mAbs

ISSN: 1942-0862 (Print) 1942-0870 (Online) Journal homepage: https://www.tandfonline.com/loi/kmab20

Discovery of high affinity, pan-allelic, and panmammalian reactive antibodies against the
myeloid checkpoint receptor SIRPαJanet Sim, Jonathan T. Sockolosky, Emma Sangalang, Shelley Izquierdo,
Darlene Pedersen, William Harriman, Ardian S. Wibowo, Josh Carter, Anup
Madan, Laura Doyle, Ons Harrabi, Steven E. Kauder, Amy Chen, Tracy C. Kuo,
Hong Wan & Jaume Pons
To cite this article: Janet Sim, Jonathan T. Sockolosky, Emma Sangalang, Shelley Izquierdo,
Darlene Pedersen, William Harriman, Ardian S. Wibowo, Josh Carter, Anup Madan, Laura Doyle,Ons Harrabi, Steven E. Kauder, Amy Chen, Tracy C. Kuo, Hong Wan & Jaume Pons (2019):
Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid
checkpoint receptor SIRPα, mAbs, DOI: 10.1080/19420862.2019.1624123
To link to this article: https://doi.org/10.1080/19420862.2019.1624123© 2019 The Author(s). Published withlicense by Taylor & Francis Group, LLC.

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MABS
https://doi.org/10.1080/19420862.2019.1624123

REPORT

Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies
against the myeloid checkpoint receptor SIRPαJanet Sim a, Jonathan T. Sockolosky a, Emma Sangalang a, Shelley Izquierdob, Darlene Pedersenb,
William Harrimanb, Ardian S. Wibowo c, Josh Carter c, Anup Madan d, Laura Doyle a, Ons Harrabia,
Steven E. Kauder a, Amy Chena, Tracy C. Kuo a, Hong Wan a, and Jaume Pons aac

ALX Oncology, Departments of Protein Sciences and Translational Biology, Burlingame, CA, USA; bLigand Pharmaceuticals, Inc, Emeryville, CA, USA;
Helix Biostructures, Columbus, IN, USA; dCovance Genomics Laboratory, Redmond, WA, USA
ABSTRACT

Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic
approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses.
Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells andneurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential
safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics
and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms
within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies fortherapeutic intervention in diverse patient populations. We immunized wild-type and human antibody
transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelicand pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200
antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies
with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity
ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, andpotentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal
structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with
SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the antiSIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive
rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced
malignancies.

ARTICLE HISTORY

Received 1 February 2019
Revised 16 April 2019
Accepted 17 May 2019
KEYWORDS

Antibody; SIRPα;macrophage; phagocytosis;
immunotherapy

Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellularphagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment
crystallizable; FcγR: Fcγ receptor; Ig: immunoglobulin; IND: investigational new drug; MDM⊘: monocytederived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe
combined immunodeficiency; SIRP: signal-regulatory protein

Introduction
Immune evasion is a hallmark of cancer, and therapeutic interventions that stimulate immune responses against tumors haveshown remarkable clinical efficacy in diverse cancers.1,2 In particular, antibodies targeting the T cell inhibitory receptors PD-1 andCTLA-4 are capable of inducing long term, durable antitumor
immunity in humans by stimulating the adaptive arm of theimmune system. However, many patients fail to respond ordevelop resistance to T cell checkpoint therapy because tumors
exploit multiple mechanisms to avoid immune detection anddestruction, including the suppression of innate immunity.3
Combination therapies targeting non-redundant pathways that
restrain antitumor immunity can dramatically improve response
rates.

Signal-regulatory protein α (SIRPα) is an innate immune
checkpoint receptor expressed primarily on myeloid cells,
including monocytes, macrophages, dendritic cells (DCs),
and neutrophils.4,5 SIRPα suppresses innate immunity uponinteraction with its ligand, CD47. CD47 is broadly expressedon normal tissues and is up-regulated by virtually all humantumors to escape macrophage recognition and programmed
cell removal.6 Inhibitory signals delivered by CD47 through
SIRPα dampen FcγR-dependent antibody effector functions,
including macrophage and neutrophil-mediated antibodydependent cellular phagocytosis (ADCP) and cytotoxicity(ADCC), limiting the induction of antibody-dependent innate
immunity and promoting resistance to antitumor antibody
therapy.6–9

CONTACT Jaume Pons
jaume@alxoncology.com
ALX Oncology, Burlingame, CA, USA
Supplemental data for this article can be accessed on the publisher’s website.
© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

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J. SIM ET AL.

Antagonizing the CD47/SIRPα interaction has a profound
impact on potentiation of macrophage and DC phagocytosis of
antibody-opsonized tumor cells, which in turn can prime antitumor CD8 + T cell responses,10–12 linking innate and adaptive
immunity to anti-CD47 therapy. Beyond augmenting phagocytosis, disrupting the CD47/SIRPα interaction triggers numerous
immunological responses that promote innate and adaptive
antitumor immunity, including enhanced DC activation,a reduction in myeloid-driven immune suppression, and stimulation of type-I and type-II interferon responses.11–13 When
combined with either tumor antigen-specific or immunomodulatory antibodies targeting T cell inhibitory receptors, CD47/
SIRPα antagonists dramatically improve response rates acrossnumerous xenogeneic and syngeneic mouse cancer models,
resulting in durable long-term tumor immunity.8,12,14–16
Collectively, these preclinical results provide compelling evidence to support clinical evaluation of CD47/SIRPα antagonists
in combination with antitumor or checkpoint antibodies.
A variety of CD47 antagonists are under evaluation in theclinic.17 Promising activity was recently reported for an antiCD47 antibody used in combination with rituximab for thetreatment of lymphoma.18 In addition, the CD47 antagonist
ALX148, a high-affinity SIRPα variant fused to an inactive Fc
domain, has demonstrated a favorable safety profile and preliminary anti-cancer activity in the clinic when used in combination with pembrolizumab and trastuzumab for the treatment ofsolid tumors.12,19
Disrupting the CD47/SIRPα interaction initially focused ontargeting CD47 due to its ubiquitous expression on humantumors;6,7 however, several limitations associated with targeting CD47 may be circumvented by targeting SIRPα. The
broad tissue distribution of CD47 creates a large antigen
sink20 and promotes target-mediated clearance of anti-CD47
antibodies. In contrast, SIRPα expression is predominately
restricted to myeloid cells; therefore, targeting SIRPα enables
reduced dosing to achieve maximal receptor occupancy andimproved pharmacokinetic and pharmacodynamic (PK/PD)
properties. Furthermore, anti-CD47 antibodies capable oftriggering Fc effector functions cause dose-limiting toxicities,
including anemia and thrombocytopenia due to CD47 expression on red blood cells and platelets.20,21 Although we previously demonstrated that safety liabilities associated with
anti-CD47 therapy can be avoided by Fc mutations that abrogate FcγR and complement (C1q) binding,12,14 it is of interest
to study similarities and differences of an orthogonal
approach to antagonize the SIRPα/CD47 axis by targeting
SIRPα.4,9,16,22
SIRPα belongs to the SIRP family of immunoreceptors that
include the highly homologous activating receptor SIRPβ1 andthe decoy receptor SIRPγ.23 Although CD47 is highly conserved,
extensive polymorphisms localized to the CD47-binding IgV
domain of SIRPα have been reported in numerous ethnic
groups.24 Initial investigation of SIRPα polymorphisms inhumans identified 10 allelic variants of SIRPα with two prevalentalleles, v1 and v2, shared between diverse ethnic groups andeight additional alleles with population-specific distributions of
unknown frequency.24 More recently, Treffers et al. quantified
SIRPα polymorphisms in Caucasians and identified only the two
prevalent SIRPα variants, v1 and v2, within this population.25

Whereas genetic variation of SIRPα does not seem to affectintrinsic phagocyte function,25 activation of a single functional
SIRPα variant in heterozygote macrophages is sufficient to inhibit antibody-dependent effector functions.9 Thus, effective therapeutic targeting of SIRPα across diverse patient populations
requires pan-allelic anti-SIRPα antibodies.
We describe the discovery and characterization of a panelof wild-type, chimeric, and fully human anti-SIRPα monoclonal antibodies from chickens that exhibit high affinity, broad
epitope coverage, and species cross-reactivity against mouse,monkey, and human SIRPα alleles. Guided by analysis of
SIRPα polymorphisms across diverse human populations, we
immunized chickens with allele and species-specific variantsof SIRPα and screened antibody-secreting B cells to identify
anti-SIRPα antibodies suitable for clinical translation. In total,
200 unique anti-SIRPα antibodies were identified andscreened for binding to human, monkey, and mouse SIRPα,human SIRPβ and SIRPγ, and for the ability to block theinteraction between CD47 and SIRPα. Epitope binning andx-ray crystallography identified six distinct SIRPα epitopes,
including epitopes that highly or partially overlap with theCD47 binding site, as well as epitopes that are distal from
CD47. A subset of anti-SIRPα antibodies were found to bindwith high affinity (nM to pM) to SIRPα v1 and v2 alleles,
cross-react with monkey and mouse SIRPα, potently antagonize the CD47/SIRPα interaction, and enhance macrophage
phagocytosis of antibody-opsonized tumor cells. Thus, theanti-SIRPα antibodies described herein represent promising
candidates for targeting the CD47/SIRPα immunoevasion
pathway for the treatment of human cancers by unleashing
innate and adaptive immune responses.

