www.sciencemag.org/cgi/content/full/science.1238856/DC1

Supplementary Materials for

Engineered SIRPα Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies

Kipp Weiskopf, Aaron M. Ring, Chia Chi M. Ho, Jens-Peter Volkmer, Aron M. Levin, Anne Kathrin Volkmer, Engin Özkan, Nathaniel B. Fernhoff,

Matt van de Rijn, Irving L. Weissman, K. Christopher Garcia*

*Corresponding author. E-mail: kcgarcia@stanford.edu

Published 30 May 2013 on Science Express DOI: 10.1126/science.1238856

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S16 Table S1 References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.1238856/DC1)

Movies S1 and S2

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Materials and Methods Protein expression and purification.

The human CD47 IgSF domain (residues 1-117), with a C15G mutation (17) and C-terminal 8× histidine tag, was cloned into pAcGP67a for secretion from Trichoplusia ni (High Five) cells using baculovirus and purified by nickel–nitrilotriacetic acid (Ni-NTA) and size exclusion chromatography with a Superdex-75 column. To generate glycan-minimized CD47 for crystallography, CD47 was co-expressed with endoglycosidase-H (endoH) in the presence of kifunensine. Monomeric wild-type human SIRPα allele 1 domain 1 (residues 1-118) and FD6 were expressed in High Five cells as described for the CD47 IgSF domain. The FD6 concatamer was constructed by site-overlap extension (SOE) PCR of two FD6 amplicons joined by a (Gly4Ser)2 linker and cloned into pAcGP67a for expression. The 2F5 leucine zipper protein was constructed by SOE PCR to generate a fusion to the GCN4 leucine zipper domain (RMKQLEDKVEELLSKNYHLENEVARLKKLVGAASGAD), then cloned into pAcGP67a. Monomeric SIRPα variant CV1 and mouse C57BL/6 SIRPα domain 1 were expressed as MBP-fusions in the periplasm of BL-21(DE3) E. coli using a modified pMal-p2X expression vector (New England Biolabs) containing a rhinovirus 3C protease cleavage site after the MBP tag and a C-terminal 8× histidine tag. Cells were induced at an OD600 of 0.8 with 1 mM IPTG and incubated with shaking at 22 °C for 24 hours. Periplasmic protein was obtained by osmotic shock and the MBP-fusion proteins were purified using Ni-NTA chromatography. Eluted proteins were digested with 3C protease at 4 °C for 12 hours to remove MBP and further purified by an additional Ni-NTA chromatography step, followed by size exclusion chromatography with a Superdex-S75 column. For in vitro phagocytosis assays and in vivo experiments, endotoxin was removed using Triton X-114 as previously described (31) and endotoxin removal confirmed using the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (Genscript). SIRPα-Fc fusions were produced by cloning SIRPα variants into a modified pFUSE-hIgG4-Fc vector (Invivogen) with an IL-2 signal sequence and engineered Ser228 Pro mutation (32). Proteins were expressed by transient transfection in Freestyle 293-F cells (Invitrogen) and purified over HiTrap Protein A columns (GE Healthcare). Chimeric anti-CD47 clone B6H12-hIgG4 was recombinantly produced by stable expression in CHO cells (Lonza).

To obtain biotinylated CD47 and SIRPα, proteins were expressed with a carboxy-terminal biotin acceptor peptide tag (GLNDIFEAQKIEWHE) and purified as described above. The purified proteins were biotinylated in vitro with BirA ligase and then repurified from the reaction mixture by size exclusion chromatography. Preparation of Fab fragments of B6H12.

B6H12 antibody was desalted into 20 mM sodium citrate pH 6.0, 25 mM cysteine, 5 mM EDTA and diluted to a concentration of 4 mg/mL. The antibody was then mixed with 250 µL immobilized ficin resin (Thermo Scientific) per mL of antibody and incubated with rotation at 37 °C for five hours. The digested fragments were purified by passing the reaction mixture over a monoQ column (B6H12 Fab resided in the flow-through), followed by gel filtration with a Superdex-200 column.

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Yeast display and construction of the first-generation library of SIRPα variants.

The N-terminal V-set domain of human SIRPα (residues 1-118) was displayed on the surface of S. cerevisiae strain EBY100 as a C-terminal fusion to Aga2 using the pCT302 vector as previously described (33). For display of mouse CD47 on yeast, the mouse CD47 IgSF domain was fused to the N-terminus of Aga2 (34) to ensure a free N-terminus for pyrogluatmic acid formation (17).

Based on the crystal structure of the wild-type human SIRPα:CD47 complex (17), we designed a pooled first-generation library that was generated by two separate assembly PCR reactions that randomized the CD47-contact residues and the hydrophobic ‘core’ residues of SIRPα, respectively, using the following primer sets with degenerate codons:

Contact residue PCR primer set, randomizing Ser29=RST, Leu30=NTT, Ile31=NTT, Pro32=CNT, Val33=NTT, Gly34=RST, Pro35=CNT, Gln52=SAW, Lys53=ARG, Glu54=SAW, Ser66=RST, Thr67=RST, Lys68=ARG, Arg69=ARG, Phe74=NTT, Lys93=ANG, Lys96=ANG, Gly97=RST, Ser98=RST, and Asp100=RAS:

5’GAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTATCAGTTGCAGCT3’,

5’GGTCACAGTGCAGTGCAGAATGGCCGACTCTCCAGCTGCAACTGATACGGA3’,

5’CTGCACTGCACTGTGACCRSTNTTNTTCNTNTTRSTCNTATCCAGTGGTTCAGAGGA3’,

5’ATTGTAGATTAATTCCCGGGCTGGTCCAGCTCCTCTGAACCACTGGAT3’,

5’CGGGAATTAATCTACAATSAWARGSAWGGCCACTTCCCCCGGGTAACAACTGTTTCAGAG3’,

5’GTTACTGATGCTGATGGAAANGTCCATGTTTTCCYTCYTASYASYCTCTGAAACAGTTGTTAC3’,

5’TCCATCAGCATCAGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTG3’,

5’TCCAGACTTAAACTCCGTWTYAGGASYASYCNTCCGGAACNTCACACAGTAGTAGGTGCC3’,

5’ACGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGTGCCAAACCCTCT3’

‘Core’ residue PCR primers, randomizing Leu4, Val6, Val27, Ile36, Phe38, Leu47, Ile49, Tyr50, Phe57, Val60, Met72, Phe74, Ile76, V92, Phe94, and Phe103 to NTT:

5’GGATCCGAGGAGGAGNTTCAGNTTATTCAGCCTGACAAGTCCGTATCAGTTGCAGCTGGAGAG3’,

5’GGGCCCCACAGGGATCAGGGAGGTAANAGTGCAGTGCAGAATGGCCGACTCTCCAGCTGCAAC3’,

5’CTGATCCCTGTGGGGCCCNTTCAGTGGNTTAGAGGAGCTGGACCAGCCCGGGAA3’,

5’GTGGCCTTCTTTTTGATTAANAANAANTTCCCGGGCTGGTCCAGC3’ 5’AATCAAAAAGAAGGCCACNTTCCCCGGNTTACAACTGTTTCAGAGTCC

ACAAAGAGAGAAAAC3’,

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5’GCCGGCATCTGCTGGGGTGATGTTACTGATGCTAANGGAAANGTCAANGTTTTCTCTCTTTGTGGA-3’,

5’ACCCCAGCAGATGCCGGCACCTACTACTGTNTTAAGNTTCGGAAAGGGAGCCCTGACACGGAG3’,

5’AGAGGGTTTGGCACGCACAGACAGCTCAGTGCCTGCTCCAGACTTAANCTCCGTGTCAGGGCTCCC3’

The PCR products were further amplified with primers containing homology to the pCT302 vector, combined with linearized pCT302 vector DNA, and co-electroporated into EBY100 yeast. The resulting library contained 4.0×108 transformants. Selection of the first-generation library.

