(2020) 13:160
Kuo et al. J Hematol Oncol
https://doi.org/10.1186/s13045-020-00989-w

Open Access

RESEARCH

Targeting the myeloid checkpoint receptor
SIRPα potentiates innate and adaptive immune
responses to promote anti‑tumor activity
Tracy C. Kuo1,3* , Amy Chen1,3, Ons Harrabi1,3, Jonathan T. Sockolosky1,4, Anli Zhang2, Emma Sangalang1,3,
Laura V. Doyle1,3, Steven E. Kauder1,5, Danielle Fontaine1,3, Sangeetha Bollini1, Bora Han1,6, Yang‑Xin Fu2,
Janet Sim1,3, Jaume Pons1 and Hong I. Wan1,3

Abstract
Background: Signal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts innate immu‑
nity through engagement of its cell surface ligand CD47. Blockade of the CD47–SIRPα interaction synergizes withtumor-specific antibodies and T-cell checkpoint inhibitors by promoting myeloid-mediated antitumor functions
leading to the induction of adaptive immunity. Inhibition of the CD47–SIRPα interaction has focused predominantly
on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47
therapeutics. Targeting SIRPα, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, andefficacy profile; however, SIRPα polymorphisms and lack of pan-allelic and species cross-reactive agents have limited
the clinical translation of antibodies against SIRPα. Here, we report the development of humanized AB21 (hAB21), a
pan-allelic anti-SIRPα antibody that binds human, cynomolgus monkey, and mouse SIRPα alleles with high affinity
and blocks the interaction with CD47.
Methods: Human macrophages derived from donors with various SIRPα v1 and v2 allelic status were used toassess the ability of hAB21 to enhance phagocytosis. HAB21_IgG subclasses were evaluated for targeted depletionof peripheral blood mononuclear cells, phagocytosis and in vivo efficacy in xenograft models. Combination therapy
with anti-PD1/anti-PD-L1 in several syngeneic models was performed. Immunophenotyping of tissues from MC38
tumor-bearing mice treated with AB21 and anti-PD-1 was evaluated. PK, PD and tolerability of hAB21 were evaluatedin cynomolgus monkeys.
Results: SIRPα blockade with hAB21 promoted macrophage-mediated antibody-dependent phagocytosis of tumor
cells in vitro and improved responses to rituximab in the Raji human tumor xenograft mouse model. Combined with
PD-1/PD-L1 blockade, AB21 improved response rates by facilitating monocyte activation, dendritic cell activation, andT cell effector functions resulting in long term, durable antitumor immunity. In cynomolgus monkeys, hAB21 has a
half-life of 5.3 days at 10 mg/kg and complete target occupancy with no hematological toxicity or adverse findings at
doses up to 30 mg/kg.
Conclusions: The in vitro and in vivo antitumor activity of hAB21 broadly recapitulates that of CD47 targeted
therapies despite differences in ligand expression, binding partners, and function, validating the CD47–SIRPα axis as a*Correspondence: tracy@tallactx.com
1
ALX Oncology, Burlingame, CA, USA
Full list of author information is available at the end of the article© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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mmons​.org/publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Kuo et al. J Hematol Oncol(2020) 13:160

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fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of
human malignancies.
Keywords: SIRPα, CD47, Macrophage, Phagocytosis, Immunotherapy, Innate immune checkpoint, Adaptive
immunity

BackgroundThe clinical development of immune checkpoint inhibitors (ICIs) has dramatically changed the landscape ofcancer treatment [1–3]. ICIs targeting T-cell inhibitory
receptors can induce complete and durable tumor immunity in patients with metastatic and treatment-refractory
cancers. Despite the clinical promise of ICIs, only a fraction of patients within the ICI responsive cancer subtypes benefit from treatment with antibodies against
PD-1, PD-L1, and CTLA4. Cancer is highly heterogeneous and complex and exploits a variety of mechanisms
to evade immune surveillance beyond suppression of
antitumor T cell responses [4]. Tumor-associated macrophages constitute a large fraction of the immune cell
infiltrates within the tumor microenvironment of many
human cancers [5]. Dendritic cells (DCs), although low infrequency within tumors, are potent and crucial mediators of antitumor immunity [6]. Given their prevalence
and immunomodulatory activities, targeting regulatorsof macrophage and DC function is an attractive strategy
to augment antitumor immunity and achieve additive orsynergistic efficacy in combination with antitumor antibodies or ICIs.
Signal regulatory protein α (SIRPα) is an immunoinhibitory receptor expressed primarily by cells of the
myeloid lineage including monocytes, macrophages,
DCs, and neutrophils [7]. Upon interaction with its
principal ligand, CD47, SIRPα transmits inhibitory signals that regulate DC homeostasis, self-recognition, andmacrophage-mediated programmed cell removal [7, 8].
During malignant transformation, many human tumors
exploit upregulation of CD47 thus activation of SIRPαsignaling to avoid phagocytic clearance, resulting in the
suppression of myeloid-mediated innate immunity andpoor induction of antigen-specific immunity. SIRPα
negatively regulates DC maturation, antigen presentation [9], and proinflammatory cytokine secretion [10].
In addition, it counteracts activating signals mediated by
antibody engagement of Fcγ receptors (FcγR), which profoundly limits antibody-dependent cellular phagocytosis(ADCP) against tumors, restricts neutrophil transmigration [11], and maintains myeloid-derived suppressor
cell (MDSC) functions [12]. Given the broad negative
regulatory roles of SIRPα on innate immunity, a varietyof CD47–SIRPα antagonists have been developed to promote the antitumor activity of phagocytes and myeloid

cells. Blockade of the CD47–SIRPα interaction synergizes
with both tumor-specific antibodies and ICIs by effectively reprogramming the myeloid compartment toward
a proinflammatory phenotype improving tumor cell
phagocytosis, antigen presentation, and T cell priming[9, 13]. A number of CD47 antagonists have entered theclinic with promising anticancer activity in both hematological and solid tumors [14–18].
Efforts to disrupt the CD47–SIRPα interaction havemainly focused on targeting CD47 due to its upregulation
and ubiquitous expression on most human tumor types.
However, CD47 is also broadly expressed on virtually
all normal cells, including red blood cells and platelets,
which creates a large antigen sink and CD47 blockers
comprising an active Fc have shown dose-dependent
cytopenia [19, 20]. We have previously shown that safetyliabilities associated with CD47 blockers with active
Fc domains can be overcome by eliminating Fc effector
function [13, 16, 17, 21]. Targeting SIRPα is an orthogonal approach to inhibit the CD47–SIRPα pathway.
Herein, we explore pre-clinical pharmacology, pharmacokinetics, and exploratory safety associated with
antibody-based blocking and antagonism of SIRPα. Utilizing an anti-SIRPα antibody we discovered in humanantibody transgenic chickens [22], we demonstrate that
pan-allelic anti-SIRPα antibody hAB21, which blocks
the CD47–SIRPα interaction, broadly recapitulates thefunctional properties of CD47 antagonists by promoting
the anticancer activity of both tumor-specific antibodies and ICIs in a macrophage and DC dependent manner. This leads to the induction of adaptive immunity,
resulting in complete and durable antitumor responses inmice. HAB21 exhibits favorable safety and pharmacokinetic (PK) profiles in monkeys with no hematological or
immune-related adverse events. Thus, targeting SIRPαwith hAB21 is a promising therapeutic approach to treat
human cancers by reprogramming innate immunity tosensitize cancers to immunotherapy.

Methods
Expression and purification

All antibodies were expressed in Expi293 cells (Invitrogen) using standard manufacturer’s protocol. Expression cultures were typically grown for 5 days at 37 °C in
8% ­CO2. Supernatants were harvested via centrifugation and sterile filtered. Proteins were affinity purified

Kuo et al. J Hematol Oncol(2020) 13:160

utilizing MabSelect Sure LX resin (GE Healthcare). For
SPR screening, the IgV domains of human SIRPα v1(NP_542970.1) and human SIRPα v2 (CAA71403.1) are
expressed in Expi293 cells as described above as either a
His-tagged or His-Avi-tagged fusions and purified using
Ni-Sepharose 6 Fast Flow affinity purification and polished via gel filtration through a superdex hi-prep resin(GE Healthcare). The anti-SIRPα v1 specific antibody
clone HEF-LB was generated as described in reference[23]. For crystallography, the IgV domain of SIRPα v1 andFab fragments was generated as described [14].
Humanization of antibodies

Parental clones of hAB21 (AB21 and AB25) were isolated from SynVH, a transgenic chicken, and have fully
human heavy chains but chicken light chains [14]. In
order to humanize the chicken-derived light chains,chicken hypervariable region (HVRs) of AB25 weregrafted onto human lambda light chain IGLV1, IGLV2
and IGLV3 frameworks, and chicken HVRs of hAB21
were only grafted onto human lambda light chain IGLV1
framework [24]. Combination of antibodies was generated from 4 humanized light chains with two heavy
chains (derived from AB21 and AB25 respectively) in
Expi293, purified and tested for SIRP binding. The top
humanized antibody, with favorable expression and binding properties, was designated hAB21 and selected forfurther testing.
Determination of KD