Results
Two SIRPα allelic variants are prevalent in diverse humanpopulations
The distribution and frequency of SIRPα variants across population and ethnic groups are not well characterized. Successful
therapeutic targeting of SIRPα in diverse patient populations
irrespective of SIRPα genotype necessitates pan-allelic antiSIRPα antibodies that cross-react with all relevant SIRPα alleles.
To aid in the design of a pan-allelic anti-SIRPα antibody discovery strategy, we first sought to determine the distribution andfrequency of SIRPα polymorphic variants in human populations.
We initially turned to the 1000 Genome Project, which has thelargest public repository of sequenced human genomes (http://
www.internationalgenome.org/home),
andperformed
a comprehensive bioinformatic analyses of the SIRPA genefrom 2535 individuals from the Phase 3 dataset of the 1000
Genome Project.26 As an orthogonal confirmation, we selected
510 individuals representing different populations from theabove Phase 3 project, procured the respective DNA samples,and performed Sanger sequencing of exon 3 the SIRPA gene ofthese individuals. In agreement with Treffers et al.,25 our bioinformatic analyses of SIRPα sequences from 2535 individuals andSanger sequencing of 510 samples identified only two SIRPαvariants, v1 and v2 (Table S1), represented as three allelic groups:
homozygous v1/v1, homozygous v2/v2, and heterozygous v1/v2.

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Examples of the detailed sequence analyses are described in Fig.
S1. This contrasts with data reported by Takenaka et al.24 who
identified eight additional allelic variants besides v1 and v2.
Since eight of these allelic variants and individuals analyzed by
Takenaka et al.24 were also coincidentally part of the Phase 3
dataset, we acquired the identical eight samples and carried out
Sanger sequencing to confirm their allelic identities. Indeed, ourresults further confirmed that these samples are either homozygotes (v1/v1 or v2/v2) or heterozygotes (v1/v2).
Next, we determined the distribution and frequency of the
SIRPα v1 and v2 allelic groups in various populations andtheir respective sub-populations (Figure 1(a,b)). The distribution of v1/v2 heterozygotes amongst the five super populations, European (EUR), American (AMR), East Asian (EAS),
African (AFR) and South Asian (SAS), is similar and ranges
from 42.0% to 47.2%. The East Asian population has
a significantly higher number of v2/v2 than v1/v1 homozygotes occurring at a frequency of 42.3% and 13.3%, respectively, whereas the African, European, American, and South
Asian populations had a moderately higher number of v1/v1
than v2/v2 homozygotes, with frequency ranges of 30.3–49.1%
and 8.9–24.2%, respectively. The distribution of the threeallelic variants within American, African, East Asian, South
Asian and European sub-populations is similar to theirrespective super population, with the exception of the
Peruvian (PEL) sub-population, which has a higher percentage of v2/v2 relative to v1/v1 homozygotes compared to theother American sub-populations (Figure 1(b)). Despite theobserved distribution and frequency, we conclude only two
SIRPα allelic variants v1 and v2 are present among the diverse
populations analyzed in this study.
Chicken immunization and GEM screen to isolate pan-allelicand -mammalian reactive anti-SIRPα antibodies
Based on our results from SIRPα polymorphism analyses, it iscritical that anti-SIRPα antibodies are capable of binding both
SIRPα v1 and v2 alleles to be useful for clinical development. In
addition, to facilitate rapid preclinical to clinical translation, wesought to discover anti-SIRPα antibodies that also cross-reactwith mouse SIRPα alleles from strains commonly used for syngeneic and xenogeneic tumor models (e.g., NOD/SCID, BALB/c,
C57BL/6), and cross-react with cynomolgus monkey SIRPα for

3

Table 1. Sequence identities of SIRPα IgV domains. The percentage amino acidsequence identities of IgV domains of SIRPα from human v1 (NP_542970.1),
human v2 (CAA71403.1), cynomolgus monkey (EHH65484.1), 129 mouse(162330193) and chicken (NP_001032920.2) sources are listed, respectively.
Percentage identity is calculated based on identical residues among all
ungapped positions between sequence pairs. % Identity
Human
v1
Human v1
Human v2
Cynomolgus Monkey
129 mouse
Chicken

Human
v2
89

Cynomolgus
Monkey91
88

129
mouse Chicken
67
46
69
41
74
47
47

toxicology studies that may enable an investigational new drug
(IND) application. Based upon SIRPα sequence homology(Table 1, Fig. S2 and Fig. S3), it may be challenging to obtainhuman, cynomolgus monkey, and mouse cross-reactive antibodies through traditional mouse immunization methods.
Chickens are an attractive alternative to mice because they are
phylogenetically distant from humans,27,28 and produce antibodies of high affinity and specificity that can recognize uniqueepitopes not accessible to mice.29 Moreover, pan-mammalianreactive anti-SIRPα antibodies are more likely to be discoveredin chickens given the low homology between chicken SIRPα andthe human and mouse orthologs (Table 1). The workflow forusing chicken for immunization and antibody discovery is
depicted in Figure 2(a).
To generate pan-allelic and species cross-reactive anti-SIRP
α antibodies, we immunized wild-type and transgenic chickens with the extracellular IgV domain of human SIRPα allele
v1, which is the sole extracellular domain that mediates interaction with CD47, followed by boosts that alternate between
human SIRPα allele (v1 vs v2) and/or species (mouse NOD or
129 vs. human SIRPα) in order to drive antibody responses
toward a cross-reactive repertoire (Table 2). Wild-type andtwo different transgenic chickens were used for immunization: SynVH chickens which contain humanized VH immunoglobulin (Ig) repertoires paired with the natural chicken
light chain repertoire, and OmniChickens®, which containhumanized VH and VL Ig repertoires.30,31 In total, eight
immunized chickens produced high titer antibodies against
SIRPα v1, v2 and mouse SIRPα (Table 2, Figure 2(b)) fromwhich splenocytes were harvested, and single antibody-

Figure 1. Frequency and distribution of SIRPα variants across human populations. The distribution and frequency of SIRPα alleles in the five super populations (a) andtheir respective subpopulations (b). In the 1000 Genome Project, the population from different locations around the globe are divided into five super populations as
African (AFR), Ad Mixed American (AMR), East Asian (EAS), European (EUR) and South Asian (SAS). These super populations are further divided into total of 26
subpopulations. The code for the subpopulation can be found at http://www.internationalgenome.org/category/population/.

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Figure 2. Isolation of anti-SIRP⍺ antibodies from wild-type and transgenic chickens. (a) Schematic depicting the generation of pan-allele and pan-mammalian antiSIRP⍺ antibodies. (b) Plasma titers to hSIRP⍺ v1, hSIRP⍺ v2, mSIRP⍺ NOD, and mSIRP⍺ m129 in wild-type (left), SynVH (middle), and OmniChicken (right) chickens.

Table 2. Summary of the chicken immunization scheme and GEM screen results. Eight chickens (two wildtype, two SynVH and four OmniChickens) were immunized
as described below. To drive response toward a cross-reactive repertoire, all the chickens were immunized by SIRPα allele v1 followed by boosts that alternate
between human SIRPα allele (v1 vs v2) and/or species (mouse NOD or 129 vs. human SIRPα). Various GEM screen combinations were used to facilitate the discoveryof anti-SIRPα antibodies with unique binding profiles. A total of 200 chicken antibodies were cloned and the variable domain region sequenced.
Immunization Scheme

GEM Screen

Chicken
WT 21288

Initial
100ug
V1

Boost 1
50ug
V2

Boost 2
50ug
V1

Boost 350ug
V2

WT 21292

V1

V2

m129

V1

SynVH 22260

V1

V2

V1

V2

SynVH 22280

V1

V2

m129

V1

Omni 22843

V1

NOD

V2

NOD

Omni 23504

V1

NOD

V2

NOD

Omni 23941

V1

NOD

V2

NOD

Omni 23975

V1

NOD

V2

NOD

Bead 1
V1
V2
V1
V2
V1
V2
V1
V2
V2
V2
V1
V1
V2
V2
V1
V1
V1
V1
V1
V1

Bead 2
V2
NOD
V2
m129
V2
NOD
V2
m129
BALBc
Complex
V2
V2
BALBc
Complex
V2
V2
V2
V2
V2
V2

Bead 3
NOD
Complexm129
Complexc
NOD
Complex
M129
Complex-b
Complex

No. of scFv-Fc
Isolated & Screened/Chickena
34
9
12
60

SIRPγ
Complex
Complex-b

50Complex SIRPγ

4Complex
SIRPγComplex SIRPγ

1021a. Total number of scFv-Fc screened by SPR from each chicken (from different GEM Screens).
b. Only two beads are used for these GEM screens.
c. Complex = CV1 and IgS domain of CD47 is pre-complexed before GEM screen.

