RESEARCH ARTICLE

ALX148 blocks CD47 and enhances innate andadaptive antitumor immunity with a favorable
safety profile
Steven E. Kauder☯, Tracy C. Kuo☯, Ons Harrabi, Amy Chen, Emma Sangalang,
Laura Doyle, Sony S. Rocha, Sangeetha Bollini, Bora Han, Janet Sim, Jaume Pons,
Hong I. Wan*
ALX Oncology, Burlingame, CA, United States of America

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OPEN ACCESS
Citation: Kauder SE, Kuo TC, Harrabi O, Chen A,
Sangalang E, Doyle L, et al. (2018) ALX148 blocks
CD47 and enhances innate and adaptive antitumor
immunity with a favorable safety profile. PLoS ONE
13(8): e0201832. https://doi.org/10.1371/journal.
pone.0201832
Editor: David D. Roberts, Center for Cancer
Research, UNITED STATES
Received: February 21, 2018
Accepted: July 23, 2018
Published: August 22, 2018
Copyright: © 2018 Kauder et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.

☯ These authors contributed equally to this work.
* hong@alxoncology.com

Abstract
CD47 is a widely expressed cell surface protein that functions as an immune checkpoint incancer. When expressed by tumor cells, CD47 can bind SIRPα on myeloid cells, leading tosuppression of tumor cell phagocytosis and other innate immune functions. CD47-SIRPαsignaling has also been implicated in the suppression of adaptive antitumor responses, but
the relevant cellular functions have yet to be elucidated. Therapeutic blockade of the CD47
pathway may stimulate antitumor immunity and improve cancer therapy. To this end, anovel CD47-blocking molecule, ALX148, was generated by fusing a modified SIRPα D1
domain to an inactive human IgG1 Fc. ALX148 binds CD47 from multiple species with highaffinity, inhibits wild type SIRPα binding, and enhances phagocytosis of tumor cells by macrophages. ALX148 has no effect on normal human blood cells in vitro or on blood cell parameters in rodent and non-human primate studies. Across several murine tumor xenograftmodels, ALX148 enhanced the antitumor activity of different targeted antitumor antibodies.
Additionally, ALX148 enhanced the antitumor activity of multiple immunotherapeutic antibodies in syngeneic tumor models. These studies revealed that CD47 blockade with
ALX148 induces multiple responses that bridge innate and adaptive immunity. ALX148 stimulates antitumor properties of innate immune cells by promoting dendritic cell activation,
macrophage phagocytosis, and a shift of tumor-associated macrophages toward an inflammatory phenotype. ALX148 also stimulated the antitumor properties of adaptive immune
cells, causing increased T cell effector function, pro-inflammatory cytokine production, anda reduction in the number of suppressive cells within the tumor microenvironment. Taken
together, these results show that ALX148 binds and blocks CD47 with high affinity, inducesa broad antitumor immune response, and has a favorable safety profile.

Funding: All authors of this manuscript are
employed by ALX Oncology. The funder provided
support in the form of salaries for authors SEK,
TCK, OH, AC, ES, LD, SSR, SB, BH, JS, JP, andHIW, but did not have any additional role in the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. The

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

specific roles of these authors are articulated in the‘author contributions’ section.
Competing interests: All authors of thismanuscript are employed by ALX Oncology. Thesestudies were sponsored by ALX Oncology. This
does not alter our adherence to PLOS ONE policies
on sharing data and materials.

Introduction
A central question in the study of cancer is why the immune system sometimes fails to mountan effective antitumor response despite possessing the components needed to do so. One
cause of this failure has become clear with the identification of checkpoint pathways, which
are co-opted by tumors to inhibit their elimination by immune cells. This phenomenon has
been best described for the adaptive component of the immune response, where cytotoxic T
cell activity is suppressed by checkpoint signals originating from tumor and other cells in thetumor microenvironment [1]. In the clinic, the CTLA-4 and PD-1 T cell checkpoint pathways
have been validated as therapeutic targets, with their blockade leading to enhancement of thepatient’s immune response and, in some cases, durable antitumor efficacy across several
tumor types [2–4].
The CD47 pathway is an additional checkpoint that can suppress antitumor immunity [5,6]. In contrast to previously identified checkpoint pathways that target the adaptive arm of theimmune response, this pathway suppresses the activity of innate immune cells [7, 8]. CD47 isexpressed on the surface of a broad range of cell types [9, 10], and this expression protects
healthy cells from macrophage-mediated phagocytosis by interacting with its receptor, signal
regulatory protein-α (SIRPα) [11, 12]. Engagement of SIRPα triggers signaling through SIRPαimmunotyrosine inhibitory motifs (ITIMs), which inhibits phagocytosis and other components of macrophage function [13–21].
Analyses of human tumor tissue have implicated CD47 in cancer. High levels of CD47
expression have been observed in a variety of hematological and solid tumors [5, 22], and elevated CD47 expression is an adverse prognostic indicator for survival [22–25]. These findings
indicate that tumor cells may utilize the CD47 pathway to evade macrophage surveillance.
One component of this surveillance is Antibody-Dependent Cellular Phagocytosis (ADCP), in
which antitumor antibodies initiate phagocytosis by binding tumor cells and engaging macrophage Fc gamma (Fcγ) receptors [26–28]. Blockade of the CD47-SIRPα interaction enhances
ADCP of tumor cells [24, 29–32], demonstrating that if unchecked, CD47 expression can protect tumor cells from macrophage phagocytosis. Similarly, CD47 blockade in mouse studies
inhibits the growth of human tumor xenografts and promotes survival [22, 24, 25, 30, 33].
Notably, these xenograft studies utilized immunocompromised mice that lack most immune
cell types other than macrophages. Thus, while these studies demonstrated that CD47 blockade
activates a macrophage-mediated antitumor response, they were incapable of identifying theroles played by other cells in the context of an intact immune system.
To better understand the full range of responses induced by CD47 blockade, CD47 function
has been disrupted in immunocompetent mice [34–36]. These studies have shown dendritic
cells (DCs) and T lymphocytes to be important components of the resultant antitumor
response. DCs express SIRPα, and inhibition of the CD47-SIRPα interaction in a model using
exogenous sheep red blood cells triggered DC activation, leading to enhanced T cell responses[37]. Furthermore, studies of syngeneic tumors in immunocompetent mice have demonstrated
that disruption of CD47 signaling can induce macrophage, DC, and T cell-mediated antitumor
responses. In fact, both DCs and T cells have been shown to be essential for the CD47-mediated antitumor response [34, 38]. Further evidence for interplay between innate and adaptive
immunity in response to CD47 blockade comes from a combination study of CD47 andPD-L1 blocking agents. The activation of T cells by PD-L1 inhibition was required to maximize the effect of CD47-SIRPα disruption [39]. Thus, the therapeutic targeting of the CD47
pathway can induce a broad antitumor response involving both innate and adaptive immunity.
While depletion studies have shown the importance of cells such as DCs and T lymphocytes
for this response, the cellular functions activated by these agents are unknown.

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Because tumor cells potentially utilize the CD47 pathway to evade both innate and adaptive
components of immune surveillance, therapeutic blockade of this pathway may activate antitumor
immunity and improve cancer therapy. For this reason, a variety of antibodies and fusion proteinstargeting the CD47 pathway are in clinical development [30, 31, 40]. Notably, the majority of thesemolecules share a common design in that they are essentially bifunctional. First, they bind CD47,
blocking its interaction with SIRPα and suppressing downstream signals that inhibit phagocytosis.
Second, they contain Fc domains that engage macrophage Fcγ receptors, inducing phagocytosis.
These molecules induce phagocytosis in vitro and are efficacious against human tumor cell xenografts in mice. However, normal healthy blood cells also express high levels of CD47, making thempotential targets for these molecules. In fact, dose-limiting toxicities related to anemia and thrombocytopenia have been reported for several of these molecules in both animal and clinical oncology
studies [31, 41, 42]. These toxicities underscore the need to ensure that CD47 blockade activates
immune responses against tumor cells while sparing normal healthy cells.
For the reasons discussed above, a safer approach to targeting CD47 is needed to achieve
the therapeutic potential of CD47 blockade. ALX148 was generated as an investigational anticancer therapeutic to safely target CD47 in humans, enhancing antitumor immunity while
having minimal effects on normal cells. ALX148 is comprised of the N-terminal D1 domain of
SIRPα, which binds CD47 [43, 44], fused to a modified human IgG1 Fc domain. The SIRPα
D1 portion contains amino acid substitutions previously shown to dramatically increase affinity for CD47 [32], thus enabling the blockade of wild-type SIRPα binding. Furthermore, thismodified SIRPα domain is designed to bind cynomolgus monkey and murine CD47 with highaffinity, allowing direct investigation of ALX148 toxicology and function in these species.
The Fc portion of ALX148 confers increased molecular mass and interaction with the neonatal Fc receptor, both of which are associated with extended pharmacokinetics. In ALX148, thisdomain has a unique set of amino acid substitutions designed to eliminate binding to humanFcγ receptors and complement C1q protein [45–48]. The absence of effector function should
prevent ALX148 from engaging Fcγ receptors and targeting normal cells for phagocytosis, differentiating ALX148 from other CD47-blocking molecules that have active Fc domains.
Here, we describe results of preclinical studies of ALX148. This investigational drug
potently blocked the CD47-SIRPα interaction and enhanced antibody dependent phagocytosis. In murine models utilizing human tumor xenografts, ALX148 improved the efficacy of targeted antitumor antibodies that are currently in clinical use. Because ALX148 binds murine
CD47 with high affinity, effects on antitumor efficacy and immune responses were evaluatedin syngeneic murine tumor models. In these models, ALX148 enhanced the antitumor activityof multiple immunotherapeutic agents. Furthermore, as a single agent and in combination
with these agents, ALX148 enhanced DC, macrophage, and T cell responses associated withantitumor immunity. These findings demonstrate that ALX148, via CD47 blockade, elicits
immune responses that bridge innate and adaptive immunity and are associated withenhanced antitumor efficacy. Consistent with the absence of Fc effector function, in rodent
and non-human primate studies no ALX148-related adverse events were observed in hematological or other parameters. These results indicate that ALX148 has the potential to be an effective anti-cancer therapeutic in humans while maintaining a favorable safety profile. Therefore,
ALX148 is currently in clinical development for multiple oncology indications.