Transformed yeast were expanded in SDCAA liquid media at 30 °C and induced in SGCAA liquid medium at 20 °C. All selection steps were carried out at 4 °C. For the first round of selection, 4.0×109 induced yeast, representing ten-fold coverage of the number of library transformants, were resuspended in 5 mL PBE (phosphate buffered saline supplemented with 0.5 % bovine serum albumin and 0.5 mM EDTA). Yeast were mixed with 500 µL paramagnetic streptavidin microbeads (Miltenyi) that were pre-coated with biotinylated CD47 and the mixture was incubated with rotation for one hour. The yeast were pelleted by centrifugation at 5,000g for five minutes and washed twice with 10 mL PBE. Magnetically-labeled yeast were resuspended in 5 mL PBE and separated with an LS MACS column according to the manufacturer’s instructions (Miltenyi). Eluted yeast were pelleted, resuspended in SDCAA medium, and expanded for the next round of selection. Four additional rounds of selection were performed similarly to the first round with the following modifications: 1.0×108 yeast were resuspended in 500 µL PBE containing FITC-labeled anti-c-Myc antibody (Miltenyi) or successively decreasing concentrations of biotinylated CD47 protein, from 1 µM to 100 nM. After incubation for one hour, yeast were washed with PBE and for selections with CD47, labeled with streptavidin-PE (Invitrogen) or streptavidin-Alexa Fluor 647 (produced in-house) for 15 minutes. Yeast were washed twice more with PBE and magnetically labeled with 50 µL of the appropriate anti-fluorophore microbeads (anti-FITC, anti-PE, or anti-Alexa Fluor 647; Miltenyi) for 15 minutes. Yeast were washed once, resuspended in 3 mL PBE, and separated with an LS column as in the first round. The resulting first-generation SIRPα variants bound CD47 with a 20-100-fold higher affinity than wild-type SIRPα as measured by surface plasmon resonance. Construction of the second-generation library.

To obtain SIRPα variants with even higher affinity, we constructed a second-generation library that randomized thirteen residues that were repeatedly mutated after the first generation of directed evolution. The second-generation library and selection strategy was designed to achieve full-coverage of mutations to these residues. The second generation library was generated and transformed identically as the first generation library, but was assembled with the following primers, randomizing Leu4=NTT, Val6=NTT, Val27=NTT, Ile31=WYT, Glu47=SWA, Lys53=ARG, Glu54=SAK, His56=CNT, Ser66=RST, Lys68=ARG, Val92=NTT, Phe94=NTT, Phe103=NTT:

5’GGATCCGAGGAGGAGNTTCAGNTTATTCAGCCTGACAAGTCCGTATC3’,

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5’GTGCAGTGCAGAATGGCCGACTCTCCAGCTGCAACTGATACGGACTTGTCAGGCTGAA3’,

5’CATTCTGCACTGCACTNTTACCTCCCTGWYTCCTGTGGGGCCCATCCAG3’,

5’CGGGCTGGTCCAGCTCCTCTGAACCACTGGATGGGCCCCACAGG3’, 5’GAGCTGGACCAGCCCGGSWATTAATCTACAATCAAARGSAKGGCCNT

TTCCCCCGGGTAACAACTGTTTCAGAG3’, 5’GAAAAGTCCATGTTTTCTCTCYTTGTASYCTCTGAAACAGTTGTTAC3’, 5’AGAGAAAACATGGACTTTTCCATCAGCATCAGTAACATCACCCCAGCA

GATGCCGGCAC3’, 5’CTCCGTGTCAGGGCTCCCTTTCCGAANCTTAANACAGTAGTAGGTGCC

GGCATCTGCTG3’, 5’GAGCCCTGACACGGAGNTTAAGTCTGGAGCAGGCACTGAGCTGTCTGT

GCGTGCCAAACCCTCT3’ The resulting library contained 2.0×108 transformants.

Selection of the second-generation library.

For the first two rounds of selection of the second-generation library, yeast were selected with monomeric, biotinylated CD47 protein, as in rounds two through five of the first generation selections. The first round was selected with 20 nM biotinylated CD47 and the second round with 1 nM biotinylated CD47, using a larger staining volume (10 mL PBE) to avoid ligand depletion. For all subsequent rounds of selection, kinetic selection was performed. Briefly, yeast were stained with 20 nM biotinylated CD47 for one hour, washed with PBE, and then resuspended in 500 µL PBE containing 1 µM non-biotinylated CD47. The cells were incubated at 25 °C for 90 minutes (round three) or 300 minutes (rounds four and five), after which they were washed with ice-cold PBE and stained with fluorescently-labeled streptavidin. For rounds one through four, yeast were separated using MACS, as described for the first generation library. For the fifth round of selection, yeast were co-labeled with FITC-labeled anti-c-Myc and streptavidin-Alexa Fluor 647 and selected with a FACSAria cell sorter (BD Biosciences). Surface plasmon resonance (SPR).

Experiments were conducted with a Biacore T100 at 25 °C. Protein concentrations were quantified by 280 nm absorbance with a Nanodrop2000 spectrometer (Thermo Scientific). A Biacore SA sensor chip (GE Healthcare) was used to capture biotinylated CD47 (Rmax ~150 RU). An unrelated biotinylated protein was immobilized with an RU value matching that of the reference surface to control for nonspecific binding. Measurements were made with serial dilutions of the SIRPα variants in HBS-P+ buffer (GE Healthcare). The CD47 surface was regenerated by three 60 second injections of 2 M MgCl2. All data were analyzed with the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model. Crystallization and structural determination of FD6:CD47 complex

Glycan-minimized CD47 and E. coli-derived FD6 were mixed at a 1:1 ratio and digested with carboxypeptidases A and B to remove their C-terminal 8x histidine tags. The digested FD6:CD47 complex was purified by gel filtration into HEPES buffered

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saline (HBS; 10 mM HEPES pH 7.4, 150 mM NaCl) with a Superdex-75 column and concentrated to 22 mg/mL. Crystals were obtained by addition of 0.1 µL protein to an equal volume of 2.0 M ammonium sulfate and 0.1 M Tris pH 7.3, and were cryoprotected in paraffin oil. Diffraction studies were performed at beamline 8.2.1 at the Advanced Light Source (Berkeley, CA, USA). An anisotropic 1.9 Å dataset was obtained and processed with HKL-2000 (35).