secreting B-lineage cells were screened for reactivity toward
SIRPα using a gel-encapsulated microenvironment (GEM)
assay.32
The GEM assay consists of a single-antibody secreting
B-cell encapsulated in a droplet containing up to threedifferent fluorescent reporter beads, each coated witha target antigen of interest (Figure 2(a)). B-cell clones
that produce antibodies that bind to the immobilized
targets were detected with a secondary antibody against
chicken IgY, visualized via fluorescent microscopy,

isolated, and sequenced. Various antigen-coated beadcombinations were used to facilitate the discovery of antiSIRPα antibodies with unique binding profiles (Table 2).
For instance, to isolate pan-allelic and species-cross reactive antibodies that bind human SIRPα v1, human SIRPαv2, and mouse SIRPα, a triple-bead combination was used
where each bead was individually coated with the respective antigen, and only B-cells producing antibodies thatreact with all three antigen-coated beads were recovered.
Additional GEM screens were designed to distinguishMABS

between antibodies that block the SIRPα/CD47 interaction
and non-blocking antibodies via beads coated with SIRPα
pre-complexed with CD47. Due to the weak affinity
between wild-type SIRPα and CD47, we used engineered,high-affinity versions of SIRPα v114 and SIRPα v2 toensure stability of the CD47/SIRPα complex and maximize
the likelihood of identifying antibodies that bind SIRPα
while in complex with CD47 (i.e., non-blocking antibodies). Conversely, blocking antibodies were identified byscreening for clones that bind SIRPα but not the CD47/
SIRPα complex, an indication that the antibody and CD47
bind overlapping epitopes on SIRPα. Through various
GEM screens of lymphocytes prepared from immunizedchickens (Table 2), over 200 unique anti-SIRPα antibody
clones were identified. The V genes from each chicken
antibody were reformatted into a single-chain variablefragment (scFv)-Fc fusion as described in Materials andMethods and produced recombinantly in HEK293FS for
characterization by surface plasmon resonance (SPR) as
described below.
Chicken immunization yields anti-SIRPα antibodies withdiverse binding properties, SIRP reactivity, and broad
epitope coverage
We first characterized the binding kinetics and specificity of antiSIRPα scFv-Fc fusions toward a panel of SIRPs including human
SIRPα v1 and v2, cynomolgus monkey SIRPα, mouse SIRPα(m129, NOD, BL6 alleles), human SIRPγ, and human SIRPβ1.
We performed a single concentration off-rate screen to triage antiSIRPα scFv-Fc fusions based on species and allelic specificity, as
well as cross-reactivity toward SIRPγ and SIRPβ1. Most anti-SIRP
α scFv-Fc fusions were found to bind both human SIRPα v1 andv2 alleles and cross-react with cynomolgus monkey and mouse
SIRPα (Table S2), in agreement with the designed immunization
strategy and GEM screens. In addition to pan-allelic and species
cross-reactive anti-SIRPα antibodies, we identified a subset ofallele-specific and species-restricted anti-SIRPα antibodies withspecificity for hSIRPα v1 (clones 1, 123, 179, and 194) or
hSIRPα v2 (clone 110). In general, most anti-SIRPα antibodies
also bound SIRPβ1 and SIRPγ, which was expected given the highdegree of homology among SIRP family proteins (Fig. S4).

5

However, a small number of anti-SIRPα antibodies were identified that bind SIRPα and SIRPγ but not SIRPβ (clones 1, 9, 93,106), SIRPα and SIRPβ1 but not SIRPγ (clones 173, 174), and one
specific for SIRPα with limited binding to SIRPβ1 and SIRPγ(clone 179). The specificity of anti-SIRP antibodies for SIRPα,
SIRPβ, and SIRPγ determined by SPR agrees with the reactivity of
the anti-SIRP antibodies to various immune cell populations
within human peripheral blood, which differ in expression of
SIRPα, SIRPβ, and SIRPγ (Fig. S7). Although outside the scopeof this research, anti-SIRPα antibodies with differential specificity
for SIRPα, β, and γ may be useful tools to further explore thebiology of the SIRP family of immunoreceptors.
Next, we determined the ability of anti-SIRPα scFv-Fc fusions
to block the interaction between SIRPα and CD47. A competitive
SPR-based binding assay was used whereby blocking and nonblocking antibodies were identified based upon their ability tobind SIRPα while in complex with CD47. Anti-SIRPα antibodies
were first captured on a chip and increasing concentrations of
CD47 (0 to 1500 nM) were added to a constant concentration of
the high-affinity SIRPα v1 variant CV114 (100 nM) to preassemble the CD47:CV1 complex prior to injection over the antibody immobilized on a chip (Figure 3). At saturating concentrations of CD47 (>100 nM), antibodies that bind overlapping
epitopes on SIRPα (blocking) fail to bind the SIRPα/CD47 complex, whereas antibodies that bind non-competitive epitopes(non-blocking) form a classical sandwich with the SIRPα/CD47
complex. A number of blocking and non-blocking antibodies
were identified using this screen, as summarized in Table S2,
and representative sensorgrams of a blocking and non-blocking
antibody are depicted in Figure 3(a and b), respectively. In addition to blockers and non-blockers, a third category of anti-SIRPα
antibodies was identified, termed kick-offs, that form a transientcomplex between antibody:SIRPα:CD47 prior to displacement of
CD47 from antibody-bound SIRPα (Figure 3(c)). These antibodies bind SIRPα at a CD47 adjacent or minimally overlapping
epitope33 and trigger antigen exchange between antibody andCD47. This mechanism is supported by structural analysis presented below.
To determine the diversity of epitopes recognized by the isolated anti-SIRPα antibodies, we preformed epitope binning using
a classical sandwich-based SPR assay.34 Binning experiments wereconducted by immobilizing each anti-SIRPα antibody (ligand)Figure 3. Surface plasmon resonance binding profiles of representative anti-SIRPα blocker, non-blocker, and kick-off antibodies. To assess if the anti-SIRPα antibodies
can block CD47 binding to SIRPα in vitro, the antibodies were first captured by anti-human IgG-Fc on GLC chip as described in Materials and Methods. Next, 100 nM
of a high-affinity SIRPα variant (CV1) pre-mixed with CD47 at concentrations of 0, 20, 55, 500, or 1500 nM were injected over the antibody-immobilized chip. The
representative SPR sensorgrams for (a) blocking antibody clone 119, (b) non-blocking antibody clone 123, and (c) kick-off antibody clone 118 are shown. All
sensorgrams were baseline-adjusted and reference cell-subtracted.

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J. SIM ET AL.

Figure 4. anti-SIRPα antibodies bind six unique epitopes on SIRPα. (a) Sorted heat map depicting the binning assignments for a representative subset of discovered
anti-SIRPα antibodies. Anti-SIRPα antibodies bound to the chip are in rows and the anti-SIRPα antibodies injected over the chip in columns. Red boxes indicate thatthe antibodies compete with each other (and considered to bind the same epitope). Green boxes indicate that the antibodies form a sandwich (and considered tobind to different epitopes). Yellow boxes indicate scenarios where the data from one orientation disagrees with the other. (b) Phylogenetic tree for the subset of antiSIRPα antibodies shown in (a). (c) Node plot illustrating the connectivity and blocking relationships between the anti-SIRPα antibodies in the six epitope bins.
Antibodies isolated from wild-type, SynVH and OmniChickens are indicated as a triangle, circle, and square, respectively. Antibodies that bind to human, cynomolgus,
and mouse SIRP (as well as SIRPβ and SIRPγ) are colored blue; those that bind to human SIRPα v1, v2, and cynomolgus SIRPα are colored yellow; and the remaining
antibodies with varied specificities are colored white. Antibodies in Bin 1 do not cross-block antibodies in other bins. However, antibodies in bins 2, 3, 4, 5, 6 harborcross-blocking properties indicated by the connecting grey lines.

followed by capture of the recombinant SIRPα antigen and the
pairwise interrogation of an in-solution antibody (analyte) binding to the antibody-captured SIRPα antigen.34 Epitope binningwas performed again by reversing the mAb ligand/analyte orientation to validate the data set. A sorted heat map representing the
epitope binning results for a panel of anti-SIRPα antibodies isshown in Figure 4(a). For simplicity, only representative antibodies within each bin are shown. Overall, the anti-SIRPα antibodies
can be divided into six distinct epitope bins: blocking antibodies(bin 1), kick-off antibodies (bin 2), and non-blocking antibodies
that subdivide into four bins (bin 3, 4, 5, and 6). The dendrogram
showed that anti-SIRPα antibodies within the respective bins havediverse VH/VL sequences (Figure 4(b)). To represent these crosscompetition matrix relationships in a different dimension, a nodeplot was generated to emphasize the interconnectivities within
and between each epitope bin, sequence diversity within each bin,
SIRP reactivity, and the respective chicken from which the antibodies were isolated (Figure 4(c)). Interestingly, certain bins contain antibodies that segregate into diverse sequence groups andoriginate from different chickens, whereas other bins are sequencefamily and source specific. For instance, blocking antibodies (bin1) were isolated from wild-type, SynVH, and OmniChickens withfive distinct sequence lineages, whereas kick-off antibodies (bin 2)
were only identified from OmniChickens and represent a single,

MABS

related sequence family (Figure 4(c)). Non-blocking antibodies
segregated into four distinct bins (bins 3, 4, 5, and 6) with diverse
sequence groups, SIRP reactivity, and interconnectivity between
the various bins.
Epitope binning experiments also indicate anti-SIRPα antibodies in bin 1 do not cross-block anti-SIRPα antibodies in
the other five bins, whereas anti-SIRPα antibodies in bins 2, 3,
4, 5, and 6 cross-block each other (Figure 4(a, c)). Therefore,anti-SIRPα antibodies in bin 1 are expected to bind nonoverlapping epitopes compared to anti-SIRPα antibodies
from the other bins. On the other hand, anti-SIRPα antibodies
in bins 2, 3, 4, 5, and 6 are expected to share binding epitopes
based on their cross-competition network. For instance, antiSIRPα antibodies in bin 3 are expected to share certain binding site residues with anti-SIRPα antibodies in bin 5 but will
share a different and unique set of overlapping binding residues with anti-SIRPα antibodies in bins 4 and 6, respectively.
These observations were subsequently confirmed by structural
analysis (Figure 5).
In summary, pan-allele and -mammalian reactive antiSIRPα blocking and non-blocking antibodies (blue nodes)
could be identified from both wild-type and transgenic chickens; the use of multiple chickens with different Ig repertoires
enabled the discovery of a collection of anti-SIRPα antibodies
with broad epitope coverage and sequence diversity.
Crystal structures of anti-SIRPα Fab fragments in complex
with SIRPα
To further confirm results from epitope binning and map thebinding site details, as well as to elucidate the structural
mechanisms underlying the effects of anti-SIRPα antibodies,
we determined the crystal structures of representative antiSIRPα Fabs from epitope bins 1–5 in complex with the IgV
domain of human SIRPα v1. The antibodies include theblocking antibody 119 (bin 1), kick-off antibody 115 (bin 2),
and non-blocking antibodies 136 (bin 3), 3 (bin 4), and 218(bin 5). High-resolution crystals (1.83–2.68 Å) were obtained
for all fiveFab:SIRPα complexes, and the corresponding structures were solved by molecular replacement35 (Figure 5(a),Table S4).
The anti-SIRPα blocking antibody clone 119 (bin 1) binds
on top of SIRPα at an epitope highly overlapping that ofCD47, in agreement with the antagonistic properties ofclone 119 (Figure 5(b)). Although the binding geometries ofCD47 and clone 119 to SIRPα are different, both proteinsinteract with a number of shared residues on SIRPα distributed throughout the binding interfaces (Figure 5(b, c)). Clone
119 binds SIRPα in a “top-down” orientation inserting its
CDR-H3 loop into a central pocket of SIRPα formed by
the BC, C’D, and DE loops (Figure 5(b)), occupying thesame site as the CD47 N-terminal pyroglutamate (pE),
which contributes significantly to interaction between CD47
and SIRPα, whereas CD47 binds SIRPα in a “lying-down”
orientation inserting its FG loop into a wide groove on thesurface of SIRPα.35
The non-blocking antibodies 3, 136, and 218 bind SIRPα at
regions distal from the CD47 binding site and do not overlap
with any residues on SIRPα involved in either CD47 or clone 119