The binding affinities of AB21 and hAB21 to human
SIPRα v1 and v2 were determined using direct immobilization of the antibodies (via GLC chip). All antibodies
and proteins were used at their nominal concentrations
determined by A280 absorbance and molar extinction
coefficient. Analytes (human SIRPα v1 and v2) were
injected in a “one-shot” kinetic mode and flowed over
AB21 and hAB21 immobilized (~ 1000 RUs) on GLC
chips using 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. AB21 and hAB21 were diluted to 80 nM concentration in 10 mM sodium acetate buffer pH 4.5 andimmobilized to the chip at 30 μL/min for 50 s. Chip wasinactivated with ethanolamine for 300 s at 25 μL/min.
The analytes (human SIRPα v1 and v2 protein) were
injected in a “one-shot” kinetic mode at nominal concentrations of 10, 3.3, 1.1, 0.37, 0.12 and 0 nM. Association times were monitored for 90 s at 100 μL/min, anddissociation times were monitored for 1200 s. The surfaces were regenerated with a 2:1 v/v blend of Pierce IgG
elution buffer/4 M NaCl. Biosensor data were doublereferenced by subtracting the interspot data (containing

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no immobilized protein) from the reaction spot data
(immobilized protein), and then subtracting the responseof a buffer “blank” analyte injection from that of an analyte injection. Double-referenced data were fit globallyto a simple Langmuir model and the KD value was calculated from the ratio of the apparent kinetic rate constants(KD = kd/ka).
Crystallization of anti‑SIRPα Fab: SIRPα complexes

A pure sample of anti-SIRPα Fab-SIRPα V1 complex at aconcentration of 11.3 mg/mL in a buffer of 10 mM Tris
pH 7.4, 50 mM NaCl was set for sitting drop vapor diffusion with sparse matrix crystallization screen kits available Qiagen. The condition and crystal form that gave
quality diffraction leading to a complete dataset was 1.
0.1 M Sodium Acetate pH 4.0, 0.2 M Ammonium Sulfate, 18% (w/v) PEG 4000 (Cryo-protectant: 5% v/v Ethylene Glycol). Even though the crystallization condition
contained a high percentage of PEG 4000, ethylene glycol was added as cryo-protectant to protect the crystal
form from deteriorating and/or forming ice during freezing. These samples were screened for protein X-ray diffraction at the NSLS-II 17-ID AMX beamline; yielding
a dataset with diffraction of 2.27 Angstroms resolution.
Further crystallography data collected are detailed in
Additional file 1: Table S1. Structure PDB ID: 7KPG.
Cell binding and blockade of CD47 binding to SIRPα cells

For detection of cell binding, AB21 was fluorescently
labeled with the Alexa Fluor 647 Protein Labeling Kit(Thermo Fisher Scientific) according to the manufacturer’s instructions. 250,000 cells per well in staining buffer(PBS, 0.5% BSA or 2% FBS) were plated in 96-well plates(Falcon). Cells were first stained with fixable Live/Dead
Stain (Invitrogen) and washed once in staining bufferprior to all binding assays.
To detect SIRPα binding to cells, 500 nM Alexa Fluor
647-labeled AB21 was titrated 1:4 for seven dilutions
and added to cells in 100 μL volume of FACS buffer(PBS + 0.5% BSA) supplemented with a cocktail of
human Fc block (Miltenyi Biotec) or mouse Fc block(Biolegend), anti-CD14 (Biolegend) for human and cynomolgus PBMCs or anti-CD11b (Biolegend) for mousesplenocytes. After a 60-min incubation on ice, cells
were washed twice in staining buffer and fixed in 0.5%
paraformaldehyde.
To block CD47 binding to SIRPα on PBMCs, Alexa
Fluor 647 labeled CD47Fc at a concentration of 500 nM
and 1:4 titration starting at 1 µM of AB21 were added tocells. After a 60-min incubation on ice, cells were washedtwice in staining buffer and fixed in 0.5% formaldehyde.

Kuo et al. J Hematol Oncol(2020) 13:160

Cells were analyzed on a FACS Canto II (BD Biosciences), with subsequent data analysis using Flowjo
10.7.
Cell lines

4T1, CT26, Raji, MDA-MB-231 and DLD-1 cells wereobtained from the American Type Culture Collection(ATCC). MC38 cells were obtained from MuriGenics. All
cell lines were cultured according to standard protocols.
Animals

All mouse experiments except for MDA-MB-231 andbatf3 KO mice were conducted according to protocols
approved by Institutional Animal Care and Use Committee (IACUC) of ALX Oncology. Tumor model with
MDA-MB-231 and batf3 KO mice were conductedin compliance with UTSW Human investigation and
UTSW Institutional Animal Care and Use Committee
protocols. Female Batf3−/− mice in the C57BL6/J background and NSG-SMG3 mice were purchased fromThe Jackson Laboratory. Female BALB/c, C57BL/6 and
NOD-SCID animals, age 6–8 weeks old, were purchasedfrom Charles River Laboratories International (Hollister,CA). All animals were housed according to institutional
IACUC guidelines.
Female naïve cynomolgus monkeys (Macaca fascicularis) were provided by Charles River Laboratories. The inlife portion of the study was conducted by CRL’s testing
facility (Reno NV), and the animals were released to the
CRL testing facility’s colony at the end of the study.
Generation of monocyte‑derived macrophages
and phagocytosis assay

Human ­CD14+ cells were purified from Trima residuals(Vitalant) with Ficoll-Paque Plus and negative selection(Monocyte Isolation Kit II, Miltenyi Biotec) according to the manufacturers’ protocols. Monocyte-derived
macrophages (MDM) were made by seeding 10 million­CD14+ cells into 150 mm tissue culture dishes (Corning) in growth medium supplemented with 10% human
AB serum (Corning) or 10% FBS and 50 ng/mL MCSF.
Cells were cultured for 7–11 days. Adherent cells weredetached from culture plates with TrypLE Select (Thermo
Fisher Scientific). Target cells (DLD-1) were labeled with
the Celltrace CFSE Cell Proliferation kit (Thermo Fisher
Scientific) according to the manufacturer’s instructions.
100,000 target cells and 50,000 MDMs were incubatedin ultra-low attachment U-bottom 96-well plates (Corning) with anti-SIRPα antibodies and the corresponding
tumor-specific antibody for 2 h at 37 °C. Cetuximab wasadded at a concentration of 0.01–0.1 µg/mL.
For flow cytometry, cells were incubated in humanFcR blocking reagent (Miltenyi Biotec) and stained with

Page 4 of 19

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. 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) wasused to identify live cells. Cells were acquired on a FACS
Canto II flow cytometer (BD Biosciences) with subsequent analysis using FlowJo software. Percent phagocytosis indicates the percentage of viable CD33+ CD206+
macrophages that stain negative for CD326 and positive
for CFSE. Where applicable, 4 parameter fit curves weregenerated with Prism 7 software (GraphPad).
In vitro PBMC culture

Peripheral blood mononuclear cells (PBMC) were isolated from Trima residuals of healthy individuals with
Ficoll-Paque Plus. 500,000 PBMCs were incubated in
U-bottom 96-well plates (Falcon) with anti-SIRP at a concentration of 10 ug/mL for 48 h at 37C.
For quantification of PBMC subsets by flow cytometry,cells were incubated in human FcR blocking reagent andstained with a cocktail of fluorochrome-labeled antibodies against lin—(CD3, CD14, CD16, CD19, CD56) and
HLADR. Fixable viability dye was used to identify live
cells. After staining, cells were washed and fixed with
0.5% paraformaldehyde in PBS. Prior to acquisition,
absolute counting beads (Thermo Fisher) were added andsamples were acquired with Canto II flow cytometer andanalyzed using FlowJo software.
Tumor studies

Isoflurane anesthesia was used on mice to eliminate orminimize pain and distress during tumor implantation.
2 × 106 MC38, 2 × 106 CT26, 5 × 105 B16F10 and 5 × 105
4T1 cells were resuspended in 100ul of PBS or RPMI
and implanted subcutaneously into the flank of female
C57BL/6 mice for MC38 and B16F10 and BALB/c for
CT26 and 4T1. 5 × 106 Raji cells were resuspended in 100
ul 1:1 PBS:Matrigel (Corning) and implanted subcutaneously in the flank of NOD-SCID mice.
When tumors reached an average of 50-180mm3, as
calculated with the formula volume = (width2 × length)/2,
mice were randomized into treatment groups. All treatments were dosed intraperitoneally (i.p.) or intratumorally. For xenograft models in NOD-SCID mice, rituximab
was dosed at 3 mg/kg and AB21 at 10 mg/kg, five times
every 3 days. In syngeneic models, mice were treatedwith 10 mg/kg for CT26, MC38, B16F10 or 30 mg/kg
for 4T1 of clone AB21, 10 mg/kg anti-PD-1 (BioXcell,clone RMP1-14) and 2 mg/kg (MC38 models) anti-PDL1 (murine IgG1, ALX Oncology). For intratumoral