Materials and methods
Molecular cloning, protein expression and purification
Wild type SIRPα, CV1 [32], human IgG1 Fc, human IgG1 Fc variants, and human IgG4 Fc
with S228P and L235E substitutions (hIgG4 PE), rituximab, hexahistidine-tagged CD47,

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

trastuzumab, daratumumab, and anti-PD-L1 (based on atezolizumab, with murine IgG1 Fc)
were synthesized (ATUM) based on publicly available sequences in CMV promoter-driven
expression plasmids for mammalian cell expression. The SIRPα domains of ALX148 incorporate amino acid substitutions that enhance affinity for CD47 [32]. This was fused to human
IgG1 Fc with modifications that eliminate effector function [45–48]. ALX377 was generated byfusing the SIRPα domains of ALX148 to unmodified human IgG1 Fc. ALX180 was generatedusing site directed mutagenesis to introduce V33R and Q52R substitutions in wild type SIRPα
fused to a modified IgG1 Fc. ALX222 was generated by fusing CV1 to human IgG1 Fc (GeneArt Seamless PLUS Cloning and Assembly Kit, Invitrogen). ALX217 was made by fusing the
SIRPα domain from ALX148 to human IgG4 S228P, S235E. Constructs were expressed in
Expi293 cells (Invitrogen) at 3 ml to 1L scale in deep well blocks or shake flasks. Expression
cultures were grown five days at 37˚C in 8% CO2 while shaking. Supernatants were harvested
via centrifugation and sterile filtered. Proteins were affinity purified utilizing either an Akta
Avant (GE Healthcare) or Amicon Pro Purification Device (Millipore Sigma). Antibodies andFc fusion proteins were bound to MabSelectSure LX resin (GE Healthcare), washed with 1x
phosphate-buffered saline (1xPBS), eluted with 0.1 M citric acid pH 3.3, neutralized with 10%
v/v 1 M sodium phosphate pH 8, then dialyzed into PBS. CD-47 His was bound to a chelatingsepharose resin, step eluted with 5, 40, and 300mM Imidazole with a running buffer of 25mM
phosphate pH 7.5, 500mM NaCl followed by a gel filtration polishing step.
The nucleotide coding sequence for ALX148 was subcloned into an expression vector
under the control of the hEF-1-alpha promoter coupled to a CMV enhancer. A stable CHO
cell line expressing ALX148 was generated and used for production of ALX148. Purification of
ALX148 was carried out using protein A affinity chromatography similar to conventional antibody purification. The final purified material was concentrated into storage buffer and stored
at -80˚C until use.

Cell lines
OE19, DLD-1, Raji, Z138, Daudi, Jurkat, MM1.R, and CT26 cells were obtained from theAmerican Type Culture Collection. DLD-1 GFP Luciferase cells [32] were obtained from K.
Christopher Garcia, Stanford University. CT26-M/H HER2 cells were generated by infecting
CT26 cells with a lentivirus vector encoding a chimera of mouse and human HER2 transmembrane and extracellular domains. MC38 cells were obtained from MuriGenics. Cell lines were
cultured according to standard protocols.

Animals
All mouse experiments were conducted according to protocols approved by Institutional Animal Care and Use Committee of Alexo Therapeutics (protocol number AL-002-2017 forhematology studies and protocol number AL-001-2017 for tumor studies). BALB/C, C57BL/6,
NOD-SCID and CD-1 animals were purchased from Charles River Laboratories International(Hollister, CA). All animals were housed in a pathogen free facility located in the vivarium at
Alexo Therapeutics, South San Francisco in accordance to IACUC guidelines. Animals used
for all studies were 6–8 weeks old.
The non-human primate CD47 occupancy study was conducted at Charles River Laboratories in accordance with standard operating procedures and with the approval of the IACUC.
Animals were housed in stainless-steel cages. Primary enclosures were as specified in the
USDA Animal Welfare Act (9 CFR, Parts 1, 2 and 3) and as described in the Guide for theCare and Use of Laboratory Animals. The targeted conditions for animal room environment
were temperature of 64F to 84F, humidity of 30% to 70%, ventilation of greater than 10 air

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ALX148 blocks CD47 with favorable safety profile and antitumor immunitychanges per hour with 100 percent fresh air, light cycle of 12 hour light/12 hour dark. Purina
Certified Primate Diet No. 5048 was provided daily in amounts appropriate for the size andage of the animals. Municipality tap water was processed through a reverse osmosis filter andpassed through UV light treatment. Animals received fruit and/or vegetable supplements aminimum of four times per week. Food distribution by means of human interaction was provided a minimum of two times per week. At least one interior (in cage) enrichment toy wasprovided for each animal at all times. Animals were socialized continuously unless prohibited
due to incompatibility, medical concerns, or study activities, up to three animals of the samesex and same dosing group together.
The non-human primate toxicity study was conducted at Covance Laboratories. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of CovanceLaboratories, Madison WI (Approval number 8333311). Procedures were also in compliance with
the Animal Welfare Act (P.L. 89–544), the Guide for the Care and Use of Laboratory Animals(National Research Council of the National Academies), and the Office of Laboratory Animal
Welfare. Animals were housed in stainless steel cages. Where possible, animals were sociallyhoused by sex: up to three animals per cage. Certified Primate Diet #5048 (PMI Nutrition International) was provided one or two times daily. Water was provided ad libitum. Environmental controls for the animal room were set to maintain 20 to 26˚C, a relative humidity of 30 to 70%, aminimum of eight air changes/hour, and a 12-hour light/12-hour dark cycle. Animals were given
various cage-enrichment devices; fruit, vegetable, or dietary enrichment (that do not require analyses). Animals were commingled in accordance with Covance standard operating procedures.

Affinity determination
All experiments were performed at 25˚C and 37˚C using a SPR-based ProteOn XPR36 biosensor (BioRad) equipped with GLC sensor chips. Running buffer was PBS pH 7.4 with 0.01%
Tween-20 (PBST+). All analytes (human, cynomolgus monkey, mouse and rat CD47-ECD)
were used at their nominal concentrations as determined by A280 absorbance and molar
extinction coefficient. Analytes were injected in a “one-shot” kinetic mode [49].
The interactions of ALX148 with human, cynomolgus monkey, mouse and rat CD47-ECD
were analyzed by flowing CD47-ECD over ALX148 immobilized (200–400 RUs) on GLC
chips using Proteon Amine Coupling Kit (Biorad). GLC chips were activated with EDAC/Sulpho-NHS 1:1 (Biorad) diluted 1:500 for 300 sec at 25 μL/min. ALX148 proteins were diluted to
80 nM concentration in 50 mM sodium acetate buffer pH 4.5 and immobilized to the chip at
30 μl/min for 50 seconds. Chips were inactivated with ethanolamine for 300 sec at 25 μL/min.
Analytes (CD47-ECD) were injected in a “one-shot” kinetic mode at nominal concentrations of 300 nM or 100 nM with 3-fold serial dilutions. Association times were monitored for
60 seconds, and dissociation times were monitored 1200 seconds for human and cynomolgus
monkey, 600 seconds for mouse and rat CD47. The surfaces were regenerated with a 2:1 v/v
blend of Pierce IgG elution buffer and 4M NaCl.
Biosensor data were double-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 Langmuir model and the KD value was calculated
from the ratio of the apparent kinetic rate constants (KD = kd/ka).

ALX148 binding on cells and blockade of SIRPα binding to CD47 cells
For detection of cell binding, ALX148, ALX180 and SIRPα-IgG1 Fc fusion protein (ALX234)
were fluorescently labeled with the Alexa Fluor 647 Protein Labeling Kit (Thermo Fisher

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Scientific) according to the manufacturer’s instructions. 100,000 cells per well in staining
buffer (PBS, 0.5% BSA or 2%FBS) were plated in 96 well plates (Falcon).
To detect ALX148 binding to cells, Alexa Fluor 647-labeled ALX148 or negative control
ALX180 at a final concentrations of 20 nM were added to cells in 50 μl volume of FACS buffer(PBS + 0.5% BSA) supplemented with a cocktail of human Fc block (Miltenyi Biotec) andhuman AB serum (Corning). After a 15 minute incubation on ice, cells were washed twice instaining buffer, stained with a fixable Live/Dead stain (Invitrogen), followed by a final wash
and fixed in 0.5% formaldehyde.
To block SIRPα binding to CD47 on Jurkat cells, ALX148 or ALX180 at a concentration of
12.5 nM were added to cells. After a 15 minute incubation on ice, cells were washed in FACS
buffer and incubated with labeled ALX234 at a concentration of 2.5 μM. Wild type SIRPαbinding is indicated by the geometric mean fluorescence for Alexa Fluor 647.
Cells were analyzed on a FACS Canto II (BD Biosciences), with subsequent data analysis
and histogram plotting using Flowjo 10.7.

Human FcRn and FcγRs binding
All experiments were performed at 25˚C using a SPR-based ProteOn XPR36 biosensor(BioRad, Inc, Hercules, CA) equipped with GLC sensor chips. The running buffer was PBS pH
7.4 with 0.01% Tween-20 (PBST+) and PBS pH 5.8 with 0.01% Tween-20 for FcRn interactions. All analytes were used at their nominal concentrations as determined by dividing the
manufacturer-provided protein amount by volume of dissolution buffer. Analytes were
injected in a “one-shot” kinetic mode.
The interactions of ALX proteins with hFcγRs and hFcRn were analyzed by flowing thereceptors over ALX proteins captured on a human CD47 GLC chip. Approximately 250 RU of
human CD47 ECD were immobilized on a GLC chip using amine chemistry (following manufacturer instructions). ALX proteins were captured for 300 seconds at 25 μl/min at 40 nM in
PBST+ (300–400 RUs). Analytes (hFcγRs and hFcRn) were injected in a “one-shot” kinetic
mode at nominal concentrations of 0, 61, 185, 555, 1666, and 5000 nM. Association times weremonitored for 200s and dissociation times were monitored for 120s (except for hFcγRI where
the dissociation time was 600s). The surfaces were regenerated with a 2:1 v/v blend of Pierce
elution buffer/4M NaCl.
Biosensor data were double-referenced by subtracting the interspot data (containing no
immobilized protein) from the reaction spot data (immobilized protein) and then subtracting
the sensograms of blank reference channel. Double-referenced data were fit to an equilibrium
analysis using a simple binding isotherm. For Fcs with strong binding to hFcγRI, data werealso fit globally to a simple Langmuir model and the KD value was calculated from the ratio of
the apparent kinetic rate constants (KD = kd/ka).

Human complement C1q binding
Immulon 4HBX ELISA plates (Thermo Fisher Scientific) were coated overnight with bovine
serum albumin (BSA, Thermo Fisher Scientific), ALX217, ALX148, or ALX222 at 5 μg/ml in
PBS. Plates were blocked in assay buffer pH 6.0 (PBS with 0.5% BSA, 0.05% Tween-20, 0.25%
CHAPS, 5mM EDTA, 0.35M NaCl, Teknova) and washed in 1x TBST (Teknova). Plates wereincubated with complement C1q (Quidel) for 1 hour at room temperature and washed withwash buffer. Plates were then incubated for 1 hour at room temperature with HRP-conjugatedsheep anti-complement C1q antibody (Thermo Fisher Scientific) at 2 μg/mL and washed, followed by the addition of 3,3’,5,5’-tetra-methylbenzidine peroxidase substrate (Thermo Fisher
Scientific) and incubation for 10 minutes. The reaction was terminated with 0.16M sulfuric

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ALX148 blocks CD47 with favorable safety profile and antitumor immunityacid (Thermo Fisher Scientific) and the resulting absorbance was measured at 450 nm with areference of 570 nm using a SpectraMax i3 plate reader (Molecular Devices).