7binding (Figure 5(a, c)), consistent with results obtained from
epitope binning (Figure 4(a, c)). Anti-SIRPα clones 3, 136, and
218 interact with three unique but interconnected epitopes on
SIRPα (Figure 5(c)). Clone 136 occupies a central SIRPα epitope
flanked on opposing sides by clones 3 and 218, which binddistinct epitopes on SIRPα that partially overlap with clone
136, but not with each other. These crystallographic findings
are consistent with the cross-competition network between
clone 136 (bin 3), clone 3 (bin 4), and clone 218 (bin 5). Clone
136 competes with both clone 3 and 218 for SIRPα binding,whereas clones 3 and 218 compete with clone 136, but not witheach other (Figure 4(a, c)).
Interestingly, the “kick-off” antibody clone 115 binds
SIRPα directly underneath CD47 at a site mostly independent
from that of CD47, with the exception of one residue localized
to the C’D loop of SIRPα, Glu54, which is shared by both 115
and CD47 (Figure 5(c, d)). Superposition of the CD47:SIRPα
and 115:SIRPα interfaces reveals remodeling of the C’D loop
to an orientation that is favorable for binding to its respective
ligand, as well as potential steric clashes between clone 115
and CD47 that could prevent CD47 from remaining bound to
SIRPα upon antibody association (Figure 5(d)). It is conceivable that clone 115 first engages SIRPα while bound toCD47, potentially initiated by interactions between clone 115
and SIRPα that are distal from CD47, which ultimately leads
to displacement of CD47 due to steric clashes between theheavy chain framework 3 regions (HC-FR3) of clone 115 andCC’ loop of CD47 (Figure 5(d)). This agrees with results fromthe SPR screen, where a transient increase in resonance wasobserved, indicating formation of the ternary complex
between clone 115:CD47:SIRPα, and subsequent decrease inresonance consistent with the displacement of CD47 from theantibody/SIRPα complex (Figure 3(c)). Similar allosteric anddisplacement effects of an anti-IL-2 antibody that modulates
cytokine–receptor interactions have been described36 as well
as for anti-EGFR antibodies that target closely adjacentepitopes.33 In addition to overlapping epitopes with CD47,
clone 115 partially overlaps with clone 218 (Figure 5(a) and
Fig. S5), consistent with results obtained from epitope binning(Figure 4(a, c)).
We solved the structures of anti-SIRP antibodies in complex with the hSIRPa v1 allele; however, the structures of
SIRPα v1 and v2 are highly conserved,35 and only 1 or 2 of
the 13 polymorphic residues contribute interactions with theanti-SIRPα antibodies (Table S3). Therefore, we suspect theantibodies will bind hSIRPα v2 in a similar orientation,although this needs to be validated through future crystallographic analyses.
Anti-SIRPα antibodies bind with high affinity to SIRPα
Based on epitope binning and mapping, SIRP reactivity, andsequence diversity, we selected a subset of antibody clones forexpression as full-length IgG molecules for further characterization. Binding affinities (KD, M) as determined by SPR of representative anti-SIRPα antibodies from each epitope bin aresummarized in Table 3. All antibodies bound to human SIRPαv1 and v2 with high affinity ranging from low nanomolar topicomolar. To facilitate clinical development, we prioritized

8

J. SIM ET AL.

Figure 5. Crystal structures of anti-SIRPα antibodies bound to the IgV-domain of hSIRPα v1. (a) Structure of the SIRPα (pink)-CD47 (teal) complex superimposed withthe anti-SIRPα blocking antibody Fab clone 119 (blue), non-blocking antibody Fabs clones 3 (pale pink), 136 (grey), and 218 (purple), and kick-off antibody Fab 115(light green). (b) Overlay of the CD47/SIRPα complex and the blocking antibody clone 119 complex with a detailed view of CDR-H3 insertion into SIRPα pocket shownon the right. (c) Depiction of SIRPα (pink) epitopes bound by CD47 (teal) and anti-SIRPα antibody clones 119 (blue), 3 (pale pink), 136 (grey), 218 (purple), and 115(light green). Residues shared between the blocking (119) and kick-off (115) antibodies and CD47 are colored yellow. Venn diagrams adjacent to each surface map
indicate the relative extent of overlap between the blocking Fab 119 anti-SIRPα antibody, SIRPα, and CD47. For non-blocking antibodies, the yellow and white in the
Venn diagrams indicate the overlap of epitopes between Fab 3, 136, and 218. (d) Overlay of the kick-off antibody clone Fab 115 (light green) and CD47 (teal) incomplex with SIRPα (pink). The inset to the right shows the position of the C’D loop of SIRPα in the CD47-bound (pink) complex or the Fab 115-bound (orange)
complex. The Glu 54 residues is modeled in the C’D loop to highlight the varying position depending on whether the binding partner is CD47 or Fab 115.

MABS

9

Table 3. Binding affinities KD (M) of representative anti-SIRPα antibodies. Representative clones from each epitope bin were selected and their binding affinities tovarious SIRP (human SIRPα v1, v2; cynomolgus SIRPα; mouse NOD, BL6, BALB/c SIRPα; human SIRPγ; human SIRPβ1) were determined by SPR. A Langmuir kinetic
model was used for fitting and calculation of KD (M). The ability of the antibody to block CD47 binding to SIRPα is indicated in the last column.
Bin
1
1
1
2
3
4
4
4
4
5
5
5
5
5
5
6

Antibody1
119
135
21
115
136
3
173
209
213
123
149
161
162
194
218
45

Source4
H
H
S
H
H
W
H
S
S
H
H
H
H
H
S
W

V1
1.83E-10
1.51E-10<1.00E-12
4,26E-10
4.58E-10
1.62E-10
9.37E-10
1.71E-10
6.05E-09
6.05E-10
8.73E-10
1.03E-09
4.50E-10
4.97E-10
1.23E-10
6.63E-11

V2
6.82E-11
2.90E-11<1.00E-12
1.86E-09
1.63E-09
7.67E-11
9.28E-09
5.01E-09
1.69E-09
NB
2.38E-10
1.27E-10
1.57E-08
NB
2.76E-10
1.34E-10

Cyno
1.12E-10
9.69E-11
3.61E-12
2.41E-09
2.15E-09
2.29E-09
4.46E-08
3.90E-08
4.49E-08
1.62E-09
7.64E-09
6.35E-09
1.26E-08
9.11E-10
5.99E-11
NB

NOD
NB2
NB
5.47E-10
NB
5.54E-10
1.63E-09
NB
NB
NB
NB
NB
NB
NB
NB
NB
NB

BL6
NB
NB
4.60E-10
NB
1.27E-08
3.65E-09
NB
NB
NB
NB
NB
NB
NB
NB
NB
NB

BALB/cNB
NB
1.05E-09
NB
3.50E-10
1.16E-09
NB
NB
NB
NB
NB
NB
NB
NB
NB
NB

SIRPγ
2.67E-10
5.39E-10<1.00E-12
B3
2.39E-08
8.36E-08
NB
8.99E-09
2.02E-09
7.47E-10
1.89E-09
2.84E-09
3.97E-09
9.47E-10
6.36E-11
2.71E-08

SIRPβ1
3.42E-10
1.10E-10<1.00E-12
B
4.35E-09
1.63E-09
B
B
1.71E-08
7.07E-08
2.06E-10
2.63E-09
3.00E-09
5.36E-08
B
1.06E-08

CD47 Blocking
Block
Block
Block
Kick-offNon-block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block

1. All antibodies are screened as full IgG1 except 173, 209, 213, 123, 149, 161, 162, 194, 218 are screened as scFv-Fc.
2. NB = no binding
3. B = binding confirmed and exact KD value was not determined.
4. W = wt, H = OmniChicken, S = SynVH chickens.