The FD6:CD47 complex was solved by molecular replacement with the individual models of CD47 and SIRPα from Protein Data Bank accession code 2JJS. Refinement was carried out using PHENIX (36) and model adjustment performed with COOT (37). Initial refinement used rigid body, coordinate, and real-space refinement, along with individual atomic displacement parameter refinement. TLS refinement was added in later refinement iterations. Cancer cell lines and GFP-luciferase+ transduction.

DLD-1 cells (ATCC), HT-29 cells (ATCC), Raji cells (ATCC), Jurkat cells (ATCC), and 639-V cells (DSMZ) were cultured in RPMI+GlutaMax (Invitrogen) supplemented with 10% fetal bovine serum (Omega Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). BT474M1 cells (38) were cultured in DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (Omega Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). GFP-luciferase+ lines were generated by transduction using a pCDH-CMV-MCS-EF1 puro HIV-based lentiviral vector (Systems Biosciences) engineered to express an eGFP-luciferase2 (pgl4) fusion protein (12). Stable lines were created by sorting for GFP expression on FACSAria II cell sorters (BD Biosciences). GFP-luciferase+ SK-BR-3 cells were a gift of B. di Robilant and M. Clarke (Stanford University). Cell-based CD47 binding assays.

Varying concentrations of biotinylated SIRPα monomers, SIRPα-hIgG4 fusion proteins, or anti-CD47 antibodies were incubated with cancer cells as indicated. Binding of biotinylated monomers was detected using 100 nM Alexa Fluor 647-conjugated streptavidin as a secondary staining reagent and was analyzed on an Accuri C6 flow cytometer (BD Biosciences). Binding of SIRPα-hIgG4 fusion proteins or anti-CD47 antibodies was detected with goat anti-human IgG antibody (Invitrogen) and was analyzed on an LSRFortessa with high-throughput sampler (BD Biosciences). Data represent the mean fluorescence intensity normalized to maximal binding for each class of reagents, and points were fit to sigmoidal dose-response curves using Prism 5 (Graphpad). Cell-based CD47 blocking assays.

Biotinylated WTa1 SIRPα was incubated with Alex Fluor 647-conjugated streptavidin to form WTa1 SIRPα tetramers. 100 nM WTa1 SIRPα tetramers were combined with titrating concentrations of CD47 antagonists and simultaneously added to 50,000 GFP-luciferase+ Raji cells. Cells were incubated for 30 min at 4 °C then washed to remove unbound tetramer. Samples were stained with DAPI (Sigma) to exclude dead cells, and fluorescence was assayed using an LSRFortessa with a high throughput sampler (BD Biosciences). Data represent the geometric mean fluorescence intensity

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analyzed using FlowJo v9.4.10 (Tree Star) normalized to maximal tetramer binding, and were fit to sigmoidal dose-response curves using Prism 5 (Graphpad). Macrophage derivation.

Leukocyte reduction system (LRS) chambers were obtained from the Stanford Blood Center from anonymous donors, and peripheral blood mononuclear cells were enriched by density gradient centrifugation over Ficoll-Paque Premium (GE Healthcare). Monocytes were purified on an autoMACS Pro Separator (Miltenyi) using anti-CD14 microbeads (Miltenyi) and differentiated to macrophages by culture for 7-10 days in IMDM+GlutaMax (Invitrogen) supplemented with 10% AB-Human Serum (Invitrogen) and 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). NSG and RFP+ mouse macrophages were generated and evaluated as previously described (12). Briefly, bone marrow cells were isolated from C57BL/Ka Rosa26 mRFP1 transgenic mice and differentiated in IMDM+GlutaMax supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin, and 10 ng/mL murine M-CSF (Peprotech). Evaluation of macrophage phagocytosis.

Phagocytosis assays were performed by co-culture of 50,000 macrophages with 100,000 GFP+ tumor cells for two hours in serum-free medium, then analyzed using an LSRFortessa cell analyzer with high throughput sampler (BD Biosciences). Antibodies used for treatment included: mouse IgG1 isotype control (eBioscience), anti-CD47 clone 2D3 (eBioscience), anti-EpCam (BioLegend), trastuzumab (Genentech), cetuximab (Bristoll-Myers Squibb), rituximab (Genentech), and the anti-hCD20 isotype collection (Invivogen). Primary human macrophages were identified by flow cytometry using anti-CD14, anti-CD45, or anti-CD206 antibodies (BioLegend). NSG macrophages were identified by anti-F4/80 (BioLegend) and RFP+ mouse macrophages were identified by intrinsic fluorescence. Dead cells were excluded from the analysis by staining with DAPI (Sigma). Phagocytosis was evaluated as the percentage of GFP+ macrophages using FlowJo v9.4.10 (Tree Star) and was normalized to the maximal response by each independent donor against each cell line. Statistical significance was determined by one-way or two-way ANOVA with Bonferroni correction, and, as indicated, data were fit to sigmoidal dose-response curves using Prism 5 (Graphpad). Sorting of macrophage populations after phagocytosis.

2.5 million human macrophages were combined with 5 million GFP+ DLD-1 cells and 100 nM CV1-hIgG4 in serum-free medium and incubated for two hours. Macrophages were identified by staining with anti-CD45, and macrophages populations were sorted on a FACSAria II cell sorter. Cells from sorted populations were centrifuged onto microscope slides and imaged on a DMI6000 B inverted microscope (Leica) for fluorescence. Slides were then stained with Modified Wright-Giemsa stain (Sigma-Aldrich) according to the manufacturer’s instructions and imaged on a DM5500 B upright light microscope (Leica). Live-cell imaging of phagocytosis.

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500,000 Raji cells were labeled with 0.5 µM CFSE (Invitrogen) and co-cultured with 50,000 RFP+ macrophages and imaged using a BioStation IMQ (Nikon) equilibrated to 37 °C and 5 % carbon dioxide. Mice.

Nod.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice were used for all in vivo experiments. Mice were engrafted with tumors at approximately 6-10 weeks of age, and experiments were performed with age and sex-matched cohorts of 8-15 mice. Mice were maintained in a barrier facility under the care of the Stanford Veterinary Services Center and handled according to protocols approved by the Stanford University Administrative Panel on Laboratory Animal Care. Tumor models.

Bladder cancer was modeled by engraftment of 1.25×105 GFP-luciferase+ 639-V cells into the dorsal subcutaneous tissue of NSG mice in 25 % Matrigel (BD Biosciences). GFP-fluorescence from tumor nodules was visualized on an M205 FA fluorescent dissecting microscope (Leica) fitted with a DFC 500 camera (Leica). 1×106 GFP-luciferase+ Raji cells were engrafted subcutaneously on the lower flank for a localized model of human lymphoma. In all models, treatment was initiated after establishment and growth of tumors and was continued as indicated. All SIRPα variants, rituximab, and alemtuzumab (Genzyme) were administered by intraperitoneal injection of 200 µg/dose. Tumor growth was monitored by bioluminescence imaging, and tumor dimensions were measured to calculate volumes according to the ellipsoid formula (π/6×length×width2). Statistical significance was determined by Mann-Whitney test or Kruskal-Wallis with Dunn’s correction. Survival was analyzed by Mantel-Cox test. Large tumor models.