ADCC reporter bioassay
The assay was performed with the ADCC Reporter Bioassay Core Kit (Promega) using Rajicells as targets according to the manufacturer’s instructions. Black walled, clear bottomed 96
well assay plates (Corning) were incubated with test proteins, 12,500 Raji cells, and 75,000 Jurkat Effector Cells for 6 hours at 37˚C. Bio-Glo luciferase assay reagent was added and luminescence was read in a SpectraMax i3 plate reader (Molecular Devices). IgG1, k antibody(Southern Biotech) was used as a negative control.

Hemagglutination assay
Human erythrocytes were isolated from whole blood buffy coats (Stanford University Blood
Center) by purification with Ficoll-Paque Plus (GE Healthcare) and subsequent washing oferythrocyte pellets in PBS. 4 million erythrocytes in PBS were plated per well in polypropylene
96 well plates (Corning). Plates were incubated with ALX148, B6H12 (Thermo Fisher Scientific), CC2C6, or PBS for four hours at room temperature. Plate images were captured as
unmodified jpeg images.

Phagocytosis assay
CD14+ cells were purified from Trima residuals (Blood Centers of the Pacific) with FicollPaque Plus and negative selection (Monocyte Isolation Kit II, Miltenyi Biotec) according to
the manufacturers’ protocols. Monocyte-derived macrophages were made by seeding 10 million CD14+ cells into 150 mm tissue culture dishes (Corning) in growth medium supplemented with 10% human AB serum (Corning). Cells were cultured for seven to eleven days.
Adherent cells were detached from culture plates with TrypLE Select (Thermo Fisher Scientific). Where necessary, target cells 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 monocyte-derived macrophages were incubated in ultra-low
attachment U-bottom 96 well plates (Corning) with ALX148 or ALX180 and the corresponding
tumor-specific antibody for two hours at 37˚ C. For DLD-1 GFP Luciferase cells, cetuximab(Absolute Antibody) was added at a concentration of 1 μg/ml. For OE19 cells, trastuzumab(Absolute Antibody) was added at a concentration of 0.01 μg/ml. For MM.1R, daratumumab(Alexo Therapeutics) was added at a concentration of 1 μg/ml. For Daudi cells, obinutuzumab(Roche) was added at a concentration of 0.1 μg/mL. IgG1, k isotype control antibody was purchased from SouthernBiotech.
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 GFP Luciferase
and OE19 cells, antibody against CD138 (clone MI15, Biolegend) was included for MM1.R
cells, and antibodies against CD20 (clone 2H7, Biolegend) was included for Daudi 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 percentage of viable CD33+CD206+
macrophages that stain negative for target cell markers and positive for GFP or CFSE. Where
applicable, 4 parameter fit curves were generated with Prism 7 software (GraphPad).

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

For fluorescence microscopy, cells were washed twice with PBS after a two hour incubationof macrophages and DLD-1 cells in the presence of various treatments. Washed cells were
resuspended in PBS and 60,000 cells in 25ul were spread on a slide (ONCYTE) to air dry atroom temperature. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by two washes with PBS. Slides were partially dried and 50ul of antifade
mount (ProLong) and coverslip were used to seal the cells. CFSE labeled target cells and macrophages were visualized by bright field and fluorescence using an Axioimager upright microscope (Zeiss) with a 470 nM eGFP filter at 20x magnification. Digital images were taken with a
AxioCam Hrm (Zeiss) and digitally merged using Fiji software [50].

Murine hematology assay
Animals were monitored for adverse clinical signs and humane endpoints were applied
according to the Institutional Animal Care and Use Committee of Alexo Therapeutics protocol number AL-002-2017. Animals were observed for a minimum of an hour following dosing,then minimally once daily, increasing if any clinical abnormalities were observed. Any
observed adverse clinical signs were described and documented in the study record andincluded time of onset relative to dose administration and if appropriate, time to recovery.
Animals with severe clinical signs, with no improvement, or the animals not anticipated torecover before the next scheduled time point or dose administration were euthanized. Body
weights were obtained pre-dose and then, minimally, once a week (increasing to daily if 15%
body weight loss is evidenced) for the duration of the study. Any animal that appeared moribund (lethargic, hunched posture, cold to touch, etc.) and/or had a loss of 20% in bodyweight as compared to age-matched, nonmanipulated cohorts was euthanized.
ALX148 and ALX377 were administered intravenously to female CD-1 mice via tail vein on
day 0. Mice were assigned to two groups of ten mice and were treated with 30 mg/kg ALX377
or 30 mg/kg ALX148. For complete blood counts (CBC) analysis, blood was collected via tailvein into K2EDTA microcapillary tubes (Heska) and hematologic parameters were evaluatedusing a HeskaView analyzer. Every time point contains four-five mice/treatment group at thefollowing time points: Day -5, 72 (Day 3), 120 (Day 5), and 240 (Day 12) hours post dose.

In vivo tumor studies
Isoflurane anesthesia were used on mice to eliminate or minimize pain and distress during
tumor impantation. CT26 and MC38 colon tumor cells (0.5×106) were resuspended in 100 μl
PBS or RPMI and implanted subcutaneously into the flank of female BALB/c and C57BL/6
mice, respectively. For the MC38 immunophenotyping study, 2 x 106 cells were implanted.
OE19 (5×106), Raji (5×106), and Z138 (5×106) tumor cells were resuspended in 100 μl 1:1 PBS:
matrigel (Corning) and implanted subcutaneously in the flank of NOD-SCID mice. Whentumors reached an average volume of 50-250mm3 (depending on the model), as calculatedwith the formula volume = (width2 x length)/2, mice were randomized into treatment groups.
All treatments were dosed intraperitoneally (i.p.). For xenograft models in NOD-SCID mice,
rituximab, obinutuzumab, and trastuzumab were dosed twice/week for three weeks. Treatment groups for syngeneic models vary and are as described in figure legends. Mice weretreated with 5 mg/kg anti-PD-1 (BioXcell, clone RMP1-14) three times, five days apart, 2 mg/
kg or 3 mg/kg anti-PD-L1 (murine IgG1, Alexo Therapeutics) three times, five days apart and
0.1 mg/kg anti-4-1BB (BioXcell, clone LOB12.3) two times, five days apart. Mice in the MC38
immunophenotyping study were dosed once a week with anti-PD-L1.
Mice were observed daily with a minimum of weekly tumor measurements when palpable
tumors were present and daily once tumor volume reaches a maximum of 1500 mm3 or if

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

animals are showing abnormal signs or body weight changes by 15%. Animals were monitored
for adverse clinical signs and humane endpoints were applied according to the Institutional
Animal Care and Use Committee of Alexo Therapeutics protocol number AL-001-2017. Animals were euthanized by CO2, followed by cervical dislocation at the end of the study or immediately during the study when weight loss 20% of baseline, tumor greater than 2000 mm3,
tumor interferes with normal bodily functions, discharge from tumor, respiratory or central
nervous system abnormalities, self-mutilation, hypothermia or veterinarian determines thatthe animal should be euthanized for humane concerns. No unexpected deaths were observed.
The length of the study depended on the model. For survival endpoints, the duration of MC38
and CT26 syngeneic tumor model studies were between 35–45 days and for Raji xenograftmodel was around 50–55 days. All animals in the studies were euthanized due to tumor size
greater than 2000 mm3 or at the end of the study. The number of mice used per study is thesame as the number of mice euthanized. All studies included 40 mice except for CT26
(ALX148 + anti-PD-1 combo study), which include 36 mice.

CD47 occupancy assays
BALB/c mice were implanted subcutaneously with 0.5 million CT26-M/H HER2 tumor cells
in the right flank. Thirteen days after implantation, mice were randomized into groups withequivalent tumor sizes and administered PBS or unlabeled ALX148 at doses of 10 or 30 mg/kg
via intraperitoneal injection. At 1, 4, or 8 days after administration, 100 μl blood was removed
by cheek bleed with a Goldenrod animal lancet (MEDIpoint) and centrifuged in serum separation tubes (Becton Dickinson) to isolate serum for ALX148 ELISA. Mice were then euthanized
and tumors and spleen removed. Tumors were approximately 500 to 900 mm3 in size at thetime of euthanasia. Single cell suspensions were made from tumors (Tumor Dissociation Kit,
mouse, Miltenyi Biotec) using 1/10 the recommended volume of enzyme R and from spleenby mechanical dissociation, with red blood cell lysis using ACK buffer (Thermo Fisher Scientific). Five million cells were incubated in 5% mouse serum (Jackson Immunoresearch), thenstained with fluorochrome-labeled antibodies against CD3 (clone 145-2C11, Biolegend), CD4(clone RM4-5, Biolegend), and trastuzumab (Alexo Therapeutics) and Fixable Viability Dye toidentify live cells. CD47 occupancy was determined by including labeled ALX148 in the stain
cocktail. Cells were analyzed on a FACS Canto II flow cytometer with subsequent analysis
using FlowJo 10.4 software.
Female cynomolgus monkeys (n = 2) were administered a single intravenous dose of
ALX148 at 10 and 30 mg/kg along with a single intravenous dose of 0.05 mg/kg rituximab.
From 75 μl blood, red blood cells were lysed using ACK buffer and remaining cells weretreated with FcR blocking reagent and stained with fluorochrome-labeled antibodies against
CD3 (clone SP34-2, BD Biosciences), CD4 (clone OKT4, Biolegend), and CD8 (clone SK1,
Biolegend). CD47 occupancy was determined by including labeled antibody against CD47
(clone B6H12).
Cells were analyzed on a FACS Canto II flow cytometer with subsequent analysis using
FlowJo 10.4 software. To calculate percent CD47 occupancy, the geometric mean fluorescencefor labeled ALX148 or B6H12 binding was determined. Values were normalized to those fromnaïve animals, with the CD47 signal from naïve animals being zero percent occupancy and theabsence of a CD47 signal being one hundred percent occupancy.