Figure 6. Anti-SIRPα blocking antibody clone 119 binds with high affinity to human and cynomolgus SIRPα and potently blocks CD47 binding to cell surface SIRPα.
(A-C) SPR analysis of the binding kinetics of anti-SIRPα antibody clone 119 binding to (a) hSIRPα v1, (b) hSIRPα v2, and (c) cynomolgus SIRPα. (d) Competition assay
depicting dose-dependent antagonism of CD47-Fc binding to human CD14+ monocytes by anti-SIRPα blocking antibody clones 119 and 135.

anti-SIRPα antibodies that bind similarly to both human SIRPαalleles and to cynomolgus SIRPα. The blocking antibody clones
21, 119, and 135 bind human SIRPα v1 and v2 alleles andcynomolgus SIRPα with a KD of ~1 pM and 100 pM, respectively(Figure 6(a, b)). Clones 119 and 135 potently antagonize the
SIRPα/CD47 interaction with an IC50 of ~0.3 nM (Figure 6(d)).
Whereas clones 119 and 135 are specific for human and cynomolgus monkey SIRPα, clone 21 cross-reacts with mouse SIRPαalleles, albeit with somewhat reduced affinity (KD ~ 0.5–1 nM)(Table 3). In addition, the non-blocking anti-SIRPα antibody

clone 136 binds human SIRPα v1 and v2 alleles and cynomolgus
SIRPα similarly with a KD of ~1 nM. Clone 136 also cross-reacts
with mouse SIRPα alleles with affinities comparable to that ofhuman SIRPα (~1 nM). Of the antibodies that bind human andcynomolgus SIRPα, three also bind with high affinity to mouse
SIRPα alleles from NOD, C57BL/6, and BALB/c: one blocking
antibody (clone 21) and two non-blocking antibodies (clones 3
and 136) (Table 3). High-affinity binding of anti-SIRPα antibodies to all three mouse SIRPα alleles are particularly attractive as
this enables the interrogation of SIRPα antagonism across10

J. SIM ET AL.

diverse preclinical mouse xenogeneic and syngeneic cancer
models in the NOD/SCID, C57BL/6, BALB/c backgrounds. In
summary, a large panel of antibodies with diverse binding specificities toward human, cynomolgus, and mouse SIRPα, as well
as SIRP isoforms (SIRPβ1, SIRPγ), broad epitope coverage, andantagonistic properties were successfully identified in this study.
Anti-SIRPα antibodies potentiate macrophage
phagocytosis of human tumor cells
Next, we evaluated the ability of anti-SIRPα antibodies topotentiate macrophage-mediated ADCP in combination withtumor-specific antibodies. We mutated anti-SIRPα antibody Fcdomains to abrogate binding to all FcγRs and complement37,38
in order to decouple potential FcγR- and complementdependent contributions of anti-SIRPα antibodies to ADCP
from direct inhibition of SIRPα signaling. Human monocytederived macrophages were incubated with carboxyfluorescein
succinimidyl ester (CFSE)-labeled DLD-1 tumor cells opsonizedwith the tumor antigen-specific (anti-epidermal growth factor
receptor (EGFR)) antibody cetuximab alone or in combination
with increasing concentrations of anti-SIRPα blocking (clone
119) or non-blocking (clone 136) antibodies and phagocytosis
was quantified by flow cytometry. Untreated DLD-1 cells werepoorly phagocytosed, but treatment with cetuximab induced
a significant increase in macrophage phagocytosis, which was

further enhanced in a dose-dependent manner by the SIRPαantagonist (blocking) antibody clone 119 (Figure 7(a)).
Somewhat surprisingly, the non-blocking anti-SIRPα antibody
clone 136 also potentiated phagocytosis in combination withcetuximab (Figure 7(a)). Enhancement of phagocytosis by nonblocking anti-SIRPα antibodies was not specific to clone 136. All
anti-SIRPα non-blocking antibodies, irrespective of epitope,
potentiated cetuximab-mediated phagocytosis, albeit with varying degrees of potency (Figure 7(b)). Potentiation of phagocytosis by both blocking and non-blocking anti-SIRPα antibodies
was not dependent on the presence of an Fc domain, as F(ab’)2
fragments of clone 21 (blocking) and clone 136 (non-blocking)similarly enhanced phagocytosis compared to their full-length
IgG counterparts (Figure 7(c)). Treatment with either intact
anti-SIRPα antibodies or F(ab’)2 fragments of the non-blocking
antibodies had little to no impact on macrophage cell surface
SIRPα levels (Fig. S6), indicating that phagocytosis by nonblocking antibodies was not due to SIRPα downregulation. AntiSIRPα blocking and non-blocking antibodies similarly enhanced
macrophage phagocytosis of OE19 cells opsonized with theantitumor (anti-human epidermal growth factor receptor 2)
antibody trastuzumab (Figure 7(b)).
SIRPβ is an activating receptor with a high degree ofhomology to SIRPα (Fig. S4), therefore many of the discovered antibodies cross-react with SIRPβ. Antibodies against
SIRPβ have been reported to enhance mouse macrophage

Figure 7. Anti-SIRPα antibodies potentiate macrophage-mediated ADCP in vitro. (a) Phagocytosis of DLD-1 cells by human monocyte-derived macrophages treatedwith the tumor-specific, anti-EGFR antibody cetuximab alone (closed square) or in combination with anti-SIRPα blocking (clone 119) or non-blocking (clone 136)
antibodies. (b) Bar graph depicting the EC50 of phagocytosis (as shown in a) of anti-SIRPα antibodies from various epitope bins. Bin 1 = blocking, bin 2 = kick-off, andbins 3–6 = non-blocking. Phagocytosis was conducted using DLD-1 cells as targets with cetuximab in combination with anti-SIRPα antibodies (black bars), or OE19
target cells with trastuzumab in combination with anti-SIRPα antibodies (grey bars). (c) Dose-dependent enhancement of DLD-1 phagocytosis by human monocytederived macrophages treated with cetuximab in combination with anti-SIRPα intact IgG antibodies (blocking clone 21 and non-blocking clone 136) or thecorresponding F(ab’)2 fragments. (d) Macrophage phagocytosis of DLD-1 cells treated with cetuximab alone or in combination with the hSIRPα v1 allele-specificantibody39 or pan-allele reactive anti-SIRPα v1/v2 antibody clone 21 as a function of macrophage SIRPα genotype. Data are normalized to the maximum
phagocytosis observed in the clone 21 group. Data in panels A, C, and D are the mean (n = 3), and error bars indicate SD. Dashed lines represent data fit to a logagonist vs. response model in Prism.

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phagocytosis of IgG-opsonized sheep red blood cells in vitro,
although phagocytosis was enhanced only in the presence of
secondary cross-linking antibodies, suggesting higher order
SIRPβ clustering is necessary to promote phagocytosis.40
Nonetheless, to investigate the contribution of SIRPβ to antiSIRP antibody-mediated macrophage phagocytosis, we profiled the human macrophages used in our assay for expressionof SIRPα, SIRPβ, and SIRPγ, and conducted phagocytosis
assays using anti-SIRPα antibodies that do not bind to
SIRPβ: clone 179 (SIRPα specific), and clone 1 (SIRPα andSIRPγ specific). SIRPβ was expressed on human CD14+
monocytes but was not expressed on the monocyte-derived
macrophages (MDM⊘) used in our assay (Fig. S8A). SIRPγ
was expressed on CD3 + T cells, but not CD14+ monocytes or
MDM⊘. The anti-SIRPα antibody clones 1 and 179, which do
not bind SIRPβ, potentiated cetuximab-mediated macrophage
phagocytosis of DLD-1 cells in a dose-dependent manner
similar to the activity of the pan-SIRP antibody clone 21(Fig. S8B). Collectively, our data indicate that the phagocytic
activity of the anti-SIRP antibodies used in this study is
mediated by SIRPα and not due to cross-reactivity with
SIRPβ.
Furthermore, SIRPα allele cross-reactivity was crucial to theefficacy of anti-SIRPα antibodies (Figure 7(d)). The anti-SIRPα
v1 allele-specific antibody potentiated tumor cell phagocytosis
by v1/v1 homozygous macrophages but failed to enhance phagocytosis mediated by v1/v2 heterozygous or v2/v2 homozygous macrophages. In contrast, the pan-allele anti-SIRPα v1/v2
antibody potentiated ADCP independent of macrophage
SIRPα genotype (Figure 7(d)). These data are consistent withthat of Zhao et al. who demonstrated that a single functional
allele of SIRPα was sufficient to inhibit neutrophil-mediatedkilling of antibody-opsonized tumor cells.9 Thus, therapeutictargeting of SIRPα requires pan-allelic antibodies in order to be
useful in diverse patient populations.

Discussion
The CD47-SIRPα axis has emerged as a myeloid checkpoint
pathway to be targeted for the treatment of human cancers,
and an ensuing plethora of agents aimed at disrupting theCD47-SIRPα interaction have entered the clinic or are inpreclinical development.17 We previously demonstrated that
ALX148, a high-affinity variant of hSIRPα fused to an inactive
Fc-domain of IgG, potently antagonizes CD47, is well tolerated in preclinical species and has a favorable safety profile inhumans due to the lack of Fc-effector function,12,19 is efficacious in numerous xenogeneic and syngeneic mouse cancer
models when used in combination with tumor-specific andcheckpoint antibodies,12 and has promising preliminary antitumor activity in humans.19 ALX148 prevents SIRPα signalingby occupying CD47, both on the tumor and in the periphery.
CD47 is broadly expressed and has numerous reported
ligands and functions beyond SIRPα-dependent regulation ofmacrophages and dendritic cells.23 The functional differences
between targeting SIRPα and CD47 are generally unknown inpart due to a lack of specific reagents that antagonize SIRPα.
Thus, we sought to generate and characterize anti-SIRPα
antibodies with diverse properties in order to investigate