Kuo et al. J Hematol Oncol(2020) 13:160

injections, 50 µg of AB21 was given four times every
3 days.
For re-challenge experiments, treated mice with complete tumor eradications were re-challenged with either
2 × 106 MC38 cells on one flank or 0.5 × 106 B16F10
tumor cells on the opposite flank. Age-matched naïve
mice were used as control.
For cellular depletion experiments, mice were dosed
intraperitoneally with 250 ug of either anti-CSF1R (BioXcell, clone AFS98), anti-CD8b (Bioxcell, clone 53-5.8) or
anti-GR1 (BioXcell, clone RB6-8C5) on days 2, 5, 10 and
15 post-tumor implantation. Confirmation of cellulardepletion was performed on tumor and spleen of sparecellular depleted mice on days 1, 3, 4 and 8 post-injection
by flow cytometry using anti-CD45 (Biolegend, clone
30-F11), anti-CD3 (Biolegend, clone 145-2C11), antiCD8 (Biolegend, clone 53.6.7), anti-CD11b (ebioscience,clone M1/70), anti-Ly6C (Biolegend, clone 145-2C11),
MHCII (ebioscience, clone M5/114.15.2) and F4/80 (Biolegend, clone BM8).
Humanized mice were generated with 4-week NSGSGM3 female mice and irradiated with 100 cGy (X-ray
irradiation with X-RAD 320 irradiator) 1 day prior to
CD34+ cells transfer. Irradiated mice were treated with
Bactrim (Aurora Pharmaceutical LLC) water for 2 weeks.
Cord blood was obtained from UT Southwestern(UTSW) Parkland Hospital. Human CD34+ cells were
purified from cord blood by density gradient centrifugation (Ficoll® Paque Plus, GE healthcare) followed by positive immunomagnetic selection with anti-human CD34
microbeads (Stem Cell). ­105 CD34+ cells were intravenously injected into recipient mice.
12 weeks after engraftment, humanized mice with over
40% human ­CD45+ cells reconstitution and age and sex
matched non-humanized mice were inoculated with
2 × 106 MDA-MB-231 tumor cells subcutaneously on
the right flank. Tumor volumes were measured by length(a), width (b) and height (c) and calculated as tumor volume = abc/2. 8 days later when tumors were around 50­mm3, mice were intratumorally treated with 50 µg AB21,
HEF-LB or PBS, four time every 3 days.
4T1 Lung metastasis quantification

BALB/c mice were implanted with 4T1 cells and lungs
were harvested 8–9 days post-last injection for metastatic
nodule quantification. In brief, lungs were harvested in
ice-cold 1xPBS, minced into small pieces then transferred
into digestion solution consisting of 2 mg/mL collagenase
type V (Worthington) supplemented with 50 ug/mL
DNAse (Sigma) and incubated for 2 h in a 37C incubatorwith end-over-end rotation. Suspension was transferred
into 70-um strainer, washed once in 1 × PBS then transferred into 10 mL selection media consisting of RMPIPage 5 of 19

1640 supplemented with 10% FBS, penicillin–streptomycin and 10 ug/mL 6-thioguanine (Sigma). Three to four
1:10 serial dilutions were plated either in 6-well platesor 10-cm dishes and cultured for 10–14 days at 37C, 5%­CO2. Metastatic plaques were then fixed in methanol
for 5 min at room temperature, re-hydrated in distilledwater then stained with 0.03% methylene blue (Sigma)
for 5 min at room temperature. Dye was then discarded,
plate was rinsed gently with distilled water and allowed
to air-dry prior to counting plaques.
Immunophenotyping

For immune response monitoring in tumor-bearing mice,anti-SIRPα was dosed either three times in combination
with anti-PD-1, or twice with anti-SIRPα and once with
anti-PD-1, all injections were administered i.p 3 days
apart at 10 mg/kg.
Spleens and tumors were harvested either 2 or 3 days
post-last injection for immunophenotyping. Spleens were
processed into single-cell suspension in ice-cold PBS,
lysed with ACK lysis buffer (Gibco), washed twice andre-suspended in PBS supplemented with 2% FBS. Tumorderived single-cell suspensions were prepared using a
cocktail of Collagenase I (Worthington), Collagenase
IV (Sigma) and DNAse (Sigma) for 45 min at 37° C. Cellcounts were performed using ViCell counter (Beckman
Coulter) for spleen and lymph node and trypan blue
exclusion with hemocytometer for tumor. Aliquots of 1–2­106 cells were either used for cell-surface antigen staining or stimulation for cytokine assessment. For surface
staining, cells were stained with LIVE/DEAD fixable dye(Thermo Fisher), followed by mouse Fc-block (Biolegend)
and subsequently stained with antibodies according tocell-type specific antibody panels for at least 30 min at
4 °C. CD4 clone GK1.5, CD8 clone 53-6.7, CD25 clone
PC61, CD3 clone 145-2C11, CD45 clone 30-F11, CD47
clone MIAP301, NKp46 clone 29A1.4, PD1 clone J43,
FoxP3 clone FJK-16s, Ki67 clone SolA15, Granzyme B
clone QA16A02, CD44 clone IM7, CD62L clone MEL-14,
TNFa clone MP6-XT22, IFNg clone XMG1.2, SIGLEC
H clone 551, CCR7 clone 4B12, CD172 clone P84, CD86
clone GL-1, MHCII clone M5/114.15.2, GR-1 clone RB68C5, 33D1 clone 33D1, CD11b clone M1/70.15, CD11cclone N418, CD103 clone 2E7, Ly6C clone NH1.4, F4/80
clone BM8, CD24 clone M1/69. All flow antibodies werepurchased from either Biolegend or Thermo Fisher.
For PMA/ionomycin ex-vivo stimulation to quantitate
IFNγ+ cells, total splenic and tumor cells were plated at
1 × 106 cells/well in complete RPMI 1640 comprised of
10% heat-inactivated FBS, 2% Pen/Strep, 1% Glutamax,
1% MEAA, 1% sodium pyruvate, 25 mM HEPES and5 μM ß-mercaptoethanol supplemented with 50 ng/ mL
PMA (Fisher Scientific) and 1 μM ionomycin (Sigma) inKuo et al. J Hematol Oncol(2020) 13:160

the presence of Golgi-Stop for at least 4 h at 37 °C, 5%­CO2, and subsequently stained with antibodies to surface
and intracellular markers. Samples were then acquired
using Attune NxT (Thermo Fisher) or Canto II (BD).
Analysis was performed using FlowJo 10.0 (BD) and tabulated using GraphPad Prism 7.3.
HAB21 and soluble SIRPα serum ELISA

Immulon 96-well ELISA plates (Thermo Fisher Scientific, 3855) were coated overnight with human wild type
SIRPα, variant 1 (ALX Oncology) in PBS. Plates were
washed with Tris-Buffered Saline Tween-20 (TBST,
25 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.5) andblocked for 1 h with assay buffer (PBS, 1% BSA, 0.05%
Tween-20, 0.25% CHAPS, 5 mM EDTA, 0.35 M NaCl).
Serum samples diluted a minimum of 1:50 in assay buffer
or hAB21 standard curve protein (two-fold serial dilutions from 160 to 1.25 ng/mL, in 1:50 normal cyno serum
diluted in assay buffer) were added to blocked plates for
1 h. Plates were washed with TBST. Standard curves andsamples were incubated for 1 h with biotinylated goatanti-human IgG (H + L) antibody (Bethyl, A80-319B),
washed with TBST, incubated for 30 min with HRPconjugated Avidin D (Vector, A2004), and washed with
TBST. All plates were incubated with 1-Step Ultra TMB
ELISA solution (Thermo Fisher Scientific, 34028) andthe reaction was stopped with 0.16 M sulfuric acid solution (Thermo Fisher Scientific, N600). Plates were readat an O.D. of 450 nm with a background reference reading at 570 nm on a SpectraMax i3 plate reader (Molecular Devices). Protein concentrations of serum samples
were interpolated from the hAB21 standard curve with a
4-parameter fit curve using Prism software (GraphPad).
For the detection of soluble SIRPα, similar assay tohAB21 serum ELISA was performed except for the following. 2 ug/mL anti-SIRPα mouse IgG1 antibody(AB136b, ALX Oncology, a non-CD47 blocking antibody
that binds both cyno and human SIRPα with high affinity) in PBS was used to coat the plates. Cyno PK serum
samples diluted 1:25 in assay buffer or SIRPα standard
curve protein (ALX135, ALX Oncology, 25 to 0.024 ng/
mL, diluted in assay buffer) were added to blocked plates
for 1 h. Pre-dose cynomolgus serum samples spiked withor without 200 ug/mL of anti-SIRPα human IgG1 antibody were run as controls since most of the PK samples
contained at least 200 ug/mL anti-SIRPα human IgG1
antibody. After incubating samples for 1 h in the ELISA,
plates were washed with TBST. 5 ug/mL anti-SIRPα
human IgG1 kick-off Fab + 6 × His tag (AB115f, ALX
Oncology, binds both cynomolgus and human SIRPαwith high affinity) was added to samples for 1 h. Plates
were washed with TBST. Samples were incubated for 1 hPage 6 of 19

with 0.15 ug/mL rabbit anti-6 × His Tag HRP conjugatedantibody (abcam cat. # ab1187).
HAB21 receptor occupancy

For the analysis of receptor occupancy, 75 μL aliquots ofwhole blood sample were washed in 1.5 mL FACS buffer(PBS, Thermo Fisher Scientific + 0.5% bovine serum
albumin, Sigma) and stained with fluorochrome-conjugated antibodies against CD14 (M5E2, Biolegend) andHLA-DR (L243, Biolegend), fixable viability dye eFluor
506 (Thermo Fisher Scientific), and FcR blocking reagent,human (Miltenyi Biotec). SIRPα occupancy was detectedusing labeled SIRPα antibodies which would competewith test article for binding to SIRPα. For detection of
SIRPα occupancy, Alexa-Fluor 647 conjugated (Thermo
Fisher Scientific) hAB21 was added to the lineage stains
and cells were incubated at 4 °C for 1 h. All stains werethen washed in 1.5 mL FACS buffer and erythrocytes
were lysed in 1.5 mL FACS lysing solution (BD Biosciences) for 10 min at room temperature. Cells were
washed in 1.5 mL FACS buffer and resuspended in FACS
buffer. Cells were analyzed by flow cytometry on a FACS
Canto II (BD Biosciences).
Data were analyzed with FlowJo 10.4 software (Becton Dickinson). To measure SIRPα occupancy on monocytes, geometric mean fluorescence intensity (MFI)
in the Alexa-Fluor 647 channel was determined for­CD14+HLADR+ cells. To calculate percent SIRPα occupancy, this value was normalized to the MFI for the
predose timepoint as follows: MFI was calculated as apercentage of MFI at the predose timepoint. This number
was subtracted from 100%, with the result being the percent occupancy. Thus, MFI greater than or equivalent to
the predose level is reported as zero percent occupancy
and MFI equivalent to background is reported as 100%
occupancy. All samples for which SIRPα staining was
equivalent to or greater than staining of predose samples
were considered to have no occupancy.
Cynomolgus monkey study