For a model of high-burden lymphoma, 1×106 GFP-luciferase+ Raji cells were engrafted subcutaneously on the lower flanks of 10 NSG mice. Tumors were allowed to grow for approximately 3 weeks, then mice were randomized into two cohorts of 5 animals for daily intraperitoneal treatment with 200 µg rituximab plus PBS or 200 µg rituximab plus 200 µg CV1 monomer. Treatment was continued for one week then mice were euthanized. Tumors were dissected out and weighed, then used for immunohistochemical analysis. For an orthotopic model of human breast cancer, 2.5×106 GFP-luciferase+ BT474M1 cells were engrafted in the mammary fat pads of female NSG mice. Tumors were allowed to grow for approximately two months, then mice were randomized into four treatment groups with 8-10 animals per cohort. Intraperitoneal treatment was initiated with vehicle control (PBS), 200 µg CV1 monomer daily, 150 µg trastuzumab bi-weekly, or the combination of CV1 monomer plus trastuzumab. For both models, tumor volumes were measured every 3-4 days and significance was determined using Mann-Whitney test with Bonferroni correction for the indicated comparisons. Experiments were piloted with independent cohorts of mice revealing similar trends. Mouse hematologic analysis.

Blood was drawn from the retro-orbital plexus and collected in dipotassium-EDTA Microtainer tubes (BD Biosciences). Hematologic parameters were evaluated using a

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HemaTrue analyzer (Heska). Statistical significance was determined by two-way ANOVA with Bonferroni correction. Binding of SIRPα-Fc variants to mouse whole blood was determined by flow cytometry using Alexa Fluor 647 goat anti-human IgG antibody (Invitrogen). Bioluminescence imaging.

Anesthetized mice were injected with 200 µL D-luciferin (firefly) potassium salt (Biosynth) reconstituted at 16.67 mg/mL in sterile PBS. Bioluminescence imaging was performed using an IVIS Spectrum (Caliper Life Sciences) over 20 minutes to record maximal radiance. Peak total flux values were assessed from the anatomical region of interest using Living Image 4.0 (Caliper Life Sciences) and were used for analysis. Immunohistochemistry analysis.

Tissue was fixed in 10% neutral buffered formalin and embedded in paraffin. Staining was performed as previously described (13). Briefly, slides were cut in 4 µm sections, deparaffinized in xylene, and hydrated with a graded series of alcohol. Slides were boiled in citrate solution (pH 6) for 12 min. Macrophages were identified by staining with rat anti-mouse F4/80 clone BM8 (Invitrogen). Staining was visualized using the EnVision+ System-HRP (DAB) for use with primary mouse antibodies (Dako). Macrophage infiltration was quantified by scoring the intensity of infiltration from 1-5 (low-high) by evaluators who were blind to the treatment conditions. Scores from each evaluator were averaged to generate an overall score for each tumor. Toxicity studies in cynomolgus macaques.

Studies were performed by Charles River Laboratories (Reno, NV) in accordance with AAALAC international guidelines. Therapeutic treatments were administered by intravenous injection into female cynomolgus macaques weighing approximately 3 kg.

Supplementary Text Author contributions

K.W. and A.M.R conceived of engineering high-affinity SIRPα variants, designed all experiments, and wrote the manuscript. A.M.R., C.C.M.H, and A.M.L. performed directed evolution of the high-affinity SIRPα variants with yeast display. K.W. and A.M.R. further engineered the CV1 variant and SIRPα-Fc fusions proteins. A.M.R. and C.C.M.H conducted SPR affinity measurements. A.M.R. crystallized the FD6:CD47 complex, and A.M.R. and E.Ö. determined and refined the structure. K.W. and A.M.R. prepared proteins for functional and in vivo studies. K.W. and A.M.R. performed binding/blocking assays on cancer cells. K.W. performed in vitro phagocytosis experiments. K.W., J.P.V., A.V., and N.B.F. performed in vivo experiments. M.v.d.R. performed pathological analysis. I.L.W. and K.C.G. supervised the research and edited the manuscript.

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Fig. S1. Library design and sequences from first-generation selections. A Schematic of CD47 blockade by soluble high-affinity SIRPα. (Left) In the basal state, CD47 expression on cancer cells activates SIRPα on macrophages, initiating an inhibitory cascade through SHP 1 and 2 tyrosine phosphatases and preventing cancer cell phagocytosis. (Right) Soluble, high-affinity SIRPα protein competitively antagonizes CD47 and prevents engagement with SIRPα on macrophages, thereby disinhibiting phagocytosis. B Schematic representation of yeast surface-display of the human SIRPα V-set Ig domain (domain 1). Yeast clones (grey cells) present different variants of SIRPα (colored bumps). Inset indicates the linkage of SIRPα to the yeast cell surface via fusion with Aga2 and selection with biotinylated CD47. C Left: Table of randomized positions of the ‘contact residue’ library with possible amino acid variants and the location of the randomized positions within SIRPα. Right: Location and description of the randomized positions for the non-contact, ‘core residue’ library. SIRPα is depicted in green, CD47 is depicted in magenta, and the randomized positions are represented as space filling side chains. D Summary of sequences of SIRPα variants obtained after the first-generation of selections. The position of the mutated residues and their corresponding sequence in wild-type allele 1 is denoted at the top of the table. Blue shading indicates ‘contact’ mutations occurring at the SIRPα:CD47 interface. Italic font indicates mutations at positions that were not randomized in the pooled library (Glu47 and His56).

Fig. S2. Library design of second-generation selections. Table of randomized positions and possible amino acid substitutions for the second-generation library and the position of the variable residues within the structure of SIRPα. SIRPα is depicted in green, CD47 is depicted in magenta, and the randomized positions are represented as space filling side chains.

Fig. S3. Representative electron density map of FD6:CD47 complex. 2mFo-DFc electron density map contoured at 2.0σ. Modeled amino acid residues are depicted as sticks, with FD6 residues in yellow and CD47 residues in green. Pyroglutamic acid residue 1 of CD47 is indicated as PCA1 above the corresponding residue and density.

Fig. S4. High-affinity SIRPα variants potently bind and block CD47 on cancer cells. A Titration curves of wild-type SIRPα allele 1 monomer (WTa1 mono, pink), wild-type SIRPα allele 1 tetramer (WTa1 tetramer, maroon), or high-affinity SIRPα variants (FD6, FA4, green) binding to Jurkat leukemia cells. Data are mean ± SD. B CD47 blocking assay on Jurkat cells. CD47 antagonists were added in competition with Alexa Fluor 647-conjugated WTa1 SIRPα tetramer. Blocking was tested with a first generation SIRPα mutant as a monomer (1A5 mono, teal), a second generation SIRPα mutant as a monomer (FD6 mono, green), a second generation SIRPα mutant as an Fc fusion with human IgG4

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(FD6-hIgG4, blue), and anti-CD47 clone B6H12 (orange). Data are mean ± SD. C Binding of wild-type SIRPα-Fc proteins (WTa1-hIgG4, pink; WTa2-hIgG4, purple), high-affinity SIRPα-Fc proteins (FD-hIgG4, CV1-hIgG4, green), and anti-CD47 antibody clone B6H12 (B6H12-hIgG4, orange) to DLD-1 colon cancer cells.