11

SIRPα biology and extend our approach of antagonizing theCD47/SIRPα interaction as a treatment modality for cancer to
SIRPα.
The development of effective SIRPα antagonists suitable forclinical translation has been challenging due to the polymorphic nature of SIRPα as well as a poor understanding of
the allelic distribution and frequency of SIRPα across humanpopulations. Zhao et al. previously demonstrated using panallelic or allele-specific anti-SIRPα blocking antibodies thatantagonism of both SIRPα alleles was necessary to enhance
trastuzumab-mediated ADCC when neutrophils from SIRPαv1/v2-heterozygous donors were used as effector cells.9 We
observed a similar requirement for antagonism of both SIRPαalleles to enhance macrophage-mediated ADCP when v1/v2heterozygous macrophages were used as effectors (Figure 7(d)),
in agreement with results by Zhao et al.9 and extending thesefindings to macrophages. Given the high percentage of SIRPαv1/v2-heterozygous individuals across human populations(Figure 1), as well as the requirement for full SIRPα antagonism
to maximize efficacy, anti-SIRPα antibodies should be capableof antagonizing both SIRPα alleles. Numerous, potent antiSIRPα antibodies that bind to both human SIRPα alleles arereported in this study.
In addition to the need for pan-allelic antibodies, to facilitate
preclinical development, antibodies directed against human
SIRPα should be cross-reactive with cynomolgus monkey SIRPα
for IND-enabling toxicology studies, and ideally cross-react withmouse SIRPα alleles to avoid the use of surrogates when investigating pharmacology. Only a few anti-human SIRPα antibodies
have been described in the scientific literature.9,16,22,41 These antibodies were raised in mice, and therefore do not cross-react withmouse SIRPα. In order to generate pan-mammalian and panallelic anti-SIRPα antibodies, we immunized chickens, which are
phylogenetically distinct from mice and humans, thereby increasing the probability of generating a productive antibody response
against mouse and human immunogens. Using a multi-species
and multi-allele immunization schedule and chickens with wildtype or human Ig repertoires,30,31 we identified a panel of wildtype, chimeric, or fully human anti-SIRPα blocking and nonblocking antibodies with broad specificities directed against theCD47-binding IgV domain of SIRPα. The isolated antibodies
exhibited high-affinity binding to SIRPα with affinities ranging
from low nM to low pM, and therefore additional affinity maturation was unnecessary. The panel of antibodies identified displayed
broad epitope coverage, enabling the interrogation of the role ofepitope on the function of anti-SIRPα antibodies.
The most well-known function of the SIRPα/CD47 interactionis the negative regulation of macrophage phagocytosis. Antibodies
that block the interaction between CD47 and SIRPα promote
macrophage phagocytosis of tumor cells by inhibiting SIRPαsignaling and tipping the balance of pro- and anti-phagocytic
signals.4,9,12,15,16 The anti-SIRPα blocking antibodies describedherein potently potentiate macrophage-mediated ADCP oftumor cells, as expected based on the known effects of antagonizing the SIRPα/CD47 axis. Interestingly, we found that nonblocking antibodies directed against SIRPα also enhance macrophage phagocytosis (Figure 7). Enhancement of phagocytosis by
non-blocking antibodies was independent of the five different
epitopes on the SIRPα IgV-domain that are targeted by the

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J. SIM ET AL.

panel of antibodies we discovered. These data are in agreementwith reports by Murata et al.41 who demonstrated that an antiSIRPα non-blocking antibody that binds the Ig-C1-like domain of
SIRPα also promoted macrophage phagocytosis, albeit to a lower
extent than blocking antibodies, suggesting that phagocytosis
mediated by non-blocking anti-SIRPα antibodies is not epitopeor domain specific, but likely an intrinsic function of antibodybased targeting of SIRPα. Phagocytosis triggered by non-blocking
antibodies directed against the SIRPα/CD47 axis appears to be
specific to targeting SIRPα, as non-blocking antibodies directedagainst CD47 have no effect on macrophage phagocytosis.15 It has
been speculated that SIRPα non-blocking antibodies promotephagocytosis through either SIRPα downregulation or the ‘scorpion effect’ where antibody binding to SIRPα permits Fc-domainbinding and activation of FcγR signaling on the same cell.41
However, we observed little to no downregulation of SIRPα during the course of phagocytosis (Fig. S6). Furthermore, the antibodies used in our experiments were mutated to abrogate FcγR
and complement binding, ruling out potential contributions of
cis-interactions with activating or inhibitory FcγRs. In addition,
F(ab’)2 fragments of non-blocking anti-SIRPα antibodies, whichlack an Fc domain, were equipotent to their corresponding fulllength IgG molecule (Figure 7(c)). The activating receptor SIRPβ
has been suggested to be a positive regulator of macrophage
phagocytosis;40 however, the macrophages used in our experiments do not express SIRPβ or SIRPγ (Fig. S8A), therefore antiSIRP antibody-mediated phagocytosis cannot be attributed tocross-reactivity with SIRPβ or SIRPγ. Collectively, these datasuggest that alternative, yet unknown mechanisms are responsible
for the potentiation of macrophage phagocytosis by anti-SIRPα
antibodies that do not block the interaction with CD47. It isconceivable that antibody binding to SIRPα may restrict CD47independent SIRPα tonic signaling, thereby promoting phagocytosis. The detailed mechanism by which SIRPα signaling influences macrophage phagocytosis is poorly understood. The broad
panel of antibodies described herein may be useful tools to interrogate such biological mechanisms and as promising therapeutics
to treat human cancers.

Materials and Methods
Bioinformatic analyses of SIRPα polymorphisms
We performed in-depth bioinformatic investigation of IgV
domain of the SIRPA gene using sequence data from 1000
Genome Project. For this, we downloaded genomic variants for2535 sequenced genomes from the Phase 3 dataset present in vcf(variant calling files) from public database (http://www.internatio
nalgenome.org/data#download) that span SIRPA gene(chr20:1,875,425–1,920,540, hg18 reference genome). Of 1722
variants in this region, 21 known variants that resulted in missense
mutations were present in the IgV domain of the SIRPA gene.
Errors in variant calls due to low coverage were fixed by customly
written perl script. cDNA sequences of various samples were
determined using fixed vcf files and translated to protein
sequences by making changes in the consensus coding sequence(CCDS13022.1) of the SIRPA gene using customly written perl
script. After which, the translated amino acid sequences in the IgV

domain were then compared with EnsEMBL_V1 sequence(ENSP00000348307.3).
Sanger sequencing of selected human samples
Sanger sequencing of 510 samples from different populations were
performed (Table S1). These samples were selected from different
populations: Mexican Ancestry from Los Angeles USA (MXL),
Yoruba in Ibadan, Nigeria (YRI), Utah Residents (CEPH) with
Northern and Western European Ancestry (CEU), Colombians
from Medellin, Colombia (CLM), British in England and Scotland(GBR), Han Chinese in Beijing, China (CHB), Bengali from
Bangladesh (BEB), and Americans of African Ancestry in SouthWest USA (ASW), Japanese in Tokyo, Japan (JPT). DNA samples
for these populations were obtained from the NHGRI Sample
Repository for Human Genetic Research at the Coriell Institutefor Medical Research. DNA was quantified using picogreen assay
and normalized to 10 ng/ml. PCR amplification of the target
region (exon 3) was carried out using PCR primers pairs forward
5’-TGTCTGGAATACCAGGCTCCCTT and reverse 5’TACCACCACACCTGATCATTGCTC (IDT Technologies,
Iowa City, USA) and KAPA Hyper polymerase (Roche Holding
AG, Basel, CH). PCR amplification was performed using thereactions conditions as follows: after preheating at 95°C for 5min, amplification consisted of 30 cycles at 98°C for 20 s, 68°C
for 30 s, and a final extension at 72°C for 5 min. PCR products
were purified using the AMpure® XP (Beckmann Coulter, Brea,
CA) and quantified using the NanoDrop® (Thermo Fisher
Scientific). While performing Sanger sequencing, we noticed thepresence of three known deletions (rs138304215-delCT,
rs749337996-delCT, and rs139878822-delCGA), and these deletions resulted in chromatograms that are not interpretable. Due tothe complexity of these regions, five different Sanger sequencing
reactions were performed using the following primers to generateconclusive Sanger sequencing data;a) 5’-GGCTCCCTTTCCGGAACTTCACACAG,b) 5’-GTGTGAAGTTCCGGAAAGGGAGCCCCGAT,c) 5’-GCTCCAGACTTAAACTCCACGTCATCGG,d) 5’-CCTGCTCCAGACTTAAACTCCACGTCAG ande) 5’-GTGTGAAGTTCCGGAAAGGGAGCCCT.
DNA (10 ng) was sequenced using the BigDye Terminator®
cycle sequencing kit (v3.1; Thermo Fisher Scientific).
Following purification using the Centri-Sep® Spin Columns(Thermo Fisher Scientific), the nucleotide sequences were
determined using an ABI3730XL Genetic Analyzer (Thermo
Fisher Scientific). Sequence data were aligned using
Sequencher® DNA sequence analysis software (v5.2.4; Gene
Codes Corporation, Ann Arbor, MI). All the Sanger sequencing results (Table S1) were found to be concordant with our
bioinformatic analysis.
Generation of antigens for immunization and screening
The IgV domains of SIRPα antigens from four respective
sources were generated for immunization as fusion tohuman IgG-Fc to increase immunogenicity. Specifically, they
are human SIRPα v1 (NP_542970.1), SIRPα v2 (CAA71403.1),