The in-life of monkey study was conducted by Charles
River Laboratories (Reno, NV). The study was performed in accordance with the standard operating procedures and Good laboratory Practices (GLP). Male
cynomolgus monkeys (n = 2/group) of Chinese origin
were administered hAB21 on Days 1 and 8 for a totalof 2 intravenous doses at doses 10 and 30 mg/kg by
slow bolus IV injection. Hematology was assessed by
Charles River Laboratories and were collected from all
animals on Days—4, 4, 8, 15, and 22. For PK bioanalysis, serum was collected from animals at the following
time points: 0 (predose), 1, 3, 8, 24, 72, and 168 h postdose on Day 1. On Day 8, serum was collected from

Kuo et al. J Hematol Oncol(2020) 13:160

animals at the following time points: 1, 3, 8, 24, 72,
168 h, Day 18 and D22 postdose. For receptor occupancy assay, blood samples were drawn and collected
for all animals at the following time points: predose(Day-4), 4, 24, and 72 h after the Day 1 dose; predose,4, 24, 72, and 168 h after the Day 8 dose; Day 18 andDay 22.

Page 7 of 19

Results
We previously described a family of anti-SIRPα antibodies that antagonize SIRPα through competition for
the CD47-binding site located on the IgV domain of
SIRPα [22]. The anti-SIRPα blocking antibody clone
AB21 was discovered from immunized SynVH chickens,which contain human ­VH immunoglobulin (Ig) repertoires paired with the natural chicken light chain repertoires [25]. The chimeric antibody AB21 binds to human
SIRPα v1 and v2 alleles with picomolar affinity (Fig. 1a),Fig. 1 Pan-allelic and species cross-reactive antibody, hAB21, binds SIRPα with high affinity and blocks CD47–SIRPα interaction. a Binding affinities(pM) of AB21 and humanized hAB21 to human SIRPα v1 and v2 were determined by SPR. b Binding curves of hCD47 on C
­ D14+ monocytes in thepresence of titrated AB21 and hAB21. PBMCs were incubated with 500 nM fluorescent hCD47-Fc and increasing concentration of AB21 and hAB21
at 4 °C for 60 min, washed and evaluated by flow cytometry. Data are shown as ratio of C
­ D14+ incubated with hCD47-Fc only. c Flow cytometry
analysis of AB21 and hAB21 binding to human, cynomolgus and mouse ­CD14+ monocytes. Fluorescent-labeled AB21 and hAB21 were incubatedin increasing concentration with cells at 4 °C for 60 min, washed and evaluated by flow cytometry. Mean fluorescent intensity is measured. d
Crystal structure of hAB21 anti-SIRPα Fab bound to the Ig-V domain of human SIRPα V1 (purple). CD47 domain (pink) was superimposed onto the
crystallized complex. The adjacent Venn diagram and surface map depict the epitopes bound by CD47 (green) and hAB21 (red); shared epitopes
bound by both CD47 and hAB21 to SIRPα V1 are colored yellow accordinglyKuo et al. J Hematol Oncol(2020) 13:160

potently blocks the interaction between CD47 and SIRPα(Fig. 1b), and cross-reacts with cynomolgus and murine
SIRPα (Fig. 1c). Since clone AB21 contained the chicken
light chain sequences, we carried out humanization as
described in the Materials and Methods to derive thehumanized version, named hAB21. Humanized AB21
(hAB21) retains the SIRPα binding and antagonistic
properties of the parental AB21 (Fig. 1a–c). The structureof a Fab version of the antibody in complex with the IgV
domain of human SIRPα v1 was solved by X-ray crystallography (Fig. 1d). The hAB21 Fab binds to SIRPα at an
epitope highly overlapping with that of CD47, with the
VL domain overlapping with the majority of the CD47
binding site while the VH domain CDR-H3 inserts into
the central pocket of SIRPα that is crucial for CD47 binding, in agreement with the ability of hAB21 to completely
block and antagonize the CD47–SIRPα interaction(Fig. 1d). AB21 and hAB21 were used interchangeably for
subsequent experiments.
Fc‑domain IgG subclass influences the in vitro and in vivo
activity of hAB21

Antibody IgG subclass dictates FcγR interactions thateither promote (wild-type IgG1 and IgG4) or limit(IgG2Δa and I­ gG1FcDead) antibody effector functions [26–29]. Competition for FcγR binding between anti-SIRPα
antibodies and anti-tumor antibodies may impact macrophage-mediated a ADCP. Therefore, we investigated
the impact of IgG subclass on anti-SIRPα antibody-mediated enhancement of macrophage phagocytosis in vitro.
HAB21 was expressed as either a human IgG1, IgG4,
IgG2Δa, or ­IgG1FcDead subclass and macrophage phagocytosis of DLD-1 tumor cells opsonized with the antiEGFR antibody cetuximab alone or in combination with
hAB21 was quantified by flow cytometry. Cetuximab
alone promoted macrophage phagocytosis of DLD-1
tumors cells, and this was further enhanced in a dosedependent manner by combination with hAB21, but only
when the Fc-domain of hAB21 was engineered to minimize binding to FcγR (Fig. 2a). HAB21 with an inactive­IgG1FcDead or attenuated IgG2Δa Fc-domain enhanced
cetuximab-mediated macrophage phagocytosis of DLD-1
tumor cells, whereas hAB21 with an active IgG1 or IgG4
Fc-domain demonstrated little to no impact on cetuximab-mediated phagocytosis (Fig. 2a). These results suggest that competition between hAB21 and cetuximab
for macrophage FcγR limits the phagocytosis enhancing
effects of anti-SIRPα antibodies or that hAB21 with anactive IgG simultaneously co-engages SIRPα and FcγR on
macrophages, resulting in a heterotrimeric interaction.
This has been described as the “scorpion effect” [30] andthis heterotrimer interaction would limit FcγR clustering by cetuximab, thereby decreasing pro-phagocytosis

Page 8 of 19

signals and reducing the enhancing effects of phagocytosis with hAB21 and cetuximab.
To further investigate the impact of IgG subclass on
hAB21 activity, we cultured primary human peripheral
blood mononuclear cells (PBMCs) with hAB21 containing different IgG subclasses for 48 h and quantified thenumbers of T, B, monocytes, NK and DCs by flow cytometry. There was no significant change in the number ofT, B, monocytes or NK cells (Additional file 1: Fig. S1A);
however, PBMCs cultured with hAB21 containing an
IgG1, IgG4, IgG2, or IgG2Δa Fc-domain had lower numbers of lineage negative ­(CD3−, ­CD19−, ­CD56−) HLADR+ DCs, whereas hAB21-IgG1FcDead had limited impact
on the numbers of respective DC lineage (Fig. 2b).
Interestingly, although monocytes express higher levels of SIRPα than DCs (Additional file 1: Fig. S1B), we
observed no changes in the number of monocytes present in PBMCs cultured with hAB21 irrespective of its
IgG subclass.
Finally, we investigated the impact of Fc-domain IgG
subclass on the in vivo antitumor activity of AB21 in
NOD SCID mice bearing subcutaneous human ­CD20+
Raji tumor cells alone or in combination with rituximab.
Treatment with rituximab alone substantially delayed
tumor growth compared to the PBS control, whereas
monotherapy with AB21 containing either an inactive (mouse IgG1 N297A) or active (mouse IgG2a) Fcdomain had minimal impact on tumor growth (Fig. 2c,d and Additional file 1: Fig. S2). The combination of
AB21 containing an inactive Fc-domain with rituximab
significantly improved anti-tumor activity compared torituximab alone (Fig. 2c and Additional file 1: Fig. S2),
consistent with the known impact of CD47–SIRPα antagonism on potentiating responses to antitumor antibody
therapy [13, 31]. However, combination therapy using
AB21 with an active Fc-domain decreased antitumor
activity when compared to rituximab alone (Fig. 2d). Collectively, these data indicate that anti-SIRPα antibodies
capable of triggering Fc effector functions limit the therapeutic activity both in vitro and in vivo through multiple
mechanisms involving macrophage-mediated ADCP and
DC depletion. We subsequently chose human I­ gG1FcDeadas the Fc-domain for hAB21 and mouse IgG1_N297A as
the Fc-domain for AB21 for further experimentation.
HAB21 potentiates macrophage phagocytosis irrespectiveof SIRPα genotype

Two allelic variants of SIRPα, v1 and v2, are present in thehuman population at various frequencies and zygosities
dependent on ethnicity [22]. Antagonism of both SIRPαalleles is essential to ensure all population can be targeted to maximize the phagocytosis promoting activities
of anti-SIRPα blocking antibodies [22, 32]. To investigate