ALX148 serum ELISA assays
Immulon 96 well ELISA plates (Thermo Fisher Scientific) were coated overnight with hexahistidine-tagged CD47 (Alexo Therapeutics) in PBS. Plates were washed with Tris-Buffered

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Saline Tween-20 (TBST, 25 mM Tris, 0.15 M Nacl, 0.05% Tween-20, pH 7.5) and blocked
for 1 hour 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 ALX148 standard curve protein (two-fold serial dilutions in assay buffer from 250 to 2 ng/ml for mouseor 500 to 3.9 ng/ml for monkey samples) were added to blocked plates for 1 hour. Plates
were washed with TBST. Mouse samples were incubated for 1 hour with HRP-conjugatedgoat anti-human IgG (H+L) antibody (Bethyl) and washed with TBST. Monkey samples
were incubated for 1 hour with biotinylated goat anti-human IgG (H+L) antibody (Bethyl),
washed with TBST, incubated for 30 minutes with HRP-conjugated Avidin D (Vector), andwashed with TBST. All plates were incubated with 1-Step Ultra TMB ELISA solution(Thermo Fisher Scientific) and the reaction was stopped with 0.16 M sulfuric acid solution(Thermo Fisher Scientific). Plates were read at an O.D. of 450nm with a background reference reading at 570nm on a SpectraMax i3 plate reader (Molecular Devices). Protein concentrations of serum samples were interpolated from the ALX148 standard curve with a 4parameter fit curve using Prism software (GraphPad).

Immune-cell phenotyping
Immune-cell phenotyping and cytokine assessment were performed 10 days post- treatment
injection in CT26 model and three days post-last dose in MC38 model. Spleens were harvested
and processed into single-cell suspension in ice-cold PBS, lysed with ACK lysis buffer (Gibco),
washed twice and re-suspended in PBS supplemented with 2% FBS. Tumor-derived single-cellsuspensions were prepared using either the Tumor Dissociation Kit, mouse and the GentleMACS Dissociator according to the manufacturer’s instructions except 1/10 of recommended
reagent R was used or a cocktail of Collagenase I (Worthington), IV (Sigma) and DNAse(Sigma) for 45 min at 37˚C.
Cell counts were performed using ViCell counter (Beckman Coulter) for spleen and trypan
blue exclusion with hemacytometer for tumor. Aliquots of 1–3 x 106 cells were either used forcell-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 to cell-type specific antibody panels for at least 30 min at 4˚C.
Tumor and splenic suspensions were stimulated ex vivo with 10 μg/mL AH1 peptide (Anaspec), a CT26 tumor antigen, in RPMI supplemented with 10% FBS for 4 hours at 37˚C, 5%
CO2 in the presence of Golgi-Stop (BD). Identification of CT26 tumor antigen-specific CD8+ T cells was performed using PE- or APC-conjugated AH-1 MHC class 1 tetramer (MBL
MuL gp70 SPSYVYHQF) on cells directly from the spleen or on AH-1 stimulated tumor andsplenic suspensions. Ex vivo cytokine assessment was either performed following AH1 peptide
or PMA/ionomycin stimulation.
For PMA/ionomycin ex-vivo stimulation, cells were plated at 1–1.5 x 106 cells/well in complete RPMI comprised of 10% heat-inactivated FBS, 2% Pen/Strep, 1% Glutamax, 1% MEAA,
1% sodium pyruvate, 25 mM HEPES and 5μM ß-mercaptoethanol supplemented with 50 ng/
mL PMA (Fisher Scientific) and 1μM ionomycin (Sigma) in the presence of Golgi-Stop for atleast 4 hours at 37˚C, 5% CO2.
For tumor infiltrating macrophage phenotyping, single-cell suspensions were rested overnight in DMEM + 10% FBS + 1% Pen/Strep and stained for flow cytometry. M1 and M2 macrophages were determined according to the following markers- M1: CD45+CD11b+CD38+
EGR2-; M2: CD45+CD11b+CD38-EGR2+. Fluorescence-minus-one (FMO) controls wereapplied for all intracellular staining.

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Flow cytometry antibodies
Antibodies used to characterize cell surface and intracellular proteins were purchased from
Biolegend, BD Biosciences or eBioscience. Live cells were separated from the dead with LIVE/DEAD Fixable Viability Dye (Thermo Fisher Scientific). The following antibodies were used:anti-CD86 (GL-1), anti-I-A/I-E (M5/114.15.2), anti-CD8a (53–6.7), anti-33D1 (33D1), antiCD11c (N418), anti-CD45 (30-F11), anti-CCR7 (4B12), anti-CD4 (RM4-5), anti-CD3e (1452C11), anti-CD25 (PC61), anti-FOXP3 (150D), anti-CTLA4 (UC10-4B9), anti-KLRG1 (2F1/
KLRG1), anti-CD62L (MEL-14), anti-Granzyme B (QA16A02), anti-KI67 (16A8), anti-CD44(IM7), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-Ly6C (HK1.4), anti-CD38 (90), antiiNOS (CXNFT), anti-EGR2 (erongr2), anti-TNF (MP6-XT22), and anti-IFNγ (XMG1.2).
Intracellular staining was performed using FoxP3 Fixation/Permeabilization Kit per manufacturer’s instructions (Thermo Fisher Scientific).

Non-human primate toxicity study
Study was conducted at Covance Laboratories (Madison, WI, USA) in accordance with thestandard operating procedures and Good Laboratory Practices (GLP). Experimentally, naïve,social housed male and female cynomolgus monkeys of Chinese origin (n = 3/sex/group, 2–4
years of age and weighing 2–5 kg at the onset of the study) were administered ALX148 on days
1, 8, 15, 22, and 29 by a slow bolus IV injection at doses of 0, 10, 30, and 100 mg/kg. For the 0,
10, and 100 mg/kg/week dose levels, reversal of potential toxicity was evaluated following a
4-week recovery phase (2/sex/group). Standard toxicological parameters were evaluatedincluding toxicokinetics, immunogenicity, cytokine analysis, and hematology parameters.
Complete histopathology was evaluated upon completion of the dosing and recovery period.
PK parameters were determined from individual animal data using noncompartmental
analysis (WinNonlin).

Statistical analysis
Results are expressed as mean +/- SEM unless otherwise noted. Statistical analyses were either
performed using GraphPad Prism 7.0 or JMP 13.0

Results
ALX148 binds CD47 with enhanced affinity and prevents its interaction
with wild-type SIRPα
ALX148 was constructed by genetically fusing an engineered version of the CD47-binding
domain from human SIRPα to an inactive human IgG1 Fc domain (Fig 1A). ALX148 is a
78-kDa disulfide-linked homodimer that has no glycosylation sites. The affinity of ALX148 forCD47 of various species was determined using surface plasmon resonance (SPR) analysis.
ALX148 bound human, cynomolgus monkey, mouse, and rat CD47 with high affinity at 25˚C
and 37˚C (Table 1). ALX148’s affinity for human CD47 is 140 pM. The affinity of wild-type
SIRPα for human CD47 is approximately 1 μM [44]. Therefore, ALX148 represents a dramaticincrease in affinity for human CD47. The modifications to human SIRPα in ALX148 also dramatically increase the affinity for mouse CD47 to 14 nM, which allows the interrogation of
ALX148 function in mice with an intact immune system.
To confirm that ALX148 binds CD47 expressed on the cell surface, a flow cytometry assay
was used to measure the binding of fluorescently labeled ALX148 to cells (Fig 1B). As a control,
the binding of ALX180, a variant of ALX148 engineered to be deficient for CD47 binding, wasalso evaluated. ALX148 bound all cell types tested, including human and mouse tumor cell

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 1. ALX148 binds human CD47 and prevents its interaction with SIRPα. (A) Structure of ALX148 molecule.
Lines indicate disulfide bonds between ALX148 monomers. (B) Flow cytometry analysis of ALX148 binding (solid
white histograms), negative control protein ALX180 binding (dashed histograms), or background fluorescence of
unstained cells (filled histograms) for indicated human and mouse tumor cell lines. Cell number is normalized tomode. Results are representative of at least three independent experiments. (C) Flow cytometry analysis of wild-type
SIRPα binding to Jurkat cells. Geometric mean fluorescence intensity is indicated on the y-axis. Cells were preincubated with proteins indicated on the x-axis prior to SIRPα incubation. Bkgd = background fluorescence in theabsence of labeled wild-type SIRPα. Error bars indicate standard deviation of triplicates. Results are representative of
three independent experiments.
https://doi.org/10.1371/journal.pone.0201832.g001

lines. Fluorescence on cells incubated with ALX180 was the same as unstained cells, indicatingthat ALX180 does not bind cells under these conditions.
To determine if ALX148 binding to tumor cells prevents their interaction with wild-type
SIRPα, the binding of SIRPα to tumor cell lines was evaluated using flow cytometry (Fig 1C).
In this assay, binding of wild-type SIRPα to the Jurkat tumor cell line was measured with fluorescently labeled wild-type SIRPα fused to human IgG1 Fc. Binding of wild-type SIRPα tothese cells was detected, with a 19-fold increase over background fluorescence at saturating

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Table 1. Apparent affinity of ALX148 for CD47 of various species.
SpeciesKD (M), 25˚CN

KD (M), 37˚C

N

Human

1.4E-10121.0E-09

12

Cynomolgous monkey

2.0E-1016

9.6E-1016

Mouse

1.4E-086

5.8E-084Rat

8.1E-098

2.6E-08

8

N, number of independent experiments
https://doi.org/10.1371/journal.pone.0201832.t001

concentrations. Preincubation with ALX148 caused a greater than 90 percent reduction influorescence, indicating that binding of the labeled wild-type SIRPα was blocked by ALX148.
The negative control protein ALX180 did not decrease wild-type SIRPα binding. These results
demonstrate that ALX148 binding to CD47 blocks the CD47-SIRPα interaction.

ALX148 lacks effector function and binds the neonatal Fc receptor
To prevent the binding of ALX148 from having any deleterious consequences for normal
CD47-expressing cells, amino acid substitutions were made within IgG1 Fc domain of ALX148
to eliminate interaction with both FcγRs and complement C1q protein [45–48]. To confirm theabsence of ALX148 binding to FcγRs, SPR analysis was performed (Table 2). There was nodetectable interaction between ALX148 and FcγRI, FcγRIIA, FcγRIIB/C, or FcγRIIIA. This wasin contrast to ALX222, a positive control protein with wild-type IgG1 fused to a high affinity
SIRPα domain. ALX222 interacted with all FcγRs tested as expected for an IgG1 molecule.
The interaction with neonatal Fc receptor (FcRn) can impact the pharmacokinetic (PK)properties of Fc containing molecules. The substitutions in the Fc domain of ALX148 should
not impact interaction with FcRn. SPR analysis confirmed binding of ALX148 to FcRn binding
was unchanged, with a KD of approximately 580 nM. This is equivalent to the 450 nM KD
observed for ALX222, the wild type IgG1 positive control (S1 Table).
All interaction with recombinant soluble FcγRs has been eliminated from ALX148. To confirm that ALX148 has no FcγR-mediated effector activity upon cells, a cell based antibody-dependent cellular cytotoxicity (ADCC) assay was used (Figure A in S1 Fig). In this assay, Fc binding
induces signaling through FcγRIIIa, causing effector cells to express a luciferase reporter protein.
Upon binding to CD47-positive Raji cells, ALX148 failed to induce luciferase expression, similar
to a negative control IgG1 antibody that does not bind Raji cells. In contrast, the positive controls
ALX222, which binds CD47, and rituximab, which binds CD20, induced robust luciferase
expression due to their wild-type Fcs. These results demonstrate that although ALX148 binds
CD47-positive cells, it lacks effector function and does not activate ADCC pathways.
To evaluate binding of ALX148 to complement C1q protein, an ELISA assay for binding ofsoluble complement C1q to immobilized proteins was performed (Figure B in S1 Fig). No
complement C1q binding was observed for ALX148 or the negative control protein ALX217,
which contains an IgG4 Fc that has no complement C1q binding activity [51]. In contrast, theTable 2. Apparent affinity ALX148 for human Fcγ receptors.
ProteinFcγRIIIA KD (M)FcγRIIA KD (M)FcγRIIB/C KD (M)FcγRI KD (M)ALX148

NB

NB

NB

NB

ALX222

1.3E-07

4.2E-07

2.2E-06

2.9E-11

NB = No binding
https://doi.org/10.1371/journal.pone.0201832.t002

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

signal for ALX222 was approximately 7-fold over background at the maximum concentration,
indicating potent binding. These results demonstrate that binding to complement C1q has
been removed from ALX148.