MABS

mouse 129 SIRPα (162330193) and NOD SIRPα sequence as
described in references42,43 and reported in Figure S2 andFigure S3. The Fc-fused proteins were expressed in Expi293
cells (Invitrogen) using standard manufacturer’s protocol.
Expression cultures were typically grown for five days at 37°
C in 8% CO2. Supernatants were harvested via centrifugationand sterile filtered. Proteins were affinity purified using
MabSelect Sure LX resin (GE Healthcare). For SPR screening,the IgV domains of the respective SIRP were expressed in
Expi293 cells as described above as either His-tagged or HisAvi-tagged fusions and purified using Ni-Sepharose 6 Fast
Flow affinity purification (GE Healthcare). The panel of
SIRP generated for SPR screening were the IgV domainfrom human SIRPα v1 (NP_542970.1), human SIRPα v2(CAA71403.1), cynomolgus SIRPα (EHH65484.1), mouse
129 SIRPα (162330193), NOD/BALBc/C57BL/6 SIRPα(sequences as described in references 41 and 42 and reported
in Figure S2 and Figure S3), and human SIRPγ (NP_061026.2)
and human SIRPβ1 (NP_006056.2) (sequences as reported in
Figure S4). The IgV domain of CD47 was generated as
described in reference 12. The high-affinity SIRPα variant
CV1 was generated as described in reference 14.
Immunization of chickens
In total, 19 chickens were immunized. Specifically, these include
fourwildtype White Leghorn chickens, 4 SynVH chickens, which
are transgenic chickens containing VH from human and VL
from chicken,31 and 11 chickens with fully human immunoglobulin loci called OmniChicken.30 The chickens were immunizedwith varied schedules having alternating doses and boosts ofhuman SIRPα v1, human SIRPα v2 and mouse SIRPα (m129or NOD). The immunization protocol was approved by theInstitutional Animal Care and Use Committee. Sera were collected bi-weekly during the immunization to determine plasma
titer by ELISA. In total, eight immunized birds with good final
titers were selected for preparation of splenocytes and lymphocyte screenings using the GEM assay (Figure 2(b)). The immunization schemes for the selected birds are described in Table 2.
For example, wildtype 21,288 bird was initially immunized with
100 µg human SIRPα v1 at week 1, boost with 50 µg of human
SIRPα v2 at week 3, draw at week 4, boost with 50 µg of human
SIRPα v1 at week 5, draw at week 6, boost with 50 µg of human
SIRPα v2 at week 7, and draw at week 8.
Screening single B cells using the GEM assay
A single lymphocyte screening and recovery method, theGEM assay was used to isolate antigen-specific mAbs from
immunized chicken. The GEM assay involves placing single
mAb-secreting lymphocytes in proximity with reporter beads.
The secreted mAb diffuses locally within the GEM and has theopportunity to bind to the reporter beads. Bound mAb isdetected directly through the use of a fluorescent secondarymAb, anti-IgY Alexa 594 (Life Technologies A-11042). There
were four beads used in this program: 1) a 5 μm latex beadcoated with hSIRPα v1; 2) a 5 μm blue latex bead coated withmSIRPα (129, NOD or BALBc); 3) a 1.4 μm latex bead coatedwith hSIRPα v2, and 4) a 5 μm latex bead coated with CD47

13

followed by a mixture of high-affinity SIRPα v1 and v2. Threeof the four beads were encapsulated with a single B cell fromthe chicken being screened depending on the objective of thescreen. For example, if the goal of the screen was to ensurespecies cross-reactivity, then the beads encapsulated were 5
um hSIPRα v1, 1.4 um hSIRPα v2 and 5 um blue mSIRPα.
Fresh beads were coated within a week of performing GEM
assays. To coat beads, protein was diluted in phosphatebuffered saline (PBS). The diluted protein was added directly
to the beads and incubated at 4°C overnight while rocking.
Beads were washed with PBS and blocked with 3% filteredmilk in PBS. Beads were washed and tested using reactive
plasma from immunized chickens. GEMs were viewed ona Leica DMI 6000 B inverted fluorescent microscope.
Cloning and expression of recombinant mAbs
Cells secreting antigen-specific mAbs were isolated from the
GEM assay, and their respective V genes were amplified bya two-step, semi-nested strategy as described.32 Briefly, theV-genes were cloned through RT PCR and cloned into themammalian expression vector pF5a (Promega) in scFv-Fc format (IgG1-Fc isotype with effector attenuating Fc
substitutions37,38,44 that ablate FcγR and complement binding).
Plasmids containing recombinant scFv-Fc from the GEM harvests were transiently transfected into HEK293F (Thermo
Fisher) and clonal supernatants were harvested. Supernatants
were tested for binding activity by SPR described below. All
clones that were confirmed as binding their respective targets
were fully sequenced to avoid redundancies. Select clones wereconverted to full-length IgG1 molecules with effector attenuating Fc substitutions and produced in HEK293F.
SPR Screening of Antibody Clones
Binding of the antibody clones to various SIRP proteins wasdetermined using SPR detection on a ProteOn XPR36 instrument(Bio-Rad, Hercules, CA) using PBS (pH 7.4) supplemented with
0.01% Tween-20 (PBS-T) as running buffer. The pre-filtered
media containing the recombinant scFv-Fc were used directly
for the assay. First, anti-human IgG Fc (BR-1008–39, GE
Healthcare) was amine-coupled onto a GLC sensor chip (BioRad, Cat. #1765011) to generate the capture surfaces for theantibodies. About 4000 RU per flow cell of immobilized antihuman IgG Fc is achieved. Each clone was screened using thesame method as follows: 5–10 μL of pre-filtered media in 10 mM
sodium acetate buffer (pH 4.5) was injected for 2 min at 30 μl/min;buffer flow for 1 min at 100 μL/min; SIRP analyte (100 nM)
injected for 1 min at 100 μL/min, followed by a dissociation
cycle of 10 min; regeneration of chip surface by flowing
3 M magnesium chloride for 1 min at 25 μL/min in both orientation; and buffer flow for 1 min at 100 μL/min. Biosensor data weredouble-referenced by subtracting the interspot data (containing
no immobilized anti-human IgG Fc) from the reaction spot data
(immobilized anti-human IgG Fc), and then subtracting the
response of a buffer “blank” analyte injection from that of ananalyte injection. Binding was fitted using a 1:1 Langmuir and koff(1/s) values calculated. All SPR assays were performed at 25°C andall SPR assays described here used PBS-T as running buffer.

14

J. SIM ET AL.

Anti-SIRPα antibody screen to identify effect on the
SIRPα/CD47 interaction
To assess if antibodies can block binding of SIRPα to CD47, an
SPR screen was carried out. Antibody capture was carried out
using an anti-human IgG-Fc immobilized GLC surface prepared
as described above. A SIRPα variant (CV1) engineered to bind
CD47 with high nM affinity14 was used for the screen rather thana wildtype SIRPα. This is because the wildtype SIRPα variant has
low μM binding affinity to CD47, which does not allow stablecomplex interaction to assess sandwich formation. First,
approximately 5–10 uL of pre-filtered media containing theantibodies in 10 mM sodium acetate buffer (pH 4.5) was injected
for 2 min at 30 μl/min and captured over the anti-human IgG-Fc
immobilized GLC surface followed by a brief buffer flow of 1
min at 100 μL/min. Next, 100 nM of a high-affinity SIRPαvariant (CV1) pre-mixed with CD47 at different concentrationsof 0, 20, 55, 500, or 1500 nM was injected separately for a minuteat 100 μL/min with a dissociation time for 10 min.

ProteOn Amine Coupling Kit. For the immobilization step,
GLC chip was activated with EDAC/Sulpho-NHS 1:1 (Biorad)
diluted 1/100 for 300 s at 25 μL/min. Anti-SIRPα antibodies werediluted to 80 nM concentration in 10 mM sodium acetate buffer
pH 4.5 and immobilized to the chip at 30 μL/min for 50 s. Chip
was inactivated with ethanolamine for 300 s at 25 μL/min. The
analytes (e.g., SIRPα from different species, SIRPβ1, SIRPγ) were
injected in a “one-shot” kinetic mode at nominal concentrationsof 100, 33, 11, 3.7, 1.2 and 0 nM. Association times were monitored for 90 s at 100 μL/min, and dissociation times were monitored for 1200 s. The surfaces were regenerated with a 2:1 v/v
blend of Pierce IgG elution buffer/4M NaCl. Biosensor data weredouble-referenced by subtracting the interspot data (containing
no immobilized protein) from the reaction spot data (immobilized protein), and then subtracting the response of a buffer“blank” analyte injection from that of an analyte injection.
Double-referenced data were fit globally to a simple Langmuirmodel, and the KD value was calculated from the ratio of theapparent kinetic rate constants (KD = kd/ka).

Epitope binning
Epitope binning was carried out using a classical sandwich
approach using methods described by Abdiche et al.34 An antiSIRPα antibody was first immobilized on a GLC chip, and thenhuman SIRPα v1 was injected followed by a second anti-SIRPα
antibody. If the second antibody was able to bind the complex
formed between the first anti-SIRPα antibody and SIRPα, thefirst and second antibodies were determined to bind different
epitopes. If the second antibody was not able to bind, the firstand second antibodies were determined to share an epitope. The
detailed components of the experiment are described as follows.
First, anti-SIRPα antibodies were immobilized on GLC chips
using Proteon Amine Coupling Kit as described before. Briefly,
for the immobilization step, GLC chip was activated with
1-ethyl-3-(−3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC)/sulfo-N-hydroxysuccinimide (Sulpho-NHS) 1:1(Biorad) diluted 1/80 for 300 s at 25 μL/min. Anti-SIRPα antibodies were diluted to 80 nM concentration in 10 mM sodium
acetate buffer pH 4.5 and immobilized to the chip at 30 μL/min
for 50 s. The chip was inactivated with ethanolamine for 300 s at
25 μL/min. After which, the IgV domain of SIRPα v1 (100 nM)
was first injected at 100 μL/min for 60 s, followed by injection of
the anti-SIRPα antibodies in testing (100–150 nM) at 100 μL/
min for 60 s. The surfaces were regenerated with a 2:1 v/v blendof Pierce IgG elution buffer/4 M NaCl. The resulting sensorgrams were used to score and group the antibodies into different
bins according to their binding profiles.
Determination of KD
The binding affinities of anti-SIRPα antibodies to SIPRα fromvarious species (human v1, human v2, cynomolgus, mouse 129,
BL6, BALBc, NOD), SIRPβ1 and SIRPγ were determined using
direct immobilization of the antibodies (via GLC chip). All analytes were used at their nominal concentrations determined by
A280 absorbance and molar extinction coefficient. Analytes were
injected in a “one-shot” kinetic mode45 and flowed over anti-SIRP
α antibodies immobilized (~1000 RUs) on GLC chips using