Kuo et al. J Hematol Oncol(2020) 13:160

Page 9 of 19

a

b

α

±

d

±

c

Fig. 2 IgG subclass influences the efficacy of anti-SIRPα antagonist antibodies in vitro and in vivo. a In vitro phagocytosis experiment with human
MDMs cultured in the presence of human AB serum and DLD-1 cells in the presence of cetuximab at a constant concentration and titrated
anti-SIRPα antibodies with different IgG subclass. Percent of macrophages that engulfed tumor cells is indicated on the y-axis. On the y-axis, closed
circle indicates background phagocytosis observed with media only and closed square indicates cells treated with cetuximab only. b Relative
dendritic cell numbers in human PBMCs after 48-h incubation with hAB21-IgG variants. Each bar indicates relative DC number as compared tomedia only. Statistics were performed using One-Way ANOVA, Dunnett’s multiple comparisons. c, d Raji B-cell lymphoma cells were implanted
subcutaneously on the right flanks of NOD-SCID mice. Mice with established tumors (average of 154 ­mm3) were randomized, n = 10/group, andtreated intraperitoneally with vehicle, rituximab, AB21 mIgG1_N297A (inactive) (c), AB21 mIgG2a (active) (d), or AB21 inactive or active + rituximab.
Mice were treated five doses every 3 days. Statistics were performed using Two-Way ANOVA, Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01,***p < 0.001 and ****p < 0.0001

the influence of macrophage SIRPα genotype on theactivity of hAB21, we derived human macrophages fromprimary human monocytes cultured in M-CSF (MDM),
determined SIRPα v1 and v2 allelic status, and performedin vitro phagocytosis assays using v1/v1 homozygous, v1/
v2 heterozygous, or v2/v2 homozygous MDMs as effectors. HAB21 potently enhanced macrophage phagocytosis of DLD-1 tumor cells in a dose-dependent manner
irrespective of SIRPα genotype with an ­EC50 of ~ 0.2 nM,

consistent with the high affinity binding of hAB21 toboth SIRPα v1 and v2 variants (Fig. 3a). In contrast, theanti-SIRPα v1 specific antibody clone HEF-LB only promoted macrophage phagocytosis of DLD-1 cells by v1/
v1 homozygous macrophages, with limited or no activity with v1/v2 heterozygous or v2/v2 homozygous macrophages, respectively (Fig. 3a). Similarly, in humanized
mice engrafted with human ­CD34+ cells from donors
with v1/v2 SIRPA genotype bearing MDA-MB-231 triple

Kuo et al. J Hematol Oncol(2020) 13:160

Page 10 of 19abFig. 3 hAB21 potentiates macrophage-mediated ADCP and delays tumor growth in SIRPα v1/v2 humanized mouse model. a In vitro phagocytosis
experiment with human M-CSF derived MDMs and DLD-1 cells in the presence of titrated hAB21, HEF-LB or isotype control (Fc inactive) antibodies.
Percent of macrophages that engulfed CFSE-labelled tumor cells is indicated on the y-axis. On the y-axis, closed circle indicates backgroundphagocytosis observed with media only. b MDA-MB-321 TNBC cells were implanted subcutaneously on the right flanks of humanized miceengrafted with human CD34+ cells from SIRPA v1/v2 donor. On day eight, mice with established tumors were randomized, n = 5 mice/group, andtreated intratumorally with PBS, clone HEF-LB, and AB21 mIgG1_N297A. Mice were treated four times every 3 days. ****p < 0.0001, statistics were
performed using Two-Way ANOVA, Tukey’s multiple comparisons test

negative breast cancer (TNBC) tumors, AB21 significantly decreased antitumor activity while clone HEF-LB
had no impact on tumor growth (Fig. 3b).
HAB21 monotherapy modestly delays tumor growthand is dependent on DC‑mediated anti‑tumor CD8+ T cell
response

We determined the anti-tumor activity of hAB21 monotherapy in C57BL/6 mice bearing syngeneic MC38
tumors. Treatment with hAB21 alone produced a modest but statistically significant delay in tumor growthcompared to the PBS control group (Fig. 4). Depletionof macrophages and myeloid cells (anti-CSF1R) had little to no impact on the anti-tumor activity of hAB21,
whereas depletion of neutrophils (anti-GR1) and ­CD8+(anti-CD8) T cells abrogated anti-tumor activity (Fig. 4)
indicating a requirement for both innate and adaptive
immunity. In b
­ atf3−/− mice that lack CD8α+ DCs [3]treatment with hAB21 had no effect on MC38 tumor
growth, indicating a requirement for CD8α+ DCs. Collectively, these results indicate that both innate andadaptive immune responses are crucial to the antitumor
activity of hAB21 monotherapy, consistent with previous
studies identifying both neutrophils and DCs as important immune effector cells responsible for the efficacy ofanti-CD47 therapy in syngeneic tumor models [9, 13, 33].

Single agent activity of hAB21 in syngeneic models was
restricted to MC38 tumors as we did not observe hAB21
activity in the more aggressive CT26, 4T1, or B16F10
tumors (Figs. 4, 5e, Additional file 1: Fig. S3).
SIRPα antagonism improves the anti‑tumor activityof T‑cell checkpoint blockade in vivo

Since hAB21 monotherapy produced a modest antitumor response requiring both innate and adaptive immunity, we determined if hAB21 activity could be further
enhanced by combination with T cell checkpoint antagonists targeting the PD-1/PD-L1 interaction. Indeed,
combination therapy with AB21 and anti-PD-L1 significantly delayed MC38 tumor growth compared to AB21
or anti-PD-L1 alone (Fig. 5a). Combination of AB21 with
anti-PD-1 also resulted in a significant delay in tumor
growth, which translated to a significant extension of survival and complete regression of MC38 tumors in 60%(6/10) of mice, whereas there were no complete curesin single agent AB21 or anti-PD-1 treatment groups(Fig. 5b). All surviving AB21 and anti-PD-1 combination therapy treated mice were protected against subsequent re-challenge with MC38 tumor cells (Fig. 5c),whereas the same surviving mice challenged contralaterally with the unrelated B16F10 tumor were not protected(Fig. 5d). These results demonstrate the development ofKuo et al. J Hematol Oncol(2020) 13:160

aPage 11 of 19bFig. 4 CD8+ T-cells, ­CD8+ DCs and granulocytes are necessary for in vivo efficacy of hAB21 monotherapy. a MC38 colon carcinoma cells were
implanted subcutaneously in C57BL/6 mice. Two days post-implant, mice were treated with anti-CSF1R, anti-GR1 or anti-CD8 depleting antibodies.
Mice with established tumors were randomized, n = 5 mice/group, and treated intraperitoneally with PBS or hAB21. Mice were treated three times
every 3 days. Depleting antibodies were dosed on days 2, 5, 10 and 15 post-implantation. Unpaired two-tailed t-test between PBS and hAB21 were
determined for days indicated. *p < 0.05, **p < 0.01, ***p < 0.001. b MC38 cells were implanted subcutaneously in wild type and Baft3-deficient
C57BL/6 mice. Eight days post mice were randomized, n = 5 mice/group, and dosed intratumorally with vehicle or AB21. Mice were treated fourtimes, every 3 days

long-term, tumor-specific immune memory as a result ofcombination therapy. The growth of contralateral B16F10
tumors in mice surviving AB21 and anti-PD-1 combination therapy was delayed compared to age-matched naïve
mice challenged with B16F10 (Fig. 5d), possibly due tothe induction of a memory immune response against theMC38 tumor that may also respond to B16F10 tumor.
We further evaluated the efficacy of AB21 in combination with anti-PD-1 in BALB/c mice bearing subcutaneous CT26 tumors, a modestly immunogenic mouse colon
carcinoma with variable responses to anti-PD-1 treatment [34]. Treatment with AB21 alone had no impact
on tumor growth while treatment with anti-PD-1 alone
trended toward a delayed tumor growth compared to
the PBS control but did not reach statistical significance(Fig. 5e). Combination of anti-PD-1 with AB21 significantly delayed CT26 tumor growth compared to the PBS
control and trended towards improved tumor control
compared to the anti-PD-1 group (Fig. 5e).
The mouse 4T1 mammary tumor is poorly immunogenic, highly metastatic, and resistant to T cell
checkpoint blockade [35]. BALB/c mice bearing subcutaneous 4T1 tumors were treated with AB21 andanti-PD-1 alone or in combination and primary tumor
growth and metastatic burden in the lungs were quantified. Neither AB21 or anti-PD-1 given as a single agent

or in combination had an effect on primary tumor
growth (Additional file 1: Fig. S3A); however, AB21
monotherapy significantly reduced metastatic burdenin the lung and a further reduction in metastatic burden in the AB21 and anti-PD-1 combination group, but
did not reach statistical significance (Fig. 5f ).
Finally, we evaluated the efficacy of AB21 in combination with an agonist antibody against the immune
stimulatory molecule 4-1BB in C57BL/6 mice bearing
subcutaneous B16F10 tumors, a poorly immunogenic,
highly aggressive mouse melanoma that is resistantto anti-PD-1 therapy but partially responsive to anti4-1BB [36]. Treatment with AB21 alone had no effecton B16F10 tumor growth, whereas anti-4-1BB significant delayed tumor growth compared to the PBS
control (Additional file 1: Fig. S3B). Combination ofanti-4-1BB with AB21 did not improve antitumor
responses against B16F10 (Additional file 1: Fig. S3B).
Collectively, the in vivo efficacy studies indicate that
AB21 combines with ICIs targeting PD-1 or PD-L1 to
improve tumor control that leads to durable antitumor
immunity against MC-38 and CT26 tumors. In moreaggressive and less immunogenic tumor types such as
B16F10 and 4T1, responses to the combination of AB21
with anti-PD-1 or anti-4-1BB are less pronounced compared to responses in MC38 and CT26 tumor models.