ALX148 does not affect hematologic parameters in vitro or in vivo
Because CD47 is expressed on erythrocytes, molecules that bind CD47 could potentially cause
hemagglutination. ALX148, however, did not induce hemagglutination at any concentration
tested in in vitro assays with human erythrocytes. The anti-CD47 antibodies B6H12 andCC2C6 were used as positive controls, and these proteins induced hemagglutination as
expected (Fig 2A).
The ADCC and C1q binding assays described above demonstrate that ALX148 lacks Fc
effector function in vitro. To confirm the absence of effector function in an animal model,
hematological parameters were evaluated in mice after administration of ALX148 or ALX377,
a control protein with the identical SIRPα domain fused to a wild-type IgG1 Fc. This experiment was feasible due to the high affinity of the ALX148 SIRPα domain for mouse CD47
(Table 1), which confers robust binding to mouse cells (Fig 1B). Hematology for mice administered 30 mg/kg ALX148 showed levels of red blood cells (RBC), platelets, and white blood cells(WBC: lymphocytes, monocytes, and granulocytes) that were similar to the predose baseline(Fig 2B). In contrast, 30 mg/kg ALX377 resulted in 34% RBC, 70% platelet and 67% WBC
reduction 3 days post dosing as compared to baseline (p<0.0001, p<0.0001 and p<0.001
respectively). By day 12, all levels in the 30mg/kg ALX377 group returned to baseline. These
results demonstrate that the removal of effector function from ALX148 eliminates any adverse
effects on normal blood cells.

ALX148 enhances antibody-dependent phagocytosis of tumor cell lines
The elimination of tumor cells by macrophage phagocytosis is an important component of the
innate antitumor immune response. In antibody-dependent cellular phagocytosis (ADCP),
naturally occurring or therapeutically administered antibodies initiate this process by binding
target cells and engaging macrophage FcγRs. CD47 blockade with ALX148 could potentiallyenhance this process by preventing the interaction of CD47 with SIRPα on macrophages. To
determine the effect of ALX148 on ADCP, a flow cytometry-based in vitro phagocytosis assay
with human monocyte-derived macrophages was used to evaluate the combination of ALX148
with multiple therapeutic antitumor antibodies. Immunofluorcence images of macrophages
confirm the phagocytosis of whole CFSE-labeled target cells inside macrophages (Figures in S2
Fig). The antitumor antibodies trastuzumab, cetuximab, daratumumab, and obinutuzumab
had ADCP activity, causing phagocytosis of OE19, DLD-1, MM1.R, and Daudi cells, respectively (Fig 3). In each case, ALX148 enhanced the phagocytosis activity of the antitumor antibody in a dose-dependent manner. ALX180, which is deficient for CD47 binding, conferred
no increase in phagocytosis over the levels observed with antitumor antibody alone. ALX148
does not induce phagocytosis in the absence of an antitumor antibody, consistent with theelimination of effector function from the ALX148 Fc domain. These results show that ALX148
enhances the ADCP activity of multiple antitumor antibodies and that CD47 binding is essential for this enhancement.

ALX148 enhances antitumor therapy in vivo
The ability of ALX148 to enhance the activity of targeted antitumor antibodies in vivo wasassessed using mouse subcutaneous xenograft models of human tumors. In the Z138 B-cellmantle cell lymphoma model, therapeutic treatment with ALX148 in combination with

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 2. ALX148 does not affect hematologic parameters in vitro or in vivo. (A) Hemagglutination of humanerythrocytes incubated with titrations of ALX148, the indicated antibodies against CD47, or PBS. Results shown arefrom the same experiment and are representative of three independent experiments. (B) CD-1 mice were administered
30mg/kg ALX377 or ALX148 intravenously. Each time point consists of 4–5 mice. RBC, platelet (PLT) and WBC
counts for each mouse were normalized to their predose values. The mean normalized values for all mice at each time
point and standard error mean (SEM) are indicated.  p<0.0001,  p<0.001. Statistics were performed using OneWay ANOVA, Tukey-Kramer.
https://doi.org/10.1371/journal.pone.0201832.g002

obinutuzumab resulted in enhanced inhibition of tumor growth compared to mice treatedwith obinutuzumab or ALX148 alone (p<0.01) (Fig 4A). Similarly, in mice harboring OE19
gastroesophageal tumors, ALX148 in combination with trastuzumab significantly inhibitedtumor growth as compared to mice treated with trastuzumab alone in a therapeutic treatment
model (p<0.01) (Fig 4B). Lastly, the combination of ALX148 with rituximab significantly
enhanced the inhibition of tumor growth compared to treatment with rituximab alone andincreased survival as compared to PBS in Raji B-cell lymphoma tumors (p<0.001 and
p<0.0001, respectively) (Figure A in S3 Fig). These results are consistent with previous studies

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 3. ALX148 enhances antibody-dependent cellular phagocytosis. In vitro phagocytosis experiments with humanmonocyte-derived macrophages and tumor cell lines. Percent of macrophages that engulfed tumor cells is indicated on
the y-axis. On y-axis, open circle indicates background phagocytosis observed with isotype control antibody and open
square indicates cells treated with antitumor antibody alone. Antitumor antibody concentrations were constant and
ALX proteins were titrated. Cells were treated with antitumor antibody plus ALX148 (solid black line) or antitumor
antibody plus ALX180 (solid grey line). Cells were then treated with control antibody plus ALX148 (dotted black line)
or control antibody plus ALX180 (dotted grey line). Tumor cell line and antibody combinations were (A) OE19 cells
and trastuzumab, (B) DLD-1 GFP Luciferase cells and cetuximab, (C) MM1.R cells and daratumumab, and (D) Daudi
cells and obinutuzumab. Error bars represent standard deviation of triplicates. For all antitumor antibodies tested, 100
nM ALX148 significantly enhanced antibody-dependent cellular phagocytosis ( p<0.0001 one-way ANOVA,
Tukey-Kramer) compared to antibody alone. Results are representative of at least three independent experiments.
https://doi.org/10.1371/journal.pone.0201832.g003

in which the inhibition of CD47 enhanced the efficacy of rituximab against established Raji
tumors [25, 32]. Thus, ALX148 mediates significant tumor growth inhibition and longer survival when used in combination with anti-cancer antibodies at concentrations with limited
single agent efficacy.
One limitation of xenograft models is the requirement for an immunodeficient host, which
prevents the full effect of ALX148 on the immune system from being addressed. Furthermore,these models may be unusually dependent upon the CD47- SIRPα interaction due to the highaffinity of NOD-SCID SIRPα for human CD47. To assess whether ALX148 engages the adaptive arm of the immune system to potentiate antitumor activity, the efficacy of ALX148 wasinvestigated as a monotherapy and in combination with checkpoint blockade in immunocompetent syngeneic tumor models. MC38 and CT26 tumor bearing mice were treated whentumors reached an average of 70–75 mm3. ALX148 alone showed minimal single agent activity
when administered twice at 30 mg/kg, ten days apart. However, ALX148 in combination with
anti-PD-L1 showed enhanced tumor growth inhibition as compared to anti-PD-L1 monotherapy in mice bearing established MC38 tumors (p<0.05) (Fig 4C). In CT26 tumor bearing
mice, combination treatment with ALX148 and anti-PD-1 resulted in enhanced tumor growth

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 4. ALX148 enhances antitumor therapy in vivo. (A) Z138 B-cell mantle cell lymphoma and (B) OE19 gastrictumor cells were implanted subcutaneously on the right flank of NOD-SCID mice. Mice with established tumors(average of 240 mm3 for Z138 and 140 mm3 for OE19) were randomized and treated i.p. with vehicle, ALX148,
obinutuzumab for Z138 or trastuzumab for OE19 or combination of ALX148 and anti-cancer antibody. Graphs show
mean tumor growth ± SEM of n = 10 mice. ALX148 in combination with obinutuzumab or trastuzumab showedsignificant inhibition of tumor growth as compared to obinutuzumab or trastuzumab monotherapy,  p<0.01, day 32
and  p<0.01, day 36 (two-tailed student’s t-test), respectively. (C) MC38 and (D) CT26 colon carcinoma cells were
implanted subcutaneously on the right flanks of C57BL/6 and BABL/C mice, respectively. When tumors reached anaverage of 70–75 mm3, mice were randomized into groups and treated. (C) Mice bearing MC38 tumors were treatedwith PBS, ALX148, anti-PD-L1 or ALX148 + anti-PD-L1. Graphs show tumor growth ± SEM of n = 10 mice (leftpanel) and survival curves (right panel). ALX148 + anti-PD-L1 shows significant inhibition of tumor growth ascompared to anti-PD-L1 monotherapy, ( p< 0.05, day 24 two-tail student’s t-test). Survival of ALX148 + anti-PD-L1
and anti-PD-L1 alone are significantly increased compared to PBS alone ( p<0.001 and  p<0.01, respectively, logrank (Mantel-Cox) test). (D) Mice bearing CT26 tumors were treated with PBS, ALX148, anti-PD-1 or ALX148 + antiPD-1. Graphs show tumor growth ± SEM of n = 9 mice (left panel) and survival curves (right panel). ALX148 + antiPD-1 shows significant inhibition of tumor growth as compared to anti-PD-1 monotherapy, ( p< 0.05, day 25 two-tailstudent’s t-test) and significant increased survival as compared to PBS alone ( p<0.05, log-rank (Mantel-Cox) test). ns,
not significant.
https://doi.org/10.1371/journal.pone.0201832.g004

inhibition as compared to anti-PD-1 treatment group (p<0.05) (Fig 4D). Furthermore,
ALX148 extended the survival of both MC38 and CT26 tumor bearing mice when used in
combination with anti-PD-1 or anti-PD-L1 (p<0.001 and p<0.05 as compared to PBS group).
ALX148 was also tested in combination with an agonistic T-cell activator, anti-4-1BB, in CT26
tumor-bearing mice. This combination showed a significant enhancement of survival as compared to PBS (Figure B in S2 Fig). These data in immunocompetent mice show that ALX148

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

enhances antitumor activity of immunotherapeutic molecules that directly enhance T cellmediated adaptive immunity.

