelopment of
thrombocytopenia and/or leukopenia), theextremely low
erythrocyte–binding profile of TTI-621 offersseveral potential
advantages over anti-CD47mAbs that stronglybind to erythrocytes.
First, treatment with TTI-621 is less likelyto result in anemia.
CD47 is thought to protect erythrocytesfrom macrophage-mediated
clearance (2), and CD47-blockingantibodies are known to trigger
anemia in non-human pri-mates, a finding that may limit their
clinical utility despite theemployment of a priming strategy (19).
Second, minimalerythrocyte binding permits the use of an IgG1-based
fusionprotein, and thus maximizes macrophage phagocytosis oftumor
cells, without concern for opsonizing red blood cellsand targeting
them for destruction. Third, CD47-targetingagents that bind
erythrocytes may interfere with transfusiontyping and
cross-matching tests, as seen with other agents thatbind
erythrocytes (35, 36). Finally, TTI-621 is likely to have asuperior
pharmacokinetic profile compared with anti-CD47mAbs by avoiding the
significant antigen sink created by densecell surface expression of
CD47 on erythrocytes, enabling morecomprehensive engagement of
tumor-expressed CD47.
We demonstrated that CD47 blockade with SIRPaFc is effi-cacious
in AML and B lymphoma xenograft models, as well asin a B lymphoma
syngeneic model. Macrophages, in additionto their direct
tumoricidal properties, function as antigen-pre-senting cells, and
thus it is possible that enhancement ofphagocytosis by TTI-621
treatment may also result in anenhanced adaptive immune response.
In support of this, CD47antibody blockade has been shown to augment
tumor antigenpresentation and priming of an antitumor cytotoxic
CD8þ T-cell response in immunocompetent mice (29). In addition,CD47
blockade using a high-affinity SIRPa-variant-human Igfusion protein
has also been shown to promote tumor-specific
CD8þ T-cell responses through a dendritic cell–based mecha-nism
(37). These studies provide compelling evidence to sup-port the
hypothesis that TTI-621 has the potential to generatean enduring
antitumor response by acting at the nexus of theinnate and adaptive
immune systems. We propose a mecha-nism in which TTI-621 blocks the
CD47 "do not eat" signal ontumor cells while simultaneously
delivering prophagocyticsignals to macrophages through FcgRs,
leading to tumor cellphagocytosis, enhanced antigen presentation,
and stimulationof a tumor antigen–specific T-cell response (Fig.
6).
In summary, these data affirm CD47 as a critical regulator
ofimmune surveillance and provide a strong rationale for
thera-peutic targeting of CD47. Simultaneous blockade of the
inhibi-tory signal of CD47 with an associated engagement of FcgR
onmacrophages form the basis for clinical development of
TTI-621.Twophase I, open label,multicenter studies are currently
ongoingto evaluate TTI-621 in patients with relapsed/refractory
hemato-logic malignancies (NCT02663518) and solid
tumors(NCT02890368).
Disclosure of Potential Conflicts of InterestE.L. Sievers holds
ownership interest (including patents) in Trillium Ther-
apeutics Inc. J. Koropatnick reports receiving commercial
research grants fromTrillium Therapeutics Inc. S. Trudel and J.C.Y.
Wang report receiving othercommercial research support from
Trillium Therapeutics Inc. No potentialconflicts of interest were
disclosed by the other authors.
Authors' ContributionsConception and design: P.S. Petrova, N.N.
Viller, M. Wong, G.H.Y. Lin,T. Mutukura, E.L. Sievers, J.
Koropatnick, S. Trudel, J.C.Y. Wang, R.A. UgerDevelopment of
methodology: P.S. Petrova, M. Wong, X. Pang, G.H.Y. Lin,K. Dodge,
V. Chai, H. Chen, V. Lee, V. House, N.T. Vigo, T. Mutukura,M.
Charbonneau, T. Truong, S. ViauAcquisition of data (provided
animals, acquired and managed patients,provided facilities, etc.):
P.S. Petrova, N.N. Viller, M. Wong, X. Pang, K. Dodge,V. Chai, H.
Chen, V. Lee, V. House, N.T. Vigo, D. Jin, T. Mutukura,M.
Charbonneau, T. Truong, E. Linderoth, S.M. Vareki, R. Figueredo,M.
Pampillo, J. Koropatnick, N. Mbong, L. Jin, J.C.Y. WangAnalysis and
interpretation of data (e.g., statistical analysis,
biostatistics,computational analysis):P.S. Petrova,N.N.
Viller,M.Wong, X. Pang, K. Dodge,V. Chai, H. Chen, V. Lee, V.
House, N.T. Vigo, M. Charbonneau, T. Truong,E. Linderoth, E.L.
Sievers, S.M. Vareki, R. Figueredo, J. Koropatnick, L. Jin,R.A.
UgerWriting, review, and/or revision of the manuscript: P.S.
Petrova, N.N. Viller,M.Wong, X. Pang, L.D. Johnson, E.L. Sievers,
S.M. Vareki, S. Trudel, J.C.Y.Wang,R.A. UgerAdministrative,
technical, or material support (i.e., reporting or organizingdata,
constructing databases): P.S. Petrova, X. PangStudy supervision:
P.S. Petrova, X. Pang, S.M. Vareki, J. Koropatnick, J.C.Y.Wang,
R.A. Uger
AcknowledgmentsEilidh Williamson provided medical writing
assistance, under the sponsor-
ship of Trillium Therapeutics Inc.
Grant SupportThese studies were sponsored by Trillium
Therapeutics Inc., Mississauga,
Canada.The costs of publication of this article were defrayed in
part by the payment
of page charges. This article must therefore be hereby marked
advertisement inaccordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Received July 5, 2016; revised October 19, 2016; accepted
October 23, 2016;published OnlineFirst November 17, 2016.
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-
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Petrova, Natasja Nielsen Viller, Mark Wong, et al. Minimal
Erythrocyte BindingCheckpoint Inhibitor with Broad Antitumor
Activity and
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