Kuo et al. J Hematol Oncol(2020) 13:160

Page 12 of 19

a

b

c

d

f

e

Fig. 5 AB21 potentiates the antitumor activity of anti-PD-L1 and anti-PD1. MC38 colon carcinoma cells were implanted subcutaneously in C57BL/6
mice. Mice with established tumors were randomized and treated intraperitoneally. a Mice were treated with PBS, AB21, anti-PD-L1 or AB21 andanti-PD-L1, two times every week for 3 weeks. Graph shows tumor curve with n = 10 mice/group. Unpaired two-tail student’s t-test on day 17.
b Mice were treated with PBS, AB21, anti-PD-1 or AB21 and anti-PD-1. Graphs show tumor growth of mice treated two times, every 3 days (left,
unpaired two-tail t-test on day 25) and survival curves of mice treated three times every 3 days (Log-rank, Mantel–Cox test) of n = 10 mice. c, dMC38 tumor-bearing mice treated with AB21 + anti-PD-1 and achieved complete eradication were rechallenged with MC38 (c) and B16F10 (d)60 days post-eradication. Concurrently, MC38 and B16F10 cells were implanted subcutaneously into age-matched naïve C57BL/6 mice. Graphs
show tumor growth of MC38 and B16F10 tumors. e Mice bearing CT26 tumors were treated with PBS, AB21, anti-PD-1 or AB21 + ant-PD-1, fourtimes, every 3 days. Graphs show tumor growth of n = 10 mice. Unpaired two-tail t-test on day 21 for growth curve and log-rank (Mantel–Cox) test
for survival curves. f Total numbers of lung metastatic colonies in 4T1 tumor-bearing mice treated with PBS, AB21, anti-PD-1 or AB21 + anti-PD-1.
The results are expressed as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates not significant

AB21 induces DC activation and promotes T cell effector
functions in combination with anti‑PD‑1

We conducted immunophenotyping using flow cytometry to directly access the cellular immune responses in
MC38 tumor bearing mice treated with AB21 alone orin combination with anti-PD-1. MC38 tumor bearing

mice were treated on days seven and ten with AB21 andone dose of anti-PD1 on day seven. Analysis of cellularresponses in the spleen and tumor was performed 4 days
post the final dose. Treatment with AB21 alone had little to no impact on the frequency of the various T cellsubsets, NK cells, monocytes, ­CD8+DCs, or monocytic

Kuo et al. J Hematol Oncol(2020) 13:160

Page 13 of 19

DCs (mDCs) in the spleen (Fig. 6, Additional file 1: Fig.
S4A), but resulted in a significant reduction of ­CD8−
DCs concomitant with an upregulation of the activation
markers MHC class II and CD86 (Fig. 6a). The combination of AB21 and anti-PD-1 also activated and reduced
the frequency of C
­ D8− DCs in the spleen, whereas treatment with anti-PD-1 alone had no effect (Fig. 6a) indicating that DC activation is a result of SIRPα blockadewith AB21. ­CD8+ DCs in the spleens of mice treatedwith AB21 alone or in combination with anti-PD-1 also
displayed an activated phenotype and combination therapy resulted in a small but significant increase in the

a

b

d

g

frequency of C
­ D8+ DCs in the spleen (Fig. 6b). Furthermore, monocytes and mDCs were activated upon treatment with AB21 either alone or in combination with
anti-PD-1 (Fig. 6d, e). Activation by AB21 was associatedwith a decrease in monocyte frequency and no change in
mDC frequency in the spleen (Fig. 6d, e).
Within the tumor microenvironment, AB21 monotherapy and combination therapy with anti-PD-1 causeda slight reduction in ­CD11c+MHC-II+ DCs; however,DC production of IL-12, a marker of activated DCs
and a potent proinflammatory cytokine necessary fortumor control in response to ICIs [37], was significantly

c

e

h

f

i

j

Fig. 6 Characterization of the lymphoid and myeloid compartments of tumors and spleen. MC38 colon carcinoma cells were implanted
subcutaneously in C57BL/6 mice. Mice with established tumors were randomized and treated intraperitoneally with PBS, AB21, anti-PD-1 orcombo (AB21 + anti-PD-1). a, b Quantifications of splenic C­ D8− and ­CD8+ ­CD11chiMHCII+ DCs as a proportion of live cells and expression of
costimulatory molecules in the spleen. c–e Quantification of granuloctyes (­ Gr1midCD11b+), monocytes ­(Gr1hiCD11b+) and monocytic DCs­(GR1hiCD11c+CD11b+MHC-II+) as a proportion of live CD45+ cells and expression of costimulatory molecules in the spleen. f Quantification of DCs­(CD11c+MHC-II+) as a proportion of live ­CD45+ cells and IL-12+ DCs in the tumor. g Quantification of iNOS+ expressing cells as a proportion of
total TAMs ­(CD11b+Gr1−MHC-II+) in the tumor. h Quantification of granulocytic monocyte derived suppressor cells, G-MDSCs, (­ CD11b+Gr1mid) as aproportion of live ­CD45+ cells in the tumor. i Quantification of ­IFNg+ cells as a proportion of CD4 and CD8 and Granzyme B as a proportion of CD8
in the tumor. j Quantification of ­Foxp3+ cells as a proportion of CD4 and ­Ki67+ cells as of F­ oxp3+CD4+ ­Tregs in the tumor. Plotted as mean ± SD andanalyzed by Ordinary one-way ANOVA, Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns is not significant

Kuo et al. J Hematol Oncol(2020) 13:160

Page 14 of 19

increased in response to combination therapy but not
AB21 or anti-PD-1 monotherapy (Fig. 6f ). Tumor-associated macrophages (TAMs) account for the majorityof leukocytes within MC38 tumors and treatment with
AB21, anti-PD-1, or the combination had no effect on
TAM frequency (Additional file 1: Fig. S4B); however,iNOS production, a marker of M1, was significantly
elevated in both the anti-PD-1 monotherapy and combination therapy groups (Fig. 6g). G-MDSC infiltration
increased upon treatment with the combination of AB21
and anti-PD-1 (Fig. 6h). Additionally, surface expressionof SIRPα on splenic and tumor myeloid cells was downregulated in response to AB21 treatment (both alone andin combination with anti-PD-1), but not in the anti-PD1
treatment group (Additional file 1: Fig. S4C). Downregulation of SIRPα by AB21 provides an additional mechanism of disrupting CD47–SIRPα signaling which likely
contributes to the antitumor activity of AB21.
Potentiation of innate immune cell functions by AB21
facilitated further improvements in T effector cell
responses to anti-PD-1 therapy with no changes to cell
frequency (Additional file 1: Fig. S4D). Combination
therapy promoted IFNγ production by ­CD4+ T cells in
the tumor and C­ D8+ T cells in the spleen and granzyme
B production by C
­ D8+ T cells in the tumor (Fig. 6i, Additional file 1: S4A). In contrast monotherapy had limited
impact on T cell effector functions. Furthermore, combination therapy significantly reduced the number of ­CD4+
regulatory T cells (­ Tregs) in the tumor and decreased their
proliferative capacity (Fig. 6j). The cellular responses
to AB21 treatment are broadly in agreement with thecell depletion studies indicating both DCs and ­CD8+ T
cells are indispensable for tumor control (Fig. 5). Collectively, these data indicate that SIRPα blockade with AB21
results in the repolarization of the myeloid-compartment, as evident by the increased activation phenotype
and IL-12 production of the DCs as well as increased
iNOS producing M1-like TAMs, resulting in an antitumorigenic phenotype.

mL and 48,635 ng h/mL, a clearance of 0.35 mL/hr/kg
and 0.29 mL/h/kg, and a half-life of 5.3 days and 8.1 days,
respectively (Fig. 7a). Complete target occupancy wasobserved in circulating monocytes within 3 h of dosing at both dose levels that persisted for 7 days post thefirst dose (Fig. 7b, c) and 14 days post the second doseat the 30 mg/kg dose (Fig. 7c). Accelerated clearance andthe concomitant loss of target occupancy were observedin monkeys that received the 10 mg/kg dose (Fig. 7b) attime points on Day 14 and beyond, suggestive of an antidrug antibody (ADA) response to human proteins.
Proteolysis and release of soluble SIRPα from cell-surface expression has been demonstrated in cultured cells[38]. To investigate the presence of soluble SIRPα andpotential impact of soluble SIRPα on PK of anti-SIRPα
antibodies in the monkey studies, we used non-crossblocking antibodies to capture and measure free andantibody-bound soluble SIRPα in the serum. In all dosedmonkeys, soluble SIRPα was detected to be present andaccumulates during the course of the study as compared
to predose levels (Fig. 7d). There is a 50-fold increase insoluble SIRPα as compared to predose after the first doseat both 10 and 30 mg/kg. Post the second dose, the foldincrease in soluble SIRPα reaches 200-fold at the 30 mg/
kg dose and back to low levels by 14 and 21 days at the
10 mg/kg dose (Fig. 7d). The concentration of soluble
SIRPα correlates with the PK of hAB21 (Fig. 7a), indicating accumulation of circulating soluble SIRPα due todecrease clearance while bound to hAB21.
No adverse findings for clinical signs, hematology andserum chemistry related to SIRPα blockade by hAB21
were observed at either dose level (Fig. 7e–h). No significant changes in red blood cells (Fig. 7e) and platelets(Fig. 7f ) were observed. In addition, monocytes and neutrophils, which express high levels of SIRPα, remained
unaffected by hAB21 treatment (Fig. 7g, h). Collectively
the data indicate that hAB21 is safe and well-tolerated incynomolgus monkeys at repeated doses up to 30 mg/kg
with linear PK and full target occupancy in blood.