Fig. S5. Phagocytosis in response to CD47-blockade is Fc-dependent. A Schematic depictions of the different SIRPα variants studied with added regions indicated in blue. SIRPα concatamers are formed as a single chain connected by a flexible linker. Dimers can be formed from two independent chains by addition of leucine zipper domains or Fc domains. B Representative S-200 gel filtration chromatograph of CV1-hIgG4 indicating the absence of significant aggregation following purification. The void peak is indicated at 8.42 mL and the CV1-hIgG4 elution peak is at 13.44 mL. C Phagocytosis assay with RFP+ mouse macrophages and GFP+ Raji cells. CD47 blockade with saturating concentrations of high-affinity SIRPα variant CV1 monomer or Fab fragments produced from anti-CD47 clone B6H12 induce equivalent levels of phagocytosis, while treatment with high-affinity SIRPα-Fc or intact anti-CD47 antibodies produced maximal phagocytosis. D Phagocytosis assay performed with primary human macrophages and GFP+ DLD-1 human colon cancer cells. Treatment with high-affinity SIRPα dimers that lack a pro-phagocytic stimulus do not induce substantial levels of phagocytosis, while treatment with high-affinity SIRPα-Fc induces maximal phagocytosis. All SIRPα variants used at 1 µM. C-D Data represent mean ± SD. ns = not significant; ****p<0.0001 versus non-Fc variants by one-way ANOVA with Bonferroni correction.

Fig. S6. Microscopy and fluorescence-activated cell sorting of macrophages demonstrates high-affinity SIRPα variants induce phagocytosis of cancer cells. A Representative images of phagocytosis assays performed with GFP+ DLD-1 colon cancer cells and primary human macrophages with vehicle control (PBS) or a high-affinity SIRPα variant fused to human IgG4 (CV1-hIgG4). Black arrows and inset show macrophages with ingested cancer cells. B Representative plots showing phagocytosis assays analyzed by flow cytometry. Phagocytosis was quantified as the percentage of macrophages (CD45+; blue gate) that became GFP+ (red gate). C Primary human macrophages and GFP+ DLD-1 colon cancer cells were co-cultured in the presence of 100 nM CV1-hIgG4. Phagocytosis was quantified as the percentage of CD45+ macrophages that became GFP+. Macrophage (MΦ) populations were sorted by flow cytometry for those that were GFP-negative (blue), GFP-low (red), or GFP-high (green). D Sorted GFP-high macrophages contained engulfed tumor cells as visualized by microscopy under brightfield transmitted light (upper image) and fluorescent light (merged lower image; red = CD45, green = GFP). E Wright-Giemsa staining of DLD-1 colon cancer cells. F Wright-Giemsa staining of sorted GFP-negative macrophage populations lacking engulfed material. G Wright-Giemsa staining of sorted GFP-low macrophages enriched for macrophages containing engulfed material. H Wright-Giemsa

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staining of sorted GFP-high macrophage populations containing engulfed tumor cells. Scale bars represent 100 µm (A, D-H).

Fig. S7. High-affinity SIRPα monomers augment phagocytosis in response to all human IgG subclasses. A Phagocytosis of GFP+ DLD-1 cells with varying concentrations of cetuximab (anti-EGFR) alone (red) or in combination with WTa1 SIRPα monomer (pink) or high-affinity SIRPα monomers (green). B Phagocytosis of GFP+ Raji cells with varying concentrations of rituximab (anti-CD20) alone (red) or in the presence of WTa1 SIRPα monomer (pink) or high-affinity SIRPα monomers (green). C Phagocytosis evaluated with primary human macrophages and GFP+ Raji lymphoma cells. Anti-CD20 isotype collection antibodies (Invivogen) contain the variable regions of rituximab and differing heavy chain isotypes (h, human; m, mouse). All antibodies were used at 10 µg/mL in the presence of vehicle control (PBS, gray bars) or 1 µM high-affinity SIRPα monomer (CV1, black bars). Data depict mean ± SD. Assays performed with human macrophages derived from at least three independent donors. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for the high-affinity SIRPα variants versus WT SIRPα, or the indicated comparisons, by two-way ANOVA with Bonferroni correction.

Fig. S8. Treatment with high-affinity SIRPα-Fc variants causes macrophage infiltration. A Dissected palpable subcutaneous tissue mass from a CV1-hIgG4 treated mouse. Left = white light, right = GFP fluorescence. Dashed ovals encircle two superficial tumor nodules, asterisks mark macrophage-rich stromal infiltrate. Scale bar = 5 mm. B Hematoxylin & eosin staining of palpable subcutaneous tissue mass from a CV1-hIgG4 treated mouse, demonstrating the presence of infiltrating macrophages (100× magnification). A tumor nodule is visible in the top right of the image with an inflammatory infiltrate surrounding it. Inset shows representative macrophages in the area outlined by the dashed box. C Immunohistochemical staining for F4/80, a mouse macrophage marker, in the subcutaneous tissue mass of a CV1-hIgG4 treated mouse (100× magnification). A tumor nodule is visible in the right portion of the image. Inset shows representative macrophages in the area outlined by the dashed box, with evidence of macrophages in the process of phagocytosis (black arrows) and successful engulfment of tumor cells (red arrows).

Fig. S9. High-affinity SIRPα-Fc variants induce red blood toxicity in mice. A Representative FACS analysis of human Fc bound to the surface of whole blood cells from treated animals. B Analysis of red blood cell parameters from treated animals showing mean ± SD from five animals per cohort on day 34 post-engraftment. Dotted lines show lower limit of normal values. C Full blood analysis of mice bearing GFP-

13

luciferase+ 639-V bladder tumors on day 34 post-engraftment. CV1-hIgG4 treatment resulted in a significant decrease in red blood cell indices (yellow). No toxicity to other blood lineages was observed. Statistical analysis performed by two-tailed Student’s t test.

Fig. S10. High-affinity SIRPα variants bind and block mouse CD47. A Binding of high-affinity SIRPα variant FD6, but not wild-type allele 1 human SIRPα, to mouse CT26 colon cancer cells. Binding of biotinylated SIRPα monomers was detected by Alexa Fluor 647-conjugated streptavidin. B Mouse CD47 blocking assay. High-affinity SIRPα variant FD6-hIgG4 blocks binding of 50 nM Alexa Fluor 647-conjugated wild-type mouse SIRPα tetramers to mouse CD47 displayed on the surface of yeast. C Binding of biotinylated high-affinity SIRPα variant CV1 to mouse CD47 displayed on the surface of yeast. Binding was detected by Alexa Fluor 647-conjugated streptavidin. Data are mean ± SD from analysis performed in triplicate.