ALX148 reduces myeloid-driven immune suppression
Given the established effect of CD47 blockade on macrophage activity, we examined the effectof ALX148 on myeloid cells within the tumor microenvironment. In the tumor, tumor-associated macrophages (TAMs) have pro-inflammatory (M1) and suppressive (M2) activities [52,53]. To identify the effect of ALX148 on TAM populations, flow cytometric analysis was conducted on CT26 tumors isolated 10 days after administration of either ALX148 (30 mg/kg),
anti-PD-1 (5 mg/kg), a combination of ALX148 and anti-PD-1, or vehicle control (Fig 5A).
ALX148 alone resulted in a nearly 3-fold increase in the ratio of M1 to M2 macrophages andthe combination of ALX148 and anti-PD-1 resulted in a 2.5-fold increase in the ratio compared to the vehicle control (p<0.01 and p<0.05, respectively) (Fig 5B). Anti-PD-1 alone had
no effect on TAMs. In addition, there was a 1.7-fold decrease in monocytic myeloid-derived
suppressor cells (mMDSC) compared to vehicle control when ALX148 was combined with
anti-PD-1 (Fig 5C). These results suggest that, through CD47 blockade, a single dose of
ALX148 can change the composition of TAM populations toward a more anti-tumor, less suppressive phenotype and reduce mMDSCs within the tumor microenvironment.

ALX148 induces dendritic cell activation
DCs and macrophages are highly effective at phagocytosis and antigen presentation, establishing them as potent regulators of innate and adaptive immune responses. Because DCs play aparticularly important role in naïve T cell activation and initiation of antigen-specific immune
responses [54, 55], we investigated the effect of ALX148 on these cells in tumor-bearing mice.
A single combination dose of ALX148 and anti-PD-1 significantly increased the percentage of
CD8+ DCs in the spleen 2.2-fold over vehicle, while neither agent affected DCs on its own (Fig
6A). Interestingly, ALX148 alone or in combination with anti-PD-1 also leads to a nearly
4-fold decrease in the percentage of CD8- DCs, while anti-PD-1 alone had no effect (Fig 6B). A
similar decrease in CD8- DCs was previously reported in non-tumor bearing mice treated with
antibodies blocking the CD47-SIRPα interaction [37]. Furthermore, ALX148 mediated anincrease in DC activation, shown by upregulation of the activation marker CD86 in both
CD8+ and CD8- DCs, while anti-PD-1 showed no such effect (Fig 6C). These studies demonstrate that ALX148 induces DC activation and a decrease in CD8- DCs as a single agent, while
an increase in CD8+ DCs is evident when ALX148 is combined with anti-PD-1.

ALX148 bridges innate and adaptive immune responses
Because of the role played by DCs in T cell activation, we reasoned that DC activation by
ALX148 might result in subsequent enhancement of T cell responses. To investigate thishypothesis, CD4+ and CD8+ T cell populations were evaluated in the spleens of tumor bearing
mice that received ALX148 alone or in combination with anti-PD-1, which activates adaptive
T cell responses by inhibition of the PD-1 checkpoint [56]. As predicted, ALX148 alone or in
combination with anti-PD-1 mediated robust increases over vehicle control in splenic effector
memory (CD44+CD62L-) and central memory (CD44+CD62L+) CD4+ T cell populations(p<0.0001 for both subsets). Anti-PD-1 had no such effect as a single agent (Fig 7A and 7B).
Similarly, CD8+ T cells revealed increased effector memory phenotype (Fig 7C) and a significant concomitant increase in central memory CD8+ T cells (Fig 7D) in the presence of
ALX148 alone or in combination with anti-PD-1 compared to the vehicle control (p<0.001
and p<0.01, respectively). This enhanced effector function was further supported by increased

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 5. ALX148 reduces myeloid-driven immune suppression in tumor. (A) Schematic of dosing schedule and tissue
harvest for immune phenotyping. CT26 colon carcinoma cells were implanted subcutaneously in BALB/c mice andtreated on day 10 post-implantation with a single dose of PBS, ALX148, anti-PD-1 or ALX148 + anti-PD-1. 10 days
post-treatment, spleen and tumor were harvested for immune phenotyping. (B) Ratio of M1/M2 TAMs in tumor. M1
is defined as CD45+CD11b+CD38+EGR2- and M2 is defined as CD45+CD11b+CD38-EGR2+. Results are
representative of two independent experiments of n = 5–7 mice/group. (C) Percent mMDSC in tumor. mMDSC is
defined as CD45+CD11b+Ly6ChiMHCII-. Results are representative of two independent experiments of n = 5–6 mice/group.  p<0.05,  p<0.01. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
https://doi.org/10.1371/journal.pone.0201832.g005

KLRG1 expression (Fig 7E). In addition, the cytotoxic potential of these CD8+ T cells was confirmed by enhanced IFNγ and Granzyme B expression upon treatment with ALX148 alone orin combination with anti-PD-1 (Fig 7F and 7G). CT26 tumor cells express the endogenous
H2-Ld restricted antigen, AH-1 [57–59]. To identify tumor-specific immune responses,
AH1-specific CD8+ T cells were interrogated with the AH1 peptide or its corresponding
H2-Ld restricted tetramer. As expected, AH1-specific CD8+ T cells were limited to CT26
tumor-bearing mice and were absent both from mice harboring MC38 tumors, which do notpresent the AH1 peptide, and from tumor-free BALB/c or C57BL/6 mice that were used as thenegative control (Figures in S4 Fig). In CT26 tumor-bearing mice, ALX148 treatment significantly enhanced the induction of AH1+CD8+ T cells compared to the vehicle control (Fig 7H,
p<0.0001) and not in mice harboring MC38 tumors regardless of treatment (Figure B in S4
Fig). These data demonstrate that ALX148 can enhance splenic memory and cytotoxic T cell
phenotypes both as a single agent and in combination with anti-PD-1.
To provide evidence for a localized antitumor immune response, the impact on tumor infiltrating T-lymphocytes (TILs) was assessed. In general, the effects on TILs were less robust
than those observed in the spleen. Interestingly, the combination treatment of ALX148 andanti-PD-1 showed a trend towards reduced CD4+Treg infiltrates and decreased proliferative
potential as evidenced by reduced Ki67 staining (Fig 8A and 8B). No changes in either effector

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 6. ALX148 induces splenic dendritic cell activation. Spleen of CT26 tumor-bearing mice, 10 days post singledose of PBS, ALX148, anti-PD1 or ALX148 in combination with one dose of anti-PD1. (A) Percent splenic CD8+ DC
of CD45+ cells. (B) Percent splenic CD8- DCs of CD45+ cells. (C) Geometric MFI of CD86+ splenic CD8+DCs (leftpanel) and CD8-DCs (right panel). Results are representative of at least two independent experiments of n = 5–7 mice/group.  p<0.05,  p<0.01,  p<0.001 and  p<0.0001. Statistics were performed using One-Way ANOVA, TukeyKramer.
https://doi.org/10.1371/journal.pone.0201832.g006

memory (not shown) or CT26 antigen-specific AH1+ CD8+ T cells (Fig 8C) were observed
across all treated groups. However, when TILs were stimulated ex vivo with PMA and ionomycin, a significant increase in IFNγ-expressing CD8+T cells was observed in mice that received
combination treatment (p<0.05 compared to anti-PD-1 only) (Fig 8D, left panel). A similar
IFNγ expression phenotype was seen in tumor infiltrating CD8+ AH1-tet+ T cells from micetreated with ALX148 and in combination with anti-PD-1. However, this trend was not statistically significant. (Fig 8D, right panel).
A similar analysis of TILs was performed in MC38 tumor bearing mice treated with two
doses of either ALX148 alone, anti-PD-L1 alone, or both. ALX148 in combination with antiPD-L1 shows significant tumor growth inhibition as compared to anti-PD-L1 treated group(p<0.0001) (Fig 9A). Two days post last treatment of ALX148, a significant increase in tumor
infiltrating CD8+ (p<0.0001) and CD4+ T cells (p<0.0001) (Fig 9B and 9C) was seen in micereceiving combination treatment compared to vehicle control. Further profiling of these T
cells revealed an enhanced effector memory phenotype (CD44+CD62L-) (p<0.0001, both CD8
and CD4) (Fig 9D and 9E) with concomitant increase in effector function and cytotoxic potential as evidenced by increased IFNγ (p<0.0001), and granzyme B (p<0.0001) within TILs ofmice receiving both ALX148 and anti-PD-L1 compared to vehicle control (Fig 9F and 9G). In
the MC38 model, ALX148 combination therapy resulted in a more robust immune response
and greater tumor growth inhibition than in the CT26 model. This association indicates that

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 7. ALX148 activates adaptive immune response in the spleen. Spleen of CT26 tumor-bearing mice, 10 days post
single-dose of PBS, ALX148, anti-PD-1 or ALX148 in combination with one dose of anti-PD-1. (A-B) Percent effector
memory (CD44+CD62L-) and central memory (CD44+CD62L+) CD4+ T cells. (C-D) Percent effector memory andcentral memory CD8+ T cells. (E) KLRG1 expression on CD8+ T cells. (F) Percent intracellular IFNγ expressing CD8+
T cells following ex vivo stimulation of splenocytes with PMA/ionomycin and Golgi-Stop for 4 hours. (G) Percent
intracellular granzyme B expressing CD8+ T cells. (H) Percent CD8+AH1-tet+ T cells following ex vivo stimulation of
splenocytes with 10 μg/mL AH1 peptide for 4 hours. Results are representative of 1–3 independent experiments ofn = 4–6 mice/group.  p<0.01,  p<0.001 and  p<0.0001. Statistics were performed using One-Way ANOVA,
Tukey-Kramer.
https://doi.org/10.1371/journal.pone.0201832.g007

enhancement of the adaptive immune response by ALX148 may drive increased antitumor
efficacy.