HAB21 demonstrates favorable PK, PD and tolerability
profiles in monkeys

Discussion
Blockade of the CD47–SIRPα interaction represents apromising approach to boost the antitumor activity ofcancer immunotherapies when used as an adjuvant in
antitumor antibody or ICI therapy. A variety of CD47
blocking antibodies or Fc-fusion proteins have shownobjective responses in patients with advanced hematologic or solid tumor malignancies [14–17, 39, 40]. Whilea multitude of agents that target CD47 have been investigated in pre-clinical models and clinical trials, only a limited number of anti-SIRPα antibodies have been reported[32, 41]. However, due to the lack of species cross-reactivity, no anti-SIRPα antibody has, to our knowledge,

HAB21 binds to both cynomolgus monkey SIRPα andhuman SIRPα with similar affinity (Fig. 1). Therefore, we
conducted an exploratory study in cynomolgus monkey
to determine the PK, PD and preclinical safety of hAB21.
Monkeys received two weekly injections of vehicle control or hAB21-IgG1FcDead on days zero and seven at dosesof either 10 mg/kg or 30 mg/kg and total serum antibody concentration, receptor occupancy, hematologic
parameters and serum chemistry were determined. Systemic exposure to hAB21 increased linearly from the 10
to 30 mg/kg dose level with an AUC​0168 of 17,783 ng h/Kuo et al. J Hematol Oncol(2020) 13:160

Page 15 of 19

a

b

c

d

e

f

g

h

Fig. 7 Pharmacokinetic and safety evaluation of hAB21 in cynomolgus monkey. Female cynomolgus monkeys were dosed twice, 1 week apart,with PBS (control), 10 or 30 mpk hAB21-Fcdead. Serum concentration of hAB21 (a) and occupancy of SIRPα on monocytes at the indicated time
points in monkeys administered 10 mpk (b) and 30 mpk (c). Increase in soluble SIRPα over time in monkeys dosed with 10 or 30 mpk (d). Verticaldashed lines indicated infusion of monkey on day 8. e–h Hematology parameters of RBC (e), platelets (f), monocytes (g) and neutrophils (h)been investigated in both mouse and monkey to evaluate safety and efficacy. The development of antibodybased SIRPα antagonists suitable for clinical translation
has been hindered in part by polymorphisms within
the CD47-binding domain of SIRPα, which necessitates
pan-allele reactive anti-SIRPα antibodies for therapeuticintervention in diverse patient populations. Therefore,
the impact of targeting SIRPα compared to CD47 wasinvestigated as differences in target expression profile andfunction may result in divergent activities of anti-CD47
versus anti-SIRPα targeted therapies that could yieldpotentially distinct pharmacokinetic and safety profiles.
We previously identified anti-SIRPα antibodies from
immunized chickens, which are phylogenetically distantfrom human, monkey, and mouse, which enabled the discovery of a diverse array of pan-allelic and species crossreactive anti-SIRPα antibodies [22]. Herein, we selected
the anti-SIRPα antibody clone AB21 for humanizationand further functional characterization due to its highaffinity binding to both human SIRPα v1 and v2 alleles,
comparable binding to cynomolgus monkey SIRPα,cross-reactivity with various mouse SIRPα alleles, andability to potently block the interaction between CD47
and SIRPα. Allele cross-reactivity is an important design
criterion for anti-SIRPα antibodies intended for clinicaluse. Several groups have reported the presence of multiple SIRPα alleles in the human population [32, 42].
However, in our analyses of 2535 individuals from 1000
Genome Project, we determined that only two dominant
SIRPα alleles, v1 and v2, are present at various frequencies dependent on ethnicity [22]. Blockade of both SIRPαalleles is necessary to maximize efficacy; a single functional SIRPα variant can compensate for the antagonizedvariant. As demonstrated in Fig. 3, the v1 allele-specificanti-SIRPα antibody HEF-LB enhanced macrophagemediated ADCP only when v1/v1 homozygous macrophages were used as effectors. HEF-LB has little to no
impact on ADCP mediated by v1/v2 heterozygous or v2/
v2 homozygous macrophages and on antitumor immunity by v1/v2 heterozygous donor (Fig. 3). In contrast,
hAB21 is efficacious independent of SIRPα genotype(Fig. 3). SIRPα v1 specific antibodies, such as HEF-LB,
would be limited to use in v1/v1 homozygote patient populations, which ranges from only 13.3–49.1% frequency
among the global sub-populations [22]. Pan-allelic antibodies such as hAB21 can potentially impact the entire
population, negating the need to stratify patients prior totherapy, a desirable property for clinical translation.
In addition to species and allele cross-reactivity, thechoice of IgG subclass is an important consideration

Kuo et al. J Hematol Oncol(2020) 13:160

when designing therapeutic antibodies. The efficacy ofboth antitumor and immunomodulatory antibodies ishighly dependent on Fc-domain engagement of FcγRs[43–45]. FcγR interactions with tumor antigen specificantibodies are crucial for the induction of antitumor
immunity [45] but can be detrimental to the activity of
immunomodulatory antibodies due to unwanted depletion of antigen-positive effector cells [43]. Toxicity may
also result from FcγR interactions that promote antibody-mediated destruction of undesirable cell types andtissues, as is the case for CD47-targeted therapies capable of engaging activating FcγRs [20]. CD47 is expressedbroadly, including on RBCs and platelets. Hematological
toxicities including thrombocytopenia and anemia areobserved in patients treated with anti-CD47 antibodies capable of triggering FcγR-dependent effector functions [19, 20, 39]. We have previously demonstrated thatsafety liabilities associated with targeting CD47 can be
mitigated by eliminating binding to FcγRs, leading to theclinical development of ALX148 [13], a fusion protein
consisting of a high-affinity SIRPα variant fused to an
inactive Fc-domain [46].
To investigate the role of FcγR interactions on antiSIRPα antibody activity, we substituted the Fc-domain
of hAB21 with various natural or engineered IgG Fcdomains that have differential binding to FcγRs. Inhibiting the CD47–SIRPα interaction generally enhances
macrophage-mediated ADCP of tumor cells. However, we found that potentiation of ADCP was highly
dependent on the hAB21 Fc-domain; hAB21 with active
Fc-domains (IgG1 and IgG4) failed to enhance ADCP,
whereas hAB21 with inactive Fc-domains potentiated
ADCP (Fig. 2a). This differential activity was demonstrated to be due to competition between the antitumor
antibody cetuximab and hAB21 for binding to effector
cell FcγR. Our data corroborate Voets et al. [32] showing abrogation of rituximab induced phagocytosis whenanti-SIRPα is expressed with an active Fc. Interestingly,
DCs numbers were reduced significantly in PBMCs cultured in vitro with hAB21 containing active Fc-domains,
whereas hAB21 with an inactive Fc-domain had no effecton DC numbers in PBMC cultures (Fig. 2b). Monocytes,
despite expressing high levels of SIRPα, were unaffectedby hAB21 irrespective of IgG subclass (Additional file 1:
Fig. S1). SIRPα expression on macrophages and DCs isdownregulated by hAB21 (Additional file 1: Fig. S4C)
and SIRPα downregulation can reduce SIRPα tonic signaling resulting in DC activation, maturation, and turnover, a mechanism previously reported in mice treatedwith an anti-SIRPα antibody [47, 48] and similar towhat we observed in mice treated with hAB21 (Fig. 6).
SIRPα downregulation by hAB21 may contribute to the
DC depleting effect of hAB21; however, all anti-SIRPαPage 16 of 19

antibodies regardless of subclass downregulate SIRPα
but only hAB21 with active Fc-domains deplete DCs. We
speculate that the DC depleting effect of hAB21 with anactive Fc-domain is due to monocyte- and/or NK cellmediated killing of DCs opsonized with hAB21; however,
we cannot rule out that the combination of SIRPα downregulation and FcγR signaling induced by hAB21 with anactive Fc-domain contributes to DC maturation and subsequent apoptosis.
A similar relationship between FcγR-binding andhAB21 activity was observed in vivo. hAB21 with amouse IgG2a Fc-domain, which preferentially binds activating FcγRs, reduced the antitumor activity of rituximab
whereas hAB21 with a mouse IgG1-N297A Fc-domain(with limited FcγR binding) enhanced rituximab antitumor activity. Although we did not investigate the in vivo
mechanism responsible for these divergent activities,
based on our in vitro results it is likely due to “scorpion
effect”, competition between rituximab and AB21 forbinding to effector cell FcγR, unwanted depletion of DCs,or all. Thus, similar to CD47-targeted therapies, ablationof FcγR-dependent effector function is desirable whentargeting SIRPα, albeit for distinct reasons. Eliminating
Fc-effector function from CD47 targeted therapies mitigates cytopenia [13], while eliminating effector function
from anti-SIRPα antibodies is necessary to enhance macrophage-mediated ADCP and to prevent DC depletionin vitro.
We and others have demonstrated that blocking theCD47–SIRPα interaction has a profound impact on both
antitumor antibody therapy and T cell checkpoint blockade, resulting in a coordinated innate and adaptive antitumor immune response mediated by multiple cell types
including macrophages, dendritic cells, neutrophils, andT cells [9, 13, 41, 49, 50]. In most reported studies thetarget is CD47, and little is known about the in vivo pharmacology of targeting SIRPα. Therefore, we investigated
the impact of SIRPα antagonism on antitumor immunity
in mouse tumor models using hAB21 as a monotherapy
or in combination with an antitumor antibody or T cell
checkpoint inhibitors. We selected hAB21 containing
an inactive Fc-domain for all in vivo studies due to theimpact of Fc-effector function on hAB21 activity in vitro.
Similar to targeting CD47 [13, 31], SIRPα blockade with
hAB21 improved responses to rituximab (anti-CD20) in
the Raji human tumor xenograft model, indicating that amajor mechanism of action of both anti-CD47 and antiSIRPα therapies in xenograft models, which lack NK, B
and T cells, is to enhance the antitumor activity of myeloid cells. Although we did not investigate the effector
cells responsible for hAB21-mediated tumor eradicationin the Raji xenograft model, Ring et al. demonstrated
that neutrophils and macrophages are the main effector