Fig. S11. High-affinity SIRPα monomers do not cause toxicity in cynomolgus macaques. A Alignment between the CD47 IgSF domains from humans (hsCD47) and cynomolgus macaques (mfCD47). Positions of amino acid similarity indicated in yellow, positions of amino acid differences indicated in red. B Surface depiction of the human CD47-FD6 complex. Amino acids that differ between human and cynomolgus CD47 are distant from the binding interface and indicated in yellow. C Blood analysis of cynomolgus macaques treated with the indicated high-affinity SIRPα variants over time. Dotted lines indicate days of treatment with the doses indicated above in mg/kg. Data depicted as percentage of pre-treatment values. FD6-hIgG4 animal #2 was pre-treated with erythropoietin prior to toxicity testing. D Hematologic analysis of cynomolgus macaques treated with high-affinity SIRPα variants. Laboratory values outside of normal limits are highlighted in yellow. E Comprehensive serum metabolic analysis from treated animals showing no detectable toxicity to other organ systems.

Fig. S12. The combination of high-affinity SIRPα monomers with therapeutic antibodies produces long-term cures. A Representative bioluminescence images of GFP-luciferase+ Raji cells on day 7 post-engraftment, demonstrating stable engraftment and intense bioluminescence signal. B Bioluminescence images of animals cured from the combined treatment of rituximab plus CV1 monomer on day 209 post-engraftment. No evidence of disease relapse was observed. Note: radiance scale is 100× more intense in A than B.

14

Fig. S13. Treatment with high-affinity SIRPα monomers does not cause red blood cell toxicity. A Measurements of red blood cell indices from five mice per cohort over the time course of treatment with the indicated therapies. Data represent mean ± SD. ns = not significant by two-way ANOVA with Bonferroni correction. Black arrows indicate the start and stop of daily treatment. B Full hematologic analysis of animals treated with rituximab versus rituximab plus CV1 monomer. Data represent mean ± SD from five animals per cohort. Statistical analysis performed by two-tailed Student’s t test.

Fig. S14. High-affinity SIRPα monomers are effective against large lymphomas and induce macrophage phagocytosis by NSG mouse macrophages. Raji lymphoma tumors were engrafted into NSG mice and treatment was initiated when tumor volumes reached a median of ~175 mm3. A Tumor volumes after one week of treatment with rituximab alone or rituximab plus CV1 monomer. Five animals were treated per group and analyzed. B Tumor weights after one week of treatment with rituximab alone or rituximab plus CV1 monomer. C Quantification of macrophage infiltration in tumors treated with the indicated therapies. Immunohistochemical staining for F4/80 was used to identify macrophages, and the intensity of infiltration was scored by evaluators who were blind to the treatment conditions. D Representative images of F4/80 staining. Areas of moderate macrophage infiltration (rituximab alone) and intense macrophage infiltration (rituximab plus CV1 combination) are depicted. Images taken at 400× magnification. E Phagocytosis assay performed with NSG mouse macrophages and GFP+ Raji lymphoma cells. Rituximab was used at 10 µg/mL and CV1 monomer was used at 1 µM. *p<0.05, **p<0.01, ****p<0.0001 determined by two-tailed Student’s t test (A-C) or one-way ANOVA with Bonferroni correction (D).

Fig. S15. High-affinity SIRPα monomers enhance the efficacy of alemtuzumab (anti-CD52). A Growth of Raji lymphoma tumors upon bi-weekly treatment with vehicle (PBS), 200 µg CV1 monomer, 200 µg alemtuzumab, or alemtuzumab plus CV1 monomer (200 µg each), as evaluated by tumor volume. Five (PBS and CV1) or ten (alemtuzumab and alemtuzumab/CV1) mice per group were treated and analyzed over the experiment, and each data point represents a measurement from an independent mouse. Bars indicate median values. B Survival of lymphoma-bearing mice from A. C Bioluminescence images of animals cured from the combined treatment of alemtuzumab plus CV1 monomer on day 136 post-engraftment. No evidence of disease relapse was observed. *p<0.05 by Mann-Whitney (A) or Mantel-Cox (B) test for antibody alone versus antibody plus CV1 monomer.

15

Fig. S16. Diagram showing macrophage responses to CD47 blockade and therapeutic antibodies. In the basal state, CD47 on cancer cells binds SIRPα on macrophages and phagocytosis does not occur. Upon CD47 blockade with high-affinity SIRPα monomers or anti-CD47 Fab fragments, macrophages are sensitized to the presence of other pro-phagocytic stimuli but do not exhibit substantial levels of phagocytosis. Treatment with tumor-specific antibodies alone induces phagocytosis, but binding of CD47 on tumor cells to SIRPα on macrophages inhibits maximal responses. Addition of high-affinity SIRPα monomers to tumor-specific antibodies results in synergy that produces maximal macrophage phagocytosis of cancer cells and provides a maximal therapeutic window due to a lack of toxicity to normal cells expressing CD47.

CD47

SIRP D1

Contact library

a.a. DiversityL4 F,I,L,VV6 F,I,L,VV27 F,I,L,VI36 F,I,L,VF39 F,I,L,VL48 F,I,L,VI49 F,I,L,VY50 F,I,L,VF57 F,I,L,VV60 F,I,L,VM72 F,I,L,VF74 F,I,L,VI76 F,I,L,VV92 F,I,L,VF94 F,I,L,VF103 F,I,L,V

a.a. DiversityS29 S,T,G,AL30 F,I,L,VI31 F,I,L,VP32 P,L,H,RV33 F,I,L,VG34 S,T,G,AP35 P,L,H,RQ52 Q,D,E,HK53 K,RE54 Q,D,E,HS66 S,T,G,AT67 S,T,G,AK68 K,RR69 K,RF74 F,I,L,VK93 K,R,M,TK96 K,R,M,TG97 S,T,G,AS98 S,T,G,A

D100 D,E,N,K

Non-contact library

4 6 27 31 47 53 54 56 66 68 92 94 103WT SIRP

allele 1L V V I E K E H S K V F F

A5 V L V R Q G R ID1 L I T D G R LD4 I R T R VE9 F R R VF6 V I R T R VH5 V L R Q R IH10 I R P A I

C

Supplementary Figure 1

D

A B

Aga1

Aga2

Yeast Surface

SIRP

CD47

N

C

Biotin

s s

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3KDJRF\WRVLV

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Cancer cell

Supplementary Figure 2

a.a. DiversityL4 F,I,L,VV6 F,I,L,VV27 F,I,L,VI31 I,T,F,SE47 E,V,Q,LK53 K,RE54 E,D,Q,HH56 H,P,L,RS66 S,G,T,AK68 K,RV92 F,I,L,VF94 F,I,L,VF103 F,I,L,V

Supplementary Figure 3

PCA1

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ndin

g (%

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) 100

75

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0

125

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C

Supplementary Figure 4

A

C

Supplementary Figure 5

D

6,53њmonomer(14 kDa)

6,53њconcatamer

(28 kDa)

6,53њ�)Ffusion

(77 kDa)

6,53њleucine zipper

(36 kDa)

5 10 15 20

0

50

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150

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PBS

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1 2 3 4��Log[cetuximab (ng/mL)]

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PBSWTa1 mono

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2 3 4 5��Log[rituximab (ng/mL)]