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 8. Combination of ALX148 and anti-PD-1 reduces suppressive tumor microenvironment and activates
adaptive immune response in CT26 tumor. (A) Percent FOXP3+CD25+ in tumor. (B) Percent Ki67+ Tregs in tumor.
(C) Percent AH1-tet+CD8+ T cells. Following antibody treatment, ex vivo tumor-derived single cell suspension
stimulation with AH1 peptide at 10 μg/mL. (D) Percent intracellular IFNγ expressing CD8+ T cells (left panel) andpercent IFNγ+ of AH1-tet+CD8+ T cells (right panel). Following antibody treatment, ex vivo tumor-derived single cellsuspension were stimulated in the presence of PMA/ionomycin or AH1 peptide at 10 μg/mL. Results are representativeof 1–3 independent experiments of n = 5–6 mice/group.
https://doi.org/10.1371/journal.pone.0201832.g008

Collectively, these data show that ALX148 strongly activates the adaptive immune response
in an antigen-specific manner within the spleen, and that such activation may lead to anincreased antitumor response.

Higher doses of ALX148 dramatically increase serum titer and lead togreater tumor penetration
To better understand the relationship between ALX148 PK and pharmacodynamics in thetumor, ALX148 serum concentration and CD47 occupancy were analyzed in mice harboringsyngeneic tumors. In mice that did not receive ALX148, staining with labeled ALX148 revealed
greater CD47 expression on tumor cells than on CD4+ cells from the spleen or tumor (Fig
10A). In mice that received ALX148, a three-fold increase in dose from 10 mg/kg to 30 mg/kg
resulted in an approximately 100-fold increase in the ALX148 serum level at 24 hours (Fig
10B). This nonlinear increase in ALX148 exposure is consistent with a saturable CD47 antigen
sink. At the four day time point, serum ALX148 was only detected at the 30 mg/kg dose, and at
the eight day time point serum ALX148 was undetectable at either dose.

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 9. Combination of ALX148 and anti-PD-L1 enhances adaptive immune response in MC38 tumor model. (A)MC38 colon carcinoma cells were implanted subcutaneously in C57BL/6 mice and treatment was initiated four days
post-implantation. Treatment consisted of PBS, ALX148, anti-PD-L1 or ALX148 + anti-PD-L1. ALX148 was dosed
twice at 30 mg/kg, ten days agart and anti-PD-L1 was dosed twice, one per week. On day 17 post-tumor challenge,
tumors were harvested for immune phenotyping. (B-C) Percent tumor infiltrating CD8+ and CD4+ T cells + (D-E)
Percent effector memory (CD44+CD62L-) CD8 and CD4 in tumor (F) Percent IFNγ expression in CD8+ T cells (G)Percent Granzyme B expression in CD8+ T cells. All values are reported as percent of CD45 with 5–6 mice/group.

p<0.001 and  p<0.0001. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
https://doi.org/10.1371/journal.pone.0201832.g009

Measurement of CD47 occupancy demonstrated dose-dependent effects on both tissue distribution and persistence. The 10 mg/kg dose resulted in 80 percent CD47 occupancy on

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Fig 10. Higher doses of ALX148 saturate CD47 antigen sink and drive tumor penetration. (A) ALX148 binding to
the indicated cells from naïve mice. (B) Serum level of ALX148 at the indicated times after administration of 10 mg/kg(grey column) or 30 mg/kg (black column). (C-E) CD47 occupancy in splenic CD4+ T cells (black columns), tumorinfiltrating CD4+ T cells (grey columns), or tumor cells (open columns) (C) one, (D) four, or (E) eight days afteradministration of the indicated dose. Mean of five mice is shown and error bars represent standard deviation. Results
are representative of two independent experiments.
https://doi.org/10.1371/journal.pone.0201832.g010

splenic T cells twenty-four hours post-dose, while only 21 and 43 percent occupancy was observed for tumor cells and T cells, respectively, in the tumor compartment. In contrast, the 30 mg/kg
dose achieved near maximal occupancy on all cell types assayed, with 87, 85, and 84 percent occupancy detected on splenic CD4+ T cells, tumor cells, and tumor-infiltrating CD4+ T cells, respectively (Fig 10C). Analysis of CD47 occupancy four days (Fig 10D) and eight days (Fig 10E) after
dosing showed that CD47 occupancy persisted longer after the 30 mg/kg dose than the 10 mg/kg
dose, consistent with levels of ALX148 in the serum. These results indicate the CD47 antigen sinkis saturable and that nonlinear increases in exposure and high coverage of CD47 in the tumor
can be attained at higher doses of ALX148. These results also corroborated the effects describedabove of ALX148 on immune cells in the spleen and tumor compartment.

ALX148 produces full target occupancy with an acceptable PK profile andhas a favorable safety profile in non-human primates
As ALX148 binds cynomolgus monkey CD47 with high affinity, this species was used to assess
the preclinical safety of ALX148. Potential toxicity of ALX148 was assessed in all tissues,
including blood cells, in a 4 week repeat dose toxicity study. Cynomolgus monkeys weretreated by weekly intravenous injection at doses of 0 (vehicle control), 10, 30, and 100 mg/kg
for 4-consecutive weeks, followed by 4 weeks of recovery period in subset of animals at 0, 10,

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ALX148 blocks CD47 with favorable safety profile and antitumor immunityand 100 mg/kg. No toxicity or adverse findings related to CD47 blockade by ALX148 wereobserved in clinical observations, body weights, ophthalmic examinations, cytokine analysis,
ECG parameters, clinical pathology including hematological analysis of red blood cells, whiteblood cells, and platelets, and anatomical pathology assessment. The no-observable-adverseeffect-level (NOAEL) in the study was the highest tested dose of 100 mg/kg. In a separate
study, pharmacokinetics and target occupancy of ALX148 were assessed after a single dose of
10 or 30 mg/kg intravenous injection when given together with a single intravenous dose of
0.05 mg/kg rituximab. Systemic exposure to ALX148 increased in a nonlinear manner with adose increase from 10 to 30 mg/kg (Figure A in S5 Fig) and complete target occupancy wasobserved in circulating T lymphocytes within 4 hours of dosing at both dose levels. The higher
dose of 30 mg/kg conferred longer persistence of CD47 occupancy (12 days) compared to 10
mg/kg (7 days) (Figure B in S5 Fig).

Discussion
The therapeutic blockade of immune checkpoints has brought dramatic advances in anticancer therapy, leading to enhanced antitumor immunity and sustained clinical responses[2–4].
The checkpoint inhibitors in clinical use act on adaptive immunity, specifically by enhancing
antitumor T cell responses. The CD47-SIRPα axis is an additional checkpoint that acts upon
innate immunity by suppression of myeloid cell function, and several molecules to block thisinteraction have entered clinical development [40]. Here, we describe the development of
ALX148, an engineered high affinity SIRPα-Fc fusion protein that blocks the CD47-SIRPαinteraction and induces antitumor immunity by bridging innate and adaptive immune
responses while maintaining a favorable preclinical safety profile.
The majority of therapeutics in development to target CD47 bind both CD47 and, to varying extents, Fcγ receptors. Thus, these molecules induce phagocytosis both by blocking theinteraction of CD47 with SIRPα and by providing a positive signal to macrophages throughtheir Fcγ receptors. Because CD47 is also expressed at high levels on normal healthy cells, suchas platelets and erythrocytes, these molecules have the potential to induce phagocytosis of nontumor cells and result in toxicity. In fact, early clinical and preclinical studies have described
thrombocytopenia and anemia subsequent to treatment with these molecules [31, 41, 42].
ALX148 was developed based upon the finding that phagocytosis can be enhanced by a
combination approach in which CD47 blockade and Fcγ receptor engagement come from different molecules [32]. We hypothesized that CD47 blockade with a molecule containing aninactive Fc domain could enhance the activity of antitumor antibodies while maintaining afavorable safety profile. ALX148 specifically binds to CD47 with picomolar affinity and blocks
the binding of wild-type SIRPα to CD47 on cells. However, the modified Fc domain in
ALX148 is devoid of binding to Fcγ receptors and complement C1q. When ALX148 is used in
combination with tumor-specific antibodies, the active Fc domains of the latter will engage
macrophage Fc receptors, thereby providing a pro-phagocytosis signal in a targeted manner.
Thus, the inadvertent targeting of normal CD47-expressing cells should be avoided and thepotential for treatment-related toxicity is minimized.
Consistent with our hypothesis, the preclinical safety profile of ALX148 differentiates itfrom other CD47-blocking molecules in development. In contrast to the anti-CD47 antibodies
5F9 [30], B6H12, or CC2C6, ALX148 does not cause hemagglutination of human erythrocytes
in vitro. ALX148 also has no in vitro ADCC or C1q binding activity. As a single agent, ALX148
did not stimulate phagocytosis by human macrophages, confirming the elimination of functional interactions with activating macrophage Fcγ receptors. Furthermore, in a mouse hematology study, no effects on RBC, WBC, or platelets were seen after ALX148 administration,

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

while a control molecule possessing an active Fc domain caused an acute depletion of thesecells. Most importantly, no toxicity or adverse findings related to CD47 blockade by ALX148
were observed up to the highest treatment dose of 100 mg/kg in a repeat-dose nonhuman primate toxicity study. This included the absence of any adverse observations related to platelets
or erythrocytes.
Although ALX148 does not itself possess an active Fc domain, it enhances the activity ofantitumor antibodies. This is similar to previously reported CD47 blocking agents that utilize
the SIRPα D1 domain [32]. In in vitro phagocytosis assays, ALX148 enhanced the ADCP activity of anti-cancer antibodies that contain an active Fc, such as trastuzumab, cetuximab, daratumumab, and obinutuzumab, in a dose-dependent manner. Furthermore, ALX148 enhanced
the antitumor activity of obinutuzumab, trastuzumab, and rituximab in murine xenograftmodels of tumorigenesis using the human Z138, OE19, and Raji cell lines, respectively.
Because this xenograft model was established in NOD-SCID mice lacking most functional
immune cell subsets except myeloid cells, these results demonstrate that ALX148 enhances theantitumor efficacy of myeloid cells in vivo. CD47 blockade can also enhance neutrophil-mediated ADCC of cells bound by an active Fc [6, 60]. Thus, ADCC may contribute to the efficacy
observed for ALX148 and antitumor antibody combinations in xenograft models. Together,
these results support the use of ALX148 in combination with a wide range of anti-cancer
antibodies.
In addition, our studies demonstrated that blocking the CD47-SIRPα interaction enhances
the adaptive immune response, which is in agreement with an increasing body of evidence.
ALX148 binds to murine CD47 with a KD of 14 nM, a high affinity that is more than 300-foldgreater than the affinity of mouse SIRPα for mouse CD47 [61]. This potent binding allows
investigation of both antitumor activity and mechanism in immunocompetent mice. Previous
studies have relied upon in vitro differentiated macrophages and DCs to demonstrate that in
response to CD47 blockade these cells process tumor antigen and present them for priming ofcytotoxic T cells [34, 36]. In contrast, the present study directly interrogates the in vivo cellularresponse to blocking CD47.
We show that ALX148 alone or in combination with anti-PD-1 results in increased activation of both CD8+ and CD8- DCs in the spleen. This is independent of Fcγ receptors, drawing
a distinction between DC activation and the enhancement of macrophage phagocytosis. DC
activation is detectable as early as four hours (data not shown) and as long as ten days after asingle treatment with ALX148. In addition, there was a significant increase in CD8+ DCs in
the spleen of mice treated with ALX148 in combination with anti-PD-1. CD8+ DCs are the primary DC subset responsible for cross-priming of cytotoxic T cells [62]. Consistent with thisrole, increased cytotoxic CD8+ T cell numbers and cytokine production were seen with
ALX148 treatment. Interestingly, treatment of mice with ALX148 resulted in a decrease insplenic CD8- DC numbers that continued ten days after treatment. The mechanism for thisdecrease remains unclear. As the up-regulation of activation markers such as CD86 wasobserved in this population, one possibility is that this DC subset is particularly sensitive toactivation-induced cell death. Conventional DCs have a half-life of 3–6 days [63], and ALX148
occupancy of CD47 on splenic CD4+ T cells persists beyond eight days when dosed at 10 mg/
kg. Therefore, it is possible that CD8- DCs would replenish at time points beyond the ten days
examined in our study. Although the role of CD8- DCs is not well characterized, they arethought to be superior to CD8+ DCs for CD4+ T cell priming [63]. In our system, however, thedecrease in CD8- DCs did not correlate with effects on the CD4+ T cell population, since anincrease in both effector and memory T cells was observed in the spleen. Collectively, our data
show that in the spleen of mice, ALX148 induces DC activation as a single agent and that the
combination of ALX148 and anti-PD-1 mediates increased numbers of CD8+ DCs, a cell