Crystallization of anti-SIRPα Fab: SIRPα complexes
The IgV domain of SIRPα v1 was generated for crystallography as previously described.46 A mutation N80A was introduced to produce a homogenous and non-glycosylated SIRPα
v1 that would be most amenable for crystallization. The antiSIRPα antibodies were generated as Fab fragments witha 6His-tag at the C-terminus of the truncated hinge domain.
Both SIRPα v1 and the Fab fragments were expressed in
Expi293 and purified using Ni-Sepharose 6 Fast Flow affinity
purification (GE Healthcare) followed by gel filtration. The
Fabs and SIRPα v1 proteins were purified individually prior tobeing complexed (with SIRPα v1 in molar excess) proceededby a final SEC step to separate unbound proteins. The final
formulation buffer is minimal with 10 mM Tris pH 7.5 and50 mM NaCl. The purified Fabs and SIRPα complex sample isstable at 4°C and was concentrated to 10–12 mg/mL for
crystallography.
The methods for the anti-SIRPα antibodies and SIRPα complex
structure solution followed well-established principles of crystallography, beginning with sparse matrix crystallization trials.
Conditions were selected from initial trials and optimized forformation of favorable crystals for harvest and synchrotron
X-ray diffraction screening. Once a crystallization regime is established for one complex, the protocol can be followed for subsequent anti-SIRPα Fab antibodies: SIRPα complexes.
Crystallization experiments used the sitting drop vapor diffusion
technique for the initial sparse matrix screening and commerciallyavailable kits from Qiagen. Crystallization trials were set in varying
ratios ranged in 1:1, 2:1, and 3:1 of anti-SIRPα Fab: SIRPα complex
to crystallant in total volume of 1 μL in the subwells of the 96-well(8 x 12) tray while 100 μL of crystallization solution was placed in
the well reservoir. The completed plates were sealed to allow vapor
diffusion to occur and stored in a 12°C incubator. Crystallizationof the five anti-SIRPα Fab: SIRPα complexes were achieved with
derivatives of two main conditions as shown in Table S5. The
detailed steps involved for crystal harvest, data collection, andprocessing, structure building and refinement are described inMABS

the supplementary materials. The PDB codes for the structuresgenerated are 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR.
Epitope mapping and superimposition analysis based on
crystal structures
Buried surface area of the antigen for the epitope was calculated as the difference between the solvent-accessible surface
area of the antigen alone and antigen in complex with the Fabfragment of the antibody. The accessible surface area wascalculated by the rolling ball method47,48 with a probe radiusof 1.4 Å. Buried surface area is reported in Å2 (Table S3). All
Fab antibody: SIRPα complexes were aligned by superimposing the carbon atoms of SIRPα from each structure with theCD47: SIRPα complex (v1 variant) structure (PDB: 4CMM).49
Superimposition was performed using PYMOL. The calculated RMSD (0.50) between the aligned SIRPα structures in
the complexes showed little variation, hence validating theoverlay (Table S6). Therefore, the alignment of the SIRPα
structures is credible to establish a reference point to analyze
the varying binding epitopes of the Fab anti-SIRPα antibodies.
Cell staining to determine binding profiles of anti-SIRP
antibodies
Peripheral blood mononuclear cells (PBMCs) were isolated
from Trima residuals of healthy individuals (purchased from
Vitalent) with Ficoll-Paque Plus (GE Cat. 17144003). CD14+
monocytes were isolated from PBMC by negative selection(Miltenyi Cat. 130–091-153) per manufacturer’s protocol.
Monocyte-derived macrophages (MDM⊘) were differentiatedfrom CD14+ monocytes using macrophage colonystimulating factor (M-CSF) (Miltenyi Cat. 130–096-491) (50ng/mL) for 9 days in culture medium. Cells were stained withFcR block (Miltenyi Cat. 130–059-901) in PBS (Gibco Cat.
10010–031) + 0.5% bovine serum albumin (BSA) (Sigma
A1933) with the addition of the following monoclonal antibodies (all from Biolegend): PerCP-Cy5.5-labeled CD3 (Cat.
317336); AF488-labeled CD4 (Cat. 344618); BV510-labeled
CD8 (Cat. 344732); PE-CY7-labeled CD14 (Cat. 325618); PElabeled LSB2.20 (Cat. 336606), B4B6 (Cat. 323906) andmatched isotype control (Cat. 400112). Purified anti-SIRP
antibodies discovered in house and un-labeled LSB2.20 and
SE5A5 (Biolegend Cat. 336602 and Cat. 323802) were aminelabeled with Alexa Fluor 647 (ThermoScientific Cat. A20173).
Fixable viability dye (eBioscience Cat. 65–0865-14) was used
to identify live cells. Samples were acquired with Canto II flow
cytometer (BD) and analyzed using FlowJo software.
Macrophage-mediated antibody-dependent cellularphagocytosis assay
CD14+ cells were purified from Trima residuals (Blood
Centers of the Pacific) with Ficoll-Paque Plus and negative
selection (Monocyte Isolation Kit II, Miltenyi Biotec) according to the manufacturers’ protocols. MDM⊘ were made byseeding six million CD14+ cells into 150 mm tissue culture
dishes (Corning) in growth medium supplemented with 10%
fetal bovine serum (Millipore) and 50 ng/mL M-CSF

15(Miltenyi Biotec). Cells were cultured for 7 to 11 days.
Adherent cells were detached from culture plates with
TrypLE Select (Thermo Fisher Scientific). Target cells werelabeled with the Celltrace CFSE Cell Proliferation kit (Thermo
Fisher Scientific) according to the manufacturer’s instructions. 100,000 target cells and 50,000 monocyte-derived
macrophages were incubated in ultra-low attachment
U-bottom 96-well plates (Corning) with anti-SIRP antibodies
and the corresponding tumor-specific antibody for 2 h at 37°
C. Cetuximab was added at a concentration of 0.01–0.1 μg/ml
for DLD-1 cells and trastuzumab was added at
a concentration of 0.01 μg/ml for OE19 cells. F(ab’)2 fragments were prepared using the Pierce F(ab’)2 purification kit
following the manufactures recommended protocol(ThermoFisher) and undigested IgG was purified away fromF(ab’)2 fragments via Protein A. Cetuximab and trastuzumab
were produced in HEK293F using standard monoclonal antibody production methods. Sequences were obtained from the
Drugbank (www.drugbank.ca), DB00072 and DB00002 fortrastuzumab and cetuximab, respectively.
For flow cytometry, cells were incubated in human FcR
Blocking Reagent (Miltenyi Biotec) and stained with fluorochrome-labeled antibodies against CD33 (clone WM53,
Biolegend) and CD206 (clone 15–2, Biolegend). To eliminate
macrophage/target cell adhesion from analyses, antibody against
CD326 (clone 9C4, Biolegend) was included for DLD-1 andOE19 cells. Furthermore, a pulse geometry gate of forward
scatter signal area vs height was used to select for single cells.
Fixable viability dye (Thermo Fisher Scientific) was used to
identify live cells. Cells were analyzed on a FACS Canto II flow
cytometer (BD Biosciences) with subsequent analysis using
FlowJo software. Percent phagocytosis indicates the percentageof viable CD33+ CD206+ macrophages that stain negative forCD326 and positive for CFSE. When applicable, four parameter
fit curves were generated with Prism 7 software (GraphPad).
Cell-based SIRPα antagonism assay
Antagonism of CD47 binding by anti-SIRPα antibodies was
performed using CD47-Fc fluorescently labeled using theAlexa Fluor 647 Protein Labeling Kit (Thermo Fisher
Scientific) according to the manufacturer’s instructions.
Alexa Fluor 647-labeled CD47-Fc at a concentration of 250
nM and fluorescently labeled anti-CD14 (Biolegend Cat.
325618) were added to 100,000 PBMCs in 100 μL volume of
FACs buffer (PBS + 0.5% BSA) supplemented with a cocktail
of human Fc block (Miltenyi Biotec). After 30 min on ice,cells were washed once in staining buffer. Anti-SIRPα wasadded at a concentration of 1.25 μM with 10-fold serial dilutions. After 30 min on ice, cells were washed twice in staining
buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed using a FACS Canto II (BD Biosciences), with subsequent data analysis and histogram plotting using FlowJo 10.7
and Prism 7 software (GraphPad).

Disclosure statement
These studies were sponsored by ALX Oncology. JS, JTS, ES, LD, OH,
SEK, AC, TCK, HW, and JP are all employed by ALX Oncology and are

16

J. SIM ET AL.

shareholders. The funder provided support in the form of salaries but did
not have any additional role in the study design, data collection, andanalysis, decision to publish, or preparation of the manuscript.

ORCID
http://orcid.org/0000-0002-6438-1995
Janet Simhttp://orcid.org/0000-0002-5441-1409
Jonathan T. Sockolosky
http://orcid.org/0000-0002-6222-6588
Emma Sangalang
http://orcid.org/0000-0002-0955-8903
Ardian S. Wibowo
http://orcid.org/0000-0001-7322-349X
Josh Carter
http://orcid.org/0000-0002-0967-7574
Anup Madan
http://orcid.org/0000-0003-1241-7017
Laura Doyle
http://orcid.org/0000-0003-4853-6544
Steven E. Kauder
http://orcid.org/0000-0001-6297-8694
Tracy C. Kuo
http://orcid.org/0000-0003-2537-8950
Hong Wan
http://orcid.org/0000-0002-0831-419X
Jaume Pons

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