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Supplementary Figure 8

C

A

Supplementary Figure 9

B

CUnit Low High Average Std Dev Average Std Dev p value

Total White Blood Cells 103�ѥ/ 2.60 10.10 6.94 4.84 5.64 2.17 0.599

Lymphocytes 1.30 8.40 2.44 1.43 1.14 0.66 0.102

Monocytes 1.10 0.30 0.72 0.48 0.54 0.11 0.440

Granulocytes 0.40 2.00 3.78 3.20 3.96 1.86 0.916

Hematocrit % 32.80 48.00 37.60 3.80 30.42 2.72 0.009

Mean Corpuscular Volume I/ 42.30 55.90 44.42 0.68 45.62 2.39 0.311

Red Cell Distribution Width % 0.00 99.90 29.40 0.73 32.78 5.11 0.181

Hemoglobin J�G/ 10.00 16.10 13.50 1.47 11.04 1.04 0.016

Mean Corpuscular Hemoglobin Conc. J�G/ 29.50 35.10 35.96 1.15 36.38 0.97 0.551

Total Red Blood Cells 6.50 10.10 8.46 0.87 6.69 0.79 0.010

Mean Corpuscular Hemoglobin pg 13.70 18.10 15.96 0.43 16.56 0.66 0.126

Platelets 250.00 1540.00 466.80 102.59 389.80 108.09 0.281

Mean Platelet Volume I/ 0.00 99.90 5.78 0.49 5.84 0.35 0.829

Normal range PBS CV1-hIgG4

103�ѥ/103�ѥ/103�ѥ/

106�ѥ/

103�ѥ/

0 102 103 104 105

100

80

60

40

20

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PBS CV1-hIgG40

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Supplementary Figure 10

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Supplementary Figure 11

D

hsCD47 QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVP 60 mfCD47 QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTAP 60 **********************************************************.*

hsCD47 TDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVS 117 mfCD47 ANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVS 117

::*******************************************************

RBC Hb Hct MCV RDW MCHC MCH Retic Plt WBC Neut LymphMono Eos Baso LUC

Treatment regimen Day 106 /uL g/dL % fL % g/dL pg 105 /uL103 /uL103 /uL /uL /uL /uL /uL /uL /uL

Animal 1: FD6-hIgG4 -4 6.02 13.8 46 76 13.6 30.3 23.0 0.48 467 7.4 4758 2279 207 81 30 44

Day 1: 1.5 mg/kg 3 5.04 11.5 36 72 14.2 31.7 22.9 0.81 477 8.6 4825 3225 215 267 26 43

5 4.75 11.0 36 75 15.3 30.9 23.1 1.38 531 13.6 7616 4814 503 517 68 82

10 4.26 10.4 36 84 16.3 28.9 24.4 2.09 941 11.5 4543 6486 253 127 46 46

13 3.98 9.7 32 80 18.2 30.6 24.5 2.27 675 9.9 5970 3188 307 366 20 50

Animal 2: FD6-hIgG4 -4 5.23 12.5 45 85.0 12.5 28.1 23.9 0.78 562 12.4 7862 4179 236 37 37 62

Day 1: 1.5 mg/kg 3 4.04 9.8 34 83.0 17.0 29.1 24.2 4.48 483 10.0 5040 4510 300 50 40 50

5 3.91 9.6 33 83.3 17.5 29.6 24.7 3.71 555 12.9 5973 6244 387 168 52 77

10 4.26 10.4 36 84 16.3 28.9 24.4 2.09 941 12 4543 6486 253 127 46 46

13 3.64 9.2 31 86 15.5 29.4 25.2 1.57 997 8.6 4661 3449 284 138 26 43

Animal 3: FD6 mono -4 5.33 12.8 40.5 76.1 12.7 31.5 24.0 0.48 318 10.8 8802 1642 313 11 22 11

Day 1: 0.3 mg/kg 3 4.75 11.4 36.0 75.7 13.0 31.7 24.0 0.95 346 8.8 5975 2376 370 18 18 44

Day 8: 1.0 mg/kg 5 4.82 11.4 37.3 77.3 13.5 30.6 23.6 1.35 469 8.6 4463 3689 292 52 34 60

Day 15: 3.0 mg/kg 10 4.67 11.2 36.6 78.4 13.9 30.6 24.0 1.54 463 10.0 6650 2910 320 50 20 50

Day 24: 10.0 mg/kg 13 4.89 12.0 38.5 78.7 14.1 31.2 24.5 1.86 475 8.0 4344 3288 264 48 24 40

17 4.48 10.8 35.0 78.0 14.0 30.8 24.0 1.52 393 9.7 7168 2105 310 49 19 39

20 5.03 12.7 39.7 79.0 14.0 32.1 25.3 2.26 452 9.0 4455 4014 333 117 36 45

27 4.80 11.6 38.3 79.7 13.4 30.3 24.2 0.96 359 8.5 4718 3128 561 60 26 17

ALT AST ALP GGT LD TBili BUN Crea Ca Phos TP

Treatment regimen Day U/L U/L U/L U/L U/L mg/dL mg/dL mg/dL mg/dL mg/dL g/dL

Animal 1: FD6-hIgG4 -4 48 39 310 44 395 0.4 18 0.8 10.4 5.6 7.5

Day 1: 1.5 mg/kg 5 65 64 255 37 693 0.3 28 0.6 9.8 4.7 7.3

13 44 35 289 40 254 0.8 25 0.6 10.3 5.2 7.3

Animal 2: FD6-hIgG4 -4 88 115 275 67 796 0.3 20 0.9 10.7 5.6 7.3

Day 1: 1.5 mg/kg 5 142 101 252 42 815 0.3 27 0.6 9.7 3.9 6.5

13 43 31 285 54 317 0.7 25 0.7 9.9 5.6 6.8

Animal 3: FD6 mono -4 39 44 419 60 496 0.3 20 0.7 9.6 4.2 6.8

Day 1: 0.3 mg/kg 5 73 42 309 50 467 0.2 26 0.6 9.5 4.0 6.7

Day 8: 1.0 mg/kg 13 46 39 364 56 338 0.2 24 0.6 9.9 5.3 7.2

Day 15: 3.0 mg/kg 20 36 37 336 50 445 0.2 24 0.6 9.8 5.3 7.2

Day 24: 10.0 mg/kg 27 37 33 376 50 367 0.2 24 0.6 9.9 4.4 7.0

E

CD47

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0 10 20 300

25

50

75

100

Day

Red

bloo

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lls (%

Max

)

0 10 20 300

25

50

75

100

Day

Hem

atoc

rit (%

Max

)

FD6-hIgG4 #2FD6-hIgG4 #1FD6 monomer

1.50.3 1.0 3.0 10.0 mg/kg

1.51.50.3 1.0 3.0 10.0 mg/kg

1.5C

A

Supplementary Figure 12

B

5.0 2.03.04.0Radiance (p/sec/cm2/sr x105)

1.0

5.0 2.03.04.0Radiance (p/sec/cm2/sr x107)

