tti-621 (sirpafc): a cd47-blocking innate immune...

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; 23(4);1068–79. �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.

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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 <0.4 EU/mgendotoxin.

AntibodiesThe anti-CD47 antibodies BRIC126 (Serotec), 2D3

(eBioscience), and CC2C6 (BioLegend) were obtained from com-mercial sources. Clones B6H12.2 (ATCCHB:9771) and 5F9 (19)were generated internally based on publicly available sequences.Rituximab (Hoffman-La Roche Ltd) was obtained from the Lon-don Health Sciences Centre pharmacy (London, Ontario,Canada).

CellsThe following human tumor cell lines were used: DLBCL (HT,

Ly1, Pfeiffer, NUDUL1, SUDHL4, SUDHL6, SUDHL8, SUDHL16,Toledo), multiple myeloma (MM1.s, 8226, H929, U266), non-DLBCL B-cell lymphomas (Raji, Namalwa, C1R, Ly5), AML (HL-60, KG-1, THP-1 and TF-1, AML-2), chronic myeloid leukemia(K562, KU812), acute T-cell leukemia (ENL-1, Jurkat), T-celllymphoma (HH, H9, SUPT1, DERL7), lung cancer (A549), epi-dermoid cancer (A431), ovarian cancer (OVCAR-3), colon cancer(DLD-1), breast cancer (SKBR3),melanoma (A375, SK-MEL-1, G-361, WM-115, SK-MEL-28), and Merkel cell carcinoma (MCC26,MKL-1). All cell lines were obtained from ATCC except MCC26and MKL-1 (Sigma-Aldrich), AML-2 (kindly provided by MarkMinden, University Health Network, Toronto, Canada), EN-1(kindly provided by Aaron Schimmer, University Health Net-work, Toronto, Canada) and Ly5 (kindly provided by DavidSpanner, Sunnybrook Research Institute, Toronto, Canada). Themouse A20 B lymphoma line was obtained from ATCC.

Translational Relevance

SIRPaFc (TTI-621) is a novel innate immune checkpointinhibitor designed to: (i) bind human CD47 on tumor cellsand prevent it from delivering inhibitory signals to macro-phages and (ii) engage Fc gamma receptors (FcgR) expressedby macrophages to further enhance phagocytosis. Here weshow that TTI-621 avidly binds to CD47 on a wide range ofhuman tumor cells and selectively promotes macrophage-mediated phagocytosis of both hematologic and solid tumorcells. Furthermore, these in vitro effects translate into signifi-cant antitumor activity in mouse models of leukemia andlymphoma. Importantly, unlike anti-CD47 antibodies, TTI-621 binds minimally to human erythrocytes, minimizingpotential toxicity related to hemolytic anemia. On the basisof the demonstrated antitumor activity in the context ofminimal erythrocyte binding, two phase I, multicenter studieshave been initiated to evaluate TTI-621 in subjects withrelapsed/refractory hematologic malignancies and solidtumors (NCT02663518 and NCT02890368, respectively).

TTI-621 Is a Novel Antitumor Immune Checkpoint Inhibitor

www.aacrjournals.org Clin Cancer Res; 23(4) February 15, 2017 1069




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.).

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.

Clin Cancer Res; 23(4) February 15, 2017 Clinical Cancer Research1070




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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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.





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.

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. Tumor

volumes 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 maximumpermissible 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-cell

(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).






acute 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 withhematologicmalignancies 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 threetimes/week for 4 weeks. Although control-treated animalsexhibited significant engraftment, particularly in the injectedbone marrow, TTI-621 treatment significantly reduced thetumor burden in bone marrow and spleen (Fig. 3A and B). Infact, tumor cells were undetectable in most animals followingTTI-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) were

constructed 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)as well as to the low-affinity receptors FcgRII (CD32) andFcgRIII (CD16). To determine whether the IgG1 Fc tail isrequired for maximum potency, we compared the in vitroactivity of TTI-621 with a variant SIRPaFc in which the IgG1Fc region of TTI-621 was replaced with an IgG4 Fc tail. IgG4 Fcregions bind well to CD64 but have weaker interactions thanIgG1 with CD32 and CD16 (25). We compared the propha-gocytic activity of both SIRPaFcs using classically activated(M1) and alternatively activated (M2) macrophages. Wehave previously shown that M1 macrophages are CD32hi

CD64hiin vitro, whereas M2 macrophages are CD32hi CD64lo

(26). TTI-621 enhanced phagocytosis by both macrophagesubsets 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 forSIRPaFc's enhancement of phagocytosis by both M1 and M2macrophages.

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 tumor

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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 byflowcytometry (%phagocytosis)in the presence of TTI-621, anti-CD47 mAbs B6H12 or 2D3, or isotype-matched controls (all at 1 mmol/L).






burden 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).

The contribution of the Fc region raises the question ofwhetherTTI-621 activity requires neutralization of the CD47 "do not eat"signal, or whether it simply opsonizes CD47-expressing cells forFc receptor–mediated destruction, similar to antibodies thattrigger classical antibody-dependent cellular phagocytosis(ADCP). To address this, we compared the in vitro activity ofTTI-621 to two isotype-matched (mouse IgG1) anti-CD47 anti-bodies: clone B6H12, which blocks the CD47–SIRPa interactionand the non-neutralizing clone 2D3. As shown in Fig. 4C, 2D3induces a low level of phagocytosis, attributable to opsonizationof CD47 without blockade of the CD47–SIRPa interaction.B6H12 is more effective than 2D3 at inducing the phagocytosisof a human B lymphoma target, indicating that efficient phago-cytosis in this system requires blockade of the CD47–SIRPainteraction. TTI-621 exhibits even greater activity than B6H12,which presumably reflects the combined effect of CD47 blockadeandmore effective Fc receptor engagement by the TTI-621 humanIgG1 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 thepotential 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 healthy

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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.

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donors 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 tohumanerythrocytes 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 cellsoutnumbered 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 criticalfor 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 obtained

through 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-specificCD8þ 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 on






tumor 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. Viau

Acquisition 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.

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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2017;23:1068-1079. Published OnlineFirst November 17, 2016.Clin Cancer Res Penka S. Petrova, Natasja Nielsen Viller, Mark Wong, et al. BindingInhibitor with Broad Antitumor Activity and Minimal Erythrocyte

Fc): A CD47-Blocking Innate Immune CheckpointαTTI-621 (SIRP

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