Kuo et al. J Hematol Oncol(2020) 13:160

cells responsible for the anti-tumor activity of a SIRPα
blocking antibody in human tumor xenograft models[41]. These data are consistent with a large body of evidence implicating phagocytes as the dominant effector
cell responsible for the efficacy of agents that block theCD47–SIRPα interaction in xenograft models [31, 46,
51].
Disrupting the CD47–SIRPα interaction bridges innate
and adaptive antitumor immune mechanisms [9, 13];
therefore, we determined the activity of hAB21 in multiple syngeneic murine tumor models as a monotherapy
or in combination with immune checkpoint inhibitors.
These models encompass various levels of immunogenicity and sensitivity towards T cell checkpoint inhibition. As monotherapy, interfering with the CD47–SIRPαinteraction generally has limited efficacy but is a powerful adjuvant to a variety of cancer immunotherapies[31, 46]. Treatment with hAB21 alone had no effecton tumor growth in syngeneic 4T1, CT26, or B16F10
tumors (Fig. 5e and Additional file 1: Fig. S3). Modest
hAB21 single agent activity was observed against MC38
tumors (Figs. 4a, 5a, b), which was dependent on thepresence of CD8α+ DCs, ­CD8+ T cells and neutrophils(Fig. 4). Depletion of neutrophils by anti-GR1 but not
macrophages by anti-CSF1R prior to hAB21 treatment in
immunocompetent mice showed no antitumor activity,
suggesting cell depletion by anti-GR1 impacted hAB21induced antitumor activity. Although, anti-GR1 has beenused extensively to deplete neutrophils in C57BL/6 mice,it does also bind GR1++ non-neutrophils, specifically­CD8+GR1+ T cells [52, 53]. The lack of antitumor activity
with anti-GR1 depletion may be due to the elimination of­CD8+GR1+ T cells, known to secrete IFNγ, neutrophils
or both cell types. CD8α+ DCs are indispensable for the
production of a ­CD8+ antitumor T cell response [3] andDCs play a critical role in the responses to anti-CD47 targeted therapies [9, 13, 33]. Administration of hAB21 toMC38 tumor bearing mice resulted in DC activation in
the spleen and tumor that when further combined with
anti-PD-1/PD-L1 therapy promoted both CD4++ and­CD8+ T cell effector functions and reduced immunosuppressive ­CD4+ ­Tregs and TAMs leading to eradication of
MC38 tumors and long-term, durable immunity. Similar
mechanisms of MC38 tumor control have been reported
for anti-CD47 antibodies [33, 54], and for ALX148, a
CD47 blocking Fc-fusion protein [13], suggesting that the
antitumor effects of targeting SIRPα broadly recapitulatethat of CD47-targeted therapies despite differences in
the ligand expression patterns and molecular and cellularfunctions of SIRPα and CD47.
Responses to the combination of hAB21 with anti-PD-1
or anti-4-1BB were less pronounced in more aggressive
and less immunogenic tumors including CT26, 4T1,

Page 17 of 19

and B16F10 (Additional file 1: Fig S3). These results are
consistent with additional studies that have investigatedthe combination of CD47 antagonists with checkpoint
regulators in syngeneic mouse tumors [49]. In moreaggressive and poorly immunogenic tumors, additional
treatment modalities may be necessary to improve tumor
control and eradication, such as triple combinations withtumor antigen specific antibodies, CD47/SIRPα antagonists, ICIs, vaccines, or chemotherapy [49, 55].
Due to the widespread expression of CD47, targeting
SIRPα may overcome some obstacles associated with targeting CD47 [32, 56] such as the large antigen sink that
promotes target mediated clearance of anti-CD47 therapies and off-tumor toxicities due to CD47 expression
on RBCs and platelets by certain anti-CD47 therapeutics. We investigated safety and PK/PD of hAB21 in an
exploratory toxicology study in cynomolgus monkey. No
dose-dependent adverse clinical observations or changes
in hematology and serum chemistry assessment wereobserved with hAB21 treatment (Fig. 7), whereas antiCD47 therapeutics capable of activating FcγR-dependent
effector functions causes dose-dependent cytopenia in
NHPs and humans [19, 20]. Administration of hAB21
with an inactive Fc at both 10 mg/kg or 30 mg/kg resulted
in complete target occupancy on circulating monocytes
and linear PK with a half-life of 5.3 days and 8.1 days,
respectively. This contrasts with anti-CD47 antibodies oranti-CD47 Fc-fusion proteins which display non-linear
PK and accelerated clearance at similar dose levels due tothe large CD47 antigen sink [20]. However, soluble SIRPα
was detected in the monkeys and accumulation observed
during the course of the study (Fig. 7d). This is indicativeof serum stabilization of the naturally produced soluble
SIRPα by binding to the anti-SIRPα antibody. Soluble
SIRPα-bound antibody is not available for blocking cellular CD47–SIRPα interaction, potentially limiting theimpact of the longer half-life of anti-SIRPα antibodies incomparison to anti-CD47 antibodies.

Conclusion
In summary, blockade of SIRPα with hAB21 potently
enhances macrophage phagocytosis of tumor cells andpromotes activation of DCs leading to the induction ofadaptive antitumor T cell responses in tumor bearing
mice, substantially improving responses to both antitumor antibody and immune checkpoint therapy. The
in vitro and in vivo antitumor activity of hAB21 broadly
recapitulates that of CD47 targeted therapies despite differences in ligand expression, binding partners, and function, validating the CD47–SIRPα axis as fundamental
myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of humanmalignancies.

Kuo et al. J Hematol Oncol(2020) 13:160

Supplementary information
Supplementary information accompanies this paper at https​://doi.
org/10.1186/s1304​5-020-00989​-w.
Additional file 1. Supplementary figures and table.

Page 18 of 19

4.
5.
6.
7.

Abbreviations
ADA: Anti-drug antibody; ADCP: Antibody-dependent cellular phagocytosis;
DC: Dendritic cell; FcγR: Fragment crystallizable gamma receptor; ICI: Immune
checkpoint inhibitor; mDC: Monocytic dendritic cell; MDSC: Myeloid-derived
suppressor cell; PBMC: Peripheral blood mononuclear cell; PK: Pharmacoki‑
netic; SIRPα: Signal regulatory protein; TAM: Tumor-associated macrophage;
TNBC: Triple negative breast cancer; Treg: Regulatory T cell.
Acknowledgements
The authors thank A.S. Wibowo and J. Carter from Helix BioStructures for help
with X-ray crystallography and Sophia Randolph for discussion and criticalreading of the manuscript.
Authors’ contributions
TCK, JS, JP, and HIW conceived and guided the research. TCK, AC, OH, AZ, ES,
LD, DF, and JS carried out the experiments. All authors analyzed and inter‑
preted the data. TCK, AC, OH, and JTS wrote the manuscript. TCK, JS, JP, andHIW critically review and provided comments on the manuscript. All authors
approved the manuscript.
Funding
The study was sponsored by ALX Oncology.
Availability of data and materials
All relevant data are within the paper and its Supplementary files. Structure
information is provided in Protein Data Bank (PDB).

8.
9.
10.

11.
12.

13.
14.
15.

Ethics approval and consent to participate
In vivo studies were approved by the Institutional Animal Care and Use Com‑mittee of ALX Oncology and UTSW.

16.

Consent for publication
Not applicable.

17.

Competing interests
TCK, AC, OH, JTS, ES, LD, SEK, DF, SB, BH, JS, and HIW were all employed by
ALX Oncology and are shareholders. JP is employed by ALX Oncology and isa shareholder.
Author details
1
ALX Oncology, Burlingame, CA, USA. 2 Department of Pathology, Universityof Texas Southwestern Medical Center, Dallas, TX, USA. 3 Present Address: Tallac
Therapeutics, Burlingame, CA, USA. 4 Present Address: Genentech, South San
Francisco, CA, USA. 5 Present Address: Coherus BioSciences, Redwood City, CA,
USA. 6 Present Address: ProLynx Inc., San Francisco, CA, USA.

18.
19.

20.
21.

Received: 1 October 2020 Accepted: 2 November 2020
22.

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