1.0

A

B

Supplementary Figure 13

Day 8 (Pre-treatment) Unit Low High Average Std Dev Average Std Dev p valueTotal White Blood Cells 103/ L 2.60 10.10 3.12 1.45 2.04 0.56 0.160Lymphocytes 103/ L 1.30 8.40 1.28 0.93 0.74 0.44 0.276Monocytes 103/ L 1.10 0.30 0.34 0.13 0.26 0.05 0.252Granulocytes 103/ L 0.40 2.00 1.50 0.51 1.04 0.24 0.108Hematocrit % 32.80 48.00 38.18 3.32 38.56 4.75 0.887Mean Corpuscular Volume fL 42.30 55.90 44.86 0.27 44.76 0.40 0.658Red Cell Distribution Width % 0.00 99.90 29.72 0.60 29.54 0.40 0.594Hemoglobin g/dL 10.00 16.10 14.10 1.17 14.32 1.95 0.834Mean Corpuscular Hemoglobin Conc. g/dL 29.50 35.10 36.90 0.42 37.08 0.76 0.654Total Red Blood Cells 106/ L 6.50 10.10 8.50 0.69 8.62 1.12 0.846Mean Corpuscular Hemoglobin pg 13.70 18.10 16.56 0.18 16.58 0.38 0.917Platelets 103/ L 250.00 1540.00 320.60 65.90 408.80 82.97 0.100Mean Platelet Volume fL 0.00 99.90 5.78 0.13 5.56 0.18 0.059

Day 16Total White Blood Cells 103/ L 2.60 10.10 4.92 2.02 4.06 1.18 0.435Lymphocytes 103/ L 1.30 8.40 2.42 1.33 1.86 1.09 0.486Monocytes 103/ L 1.10 0.30 0.44 0.13 0.40 0.10 0.608Granulocytes 103/ L 0.40 2.00 2.06 0.73 1.80 0.40 0.503Hematocrit % 32.80 48.00 37.44 2.63 37.06 1.87 0.799Mean Corpuscular Volume fL 42.30 55.90 45.56 0.33 45.28 0.48 0.311Red Cell Distribution Width % 0.00 99.90 29.60 0.12 29.84 0.59 0.402Hemoglobin g/dL 10.00 16.10 13.60 1.06 13.46 0.69 0.810Mean Corpuscular Hemoglobin Conc. g/dL 29.50 35.10 36.30 0.95 36.38 0.31 0.863Total Red Blood Cells 106/ L 6.50 10.10 8.22 0.63 8.19 0.47 0.916Mean Corpuscular Hemoglobin pg 13.70 18.10 16.54 0.38 16.44 0.19 0.618Platelets 103/ L 250.00 1540.00 327.60 58.59 314.80 95.88 0.805Mean Platelet Volume fL 0.00 99.90 5.92 0.33 5.98 0.44 0.814

Day 30Total White Blood Cells 103/ L 2.60 10.10 4.36 2.29 4.02 1.91 0.805Lymphocytes 103/ L 1.30 8.40 1.96 1.13 2.32 1.05 0.616Monocytes 103/ L 1.10 0.30 0.46 0.21 0.38 0.13 0.486Granulocytes 103/ L 0.40 2.00 1.94 0.98 1.32 0.74 0.291Hematocrit % 32.80 48.00 36.82 1.03 36.82 2.65 1.000Mean Corpuscular Volume fL 42.30 55.90 44.50 0.24 44.48 0.31 0.913Red Cell Distribution Width % 0.00 99.90 29.04 0.42 29.18 0.55 0.663Hemoglobin g/dL 10.00 16.10 13.30 0.57 13.28 1.06 0.971Mean Corpuscular Hemoglobin Conc. g/dL 29.50 35.10 36.16 1.62 36.08 0.35 0.917Total Red Blood Cells 106/ L 6.50 10.10 8.27 0.25 8.27 0.58 0.978Mean Corpuscular Hemoglobin pg 13.70 18.10 16.06 0.69 16.04 0.17 0.951Platelets 103/ L 250.00 1540.00 416.40 95.29 328.40 72.20 0.138Mean Platelet Volume fL 0.00 99.90 5.88 0.18 6.00 0.20 0.347

Normal range Rituximab Rituximab+CV1

0 10 20 300

10

20

30

40

50

Days

Hem

atoc

rit (%

)

0 10 20 300

5

10

15

20

Days

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(g/d

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0 10 20 300

3

6

9

12

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Red

bloo

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06 �ѥ/� PBS

CV1 monoRituximabRituximab+CV1 mono

ns nsns ns ns

nsns ns

ns

A B

Supplementary Figure 14

D

0.0

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0.8

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1.0

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2.5

Tum

or w

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t (g)

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Rituximab + PBS Rituximab + CV1 monomer

0

10

20

30

40

Pha

gocy

tosi

s (%

)

PBS CV1mono

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****

*

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C

0

1

2

3

4

5

Mac

roph

age

infilt

ratio

n sc

ore

Ritux+PBS

Ritux+CV1

**

E

A B

Supplementary Figure 15

C

5.0 2.03.04.0Radiance (p/sec/cm2/sr x105)

1.0

PBSCV1 monoAlemtuzumabAlemtuzumab+CV1 mono

0 20 30 40 50 600.0

0.5

1.0

1.5

2.0

2.5

Days

Tum

or v

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10

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0 25 50 75 100 125 1750

25

50

75

100

Days

Surv

ival (

%)

PBSCV1 monoAlemtuzumabAlemtuzumab+CV1 mono

*

Supplementary Figure 16

sensitizedunstimulated PHAGOCYTOSIS

6,53њ CD47 +LJK�DIÀQLW\�6,53њ TXPRU�DQWLJHQ $QWL�WXPRU�DQWLERG\)F�UHFHSWRU

Basal state 6,53њ�PRQRPHU�)DE&'���EORFNDGH

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32

Table S1. Data collection and refinement of the FD6:CD47 crystal structure. FD6:CD47 complex Data collection Space group P6522 Cell dimensions a, b, c (Å) 71.34, 71.34, 399.18 α, β, γ (°) 90.0, 90.0, 120.0 Resolution (Å) 50-1.90 (1.93-1.90) Rsym 8.9 (83.4) I/σI 21.4 (0.8) Completeness (%) 83.1 (30.3) Redundancy 8.3 (2.2) Refinement

Resolution (Å) 50-1.93 (1.99-1.93) No. reflections 36,706 Rwork/Rfree 22.0/26.2 (36.1/39.9) No. atoms Protein 3,513 Ligand/ion 127 Water 378 B-factors Protein 47.6 Ligand/ion 53.8 Water 41.1 R.m.s deviations Bond lengths (Å) 0.004 Bond angles (º) 0.831

33

Movie S1. Control treatment of Raji lymphoma cells cultured with macrophages results in minimal phagocytosis. CFSE-labeled Raji cells (green) were treated with vehicle control (PBS) and co-cultured with RFP+ macrophages (red). Movie depicts 1.5 hours of elapsed time. Scale bar indicates 10 µm.

Movie S2. High-affinity SIRPα-Fc treatment of Raji lymphoma cells cultured with macrophages results in substantial phagocytosis. CFSE-labeled Raji cells (green) were treated with 100 nM CV1-hIgG4 and co-cultured with RFP+ macrophages (red). Movie depicts 1.5 hours of elapsed time. Scale bar indicates 10 µm.

34

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