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

population that primes cytotoxic T cells for antitumor activity. A topic of current interest is
the mechanism by which inhibition of the CD47-SIRPα interaction mediates DC activation.
Cytosolic DNA sensing by the STING pathway has been implicated in this process [34, 38],
and investigations of the effect of ALX148 on this pathway are ongoing.
Within the tumor microenvironment, macrophages are phenotypically heterogeneous andhave different functional properties with regard to tumorigenesis. While the functional classification of tissue-resident macrophages represents extremes of a continuum that is shaped by
the tumor microenvironment, there is evidence to support broad definitions of function. M1(classically activated) macrophages have been shown to exhibit strong tumoricidal activity,
while M2 (alternatively activated) macrophages are thought to have immunosuppressive functions and to mediate tumor progression [64–67]. To extend our understanding of ALX148
function beyond confirming the enhancement of macrophage phagocytosis, effects on TAM
populations were investigated in detail. Treatment with ALX148 and anti-PD-1 resulted in an
increased M1/M2 ratio among TAMs. A similar skewing of TAMs toward an M1-like phenotype subsequent to antibody-mediated CD47-SIRPα disruption has been described previously[68]. TAM repolarization is apparently independent of which arm of the CD47-SIRPα interaction is targeted, as this phenotype was also described for mice treated with an antibody against
SIRPα in a RENCA tumor model [69]. This less suppressive phenotype is consistent withenhanced tumoricidal activity. Furthermore, in the tumors of mice treated with ALX148 andanti-PD-1, we observed decreases in both mMDSC and Tregs that were not evident undermonotherapy. This reduction in immunosuppressive cells establishes a milieu for effective
tumor elimination by T cells. The combination leads to an increase in IFNγ expressing CD8+
T cells upon stimulation while the number of tumor-specific CD8+ T cells remained similar,
as in a previously reported study of checkpoint inhibitor combinations [57].
In the CT26 colon carcinoma model, TIL phenotyping was conducted following a single
dose of test articles with the goal of elucidating ALX148-driven cellular responses. These
results demonstrate that in combination with anti-PD-1, ALX148 enhances antitumor efficacy
and the persistence of antitumor immune responses. In the MC38 colon carcinoma model,
multiple doses of ALX148 and anti-PD-L1 demonstrated even stronger antitumor efficacy anda more robust cellular immune response within the tumor than that produced by a single doseof ALX148 and anti-PD-1 in the CT26 model. Additional studies are needed to elucidate thebasis for the heightened cellular response observed in the MC38 model. The second dose of
ALX148 in combination with anti-PD-L1, the decreased interval between dosing and tumor
harvest, or the variability in anti-tumor activity observed in different models may be responsible for the improved response. Alternatively, the murine IgG1 Fc of the anti-PD-L1 antibody
used here could contribute to efficacy by opsonizing tumor cells for killing. Further exploration of this would be necessary, as there have been differing reports on the effector activity ofthe murine IgG1 Fc [70, 71]. Nevertheless, ALX148 in combination with either anti-PD-1 oranti-PD-L1 is capable of driving an enhanced anti-tumor cellular response.
The widespread expression of CD47 poses a challenge to the therapeutic targeting of thismolecule because of the effect this large antigen sink will have on pharmacokinetics. Rapid
clearance of the 17 kD SIRPα D1 domain would largely hinder its therapeutic use in humans,
despite the ability to potently block the CD47-SIRPα interaction. With ALX148, fusion of thisSIRPα domain to a modified human IgG1 Fc confers increased molecular mass and an interaction with the neonatal Fc receptor, both of which are expected to extend pharmacokinetics.
Studies of ALX148 pharmacokinetics in mice and cynomolgus monkeys demonstrated anonlinear increase in ALX148 levels with increasing doses, consistent with the presence of asaturable CD47 antigen sink. In mice, higher doses of ALX148 led to increased tumor penetration, with more CD47 occupancy on tumor cells and tumor-infiltrating lymphocytes.

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Comparison of CD47 occupancy at 10 and 30 mg/kg demonstrated that greater levels of
ALX148 are required to attain full occupancy in the tumor than are required for the periphery.
Importantly, these effects were observed at well-tolerated doses of ALX148.
In both mice and monkeys, higher doses of ALX148 also led to a longer duration of CD47
occupancy. In fact, full CD47 occupancy on peripheral blood cells was maintained for twelvedays after administration of cynomolgus monkeys with a single ALX148 dose at 30 mg/kg,
with seven day occupancy observed after a 10 mg/kg dose. The pharmacokinetic and pharmacodynamic properties of ALX148 are consistent with that of an anti-CD47 antibody observedin NHP [31] and support a weekly or biweekly dosing frequency in human patients. Notably,
ALX148 is smaller than a typical antibody, with about one-half the molecular weight. This
small size may provide the additional advantages of better tissue penetration and twice thenumber of molecules at the same dosage level.
The CD47-SIRPα axis is now recognized as an important innate immune checkpoint incancer. Here, we describe the therapeutic targeting of this pathway with ALX148, which was
designed to potently block the CD47-SIRPα while avoiding toxicity to normal cells. ALX148
induces innate and adaptive immune responses, enhances preclinical anti-tumor activity in
combination with a variety of cancer therapies, and has a favorable preclinical safety profile. It
is currently being investigated in a first-in-patient, phase 1 dose escalation/expansion, multicenter study in patients with advanced solid tumors and lymphoma. (NCT03013218).

Supporting information
S1 Fig. ALX148 has no activity in assays for ADCC activity and C1q binding. (A) Cellbased ADCC activity assay. Target and effector cells were incubated with the indicatedamounts (x-axis) of different proteins. Luminescence, resulting from FcγRIIIa receptor signaling, is indicated on the y-axis. Samples were run in duplicate and averages of the duplicate values were graphed with error bars displaying standard deviation. (B) ELISA assay to measurebinding of recombinant human C1q to the indicated proteins. Quantity of C1q protein is indicated on the x-axis. Optical density resulting from C1q binding is indicated on the y-axis. Samples were run in duplicate and the average of the duplicate values is graphed with error bars
showing standard deviation.
(TIF)
S2 Fig. Fluorescence microscopy to detect phagocytosis. In vitro phagocytosis experimentwith human monocyte-derived macrophages and CFSE-labeled DLD-1 cells treated with
100nM ALX148 and 100 ng/mL of cetuximab for two hours were washed with PBS and fixedon slides. Cells were imaged using immunofluorescence microscopy to detect phagocytosis.
Bright field (A), CFSE-immunofluorescence (B), and merged images showing CFSE-labeled
DLD-1 inside macrophages as indicated by arrows (C).
(TIF)
S3 Fig. ALX148 enhances antitumor therapy in vivo. (A) Raji B-cell lymphoma cells were
implanted subcutaneously on the right flanks of NOD-SCID mice. Mice with establishedtumors (average of 85 mm3) were randomized and treated intraperitoneally with vehicle,
ALX148, rituximab, or ALX148 + rituximab. Left panel shows mean tumor growth ± SEM ofn = 10 mice and right panel shows survival curves. ALX148 in combination with rituximab
showed significant inhibition of tumor growth as compared to rituximab alone,  p<0.0001
on day 26 and significant increased survival as compared to PBS alone ( p<0.0001, logrank (Mantel-Cox) test). Mice treated with rituximab alone also had increased survival as compared to PBS, ( p<0.01, log-rank (Mantel-Cox) test). (B) CT26 colon carcinoma cells were

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

implanted subcutaneously on the right flanks of BALB/C mice. When tumors reached an average of 77 mm3, mice were randomized into groups and treated i.p. with PBS, ALX148, anti-41BB or ALX148 + anti-4-1BB. Graph shows survival curves of n = 10 mice per group. ALX148
in combination with anti-4-1BB and anti-4-1BB groups showed significant increased survival
as compared to PBS alone ( p<0.01 and  p<0.05, log-rank (Mantel-Cox) test). Results are
representative of two independent experiments.
(TIF)
S4 Fig. AH-1 specific CD8+ T- cell response in CT26 and not MC38 tumor-bearing mice.
Spleen of naïve BALB/c, CT26 and MC38 tumor bearing mice, 15 days post implantation. Cellsuspension were stained directly with AH-1 MHC I tetramer to identify antigen-specific CD8+
T cells and percent AH-1+CD8+ of CD3+ T cells are shown. (A) Spleens were harvested 10days post single dose of PBS from naïve BALB/c, CT26 and MC38 tumor-bearing mice. CT26
tumor-bearing mice show significant increase in AH-1 specific CD8+ T cells compared toboth naive BALB/c and MC38 tumor-bearing mice. (B) Spleens were harvested 10 days post
single dose of PBS or ALX148 + anti-PD-1 from naive C57BL/6 and MC38 tumor-bearing
mice. Mice treated with ALX148 in combination with anti-PD-1 do not show AH-1 specificCD8+ T cells. Results are representative of one experiment, n = 4–5 mice/group,  p<0.01. Statistics were performed using One-Way ANOVA, Tukey-Kramer.
(TIF)
S5 Fig. Pharmacokinetic and pharmacodynamic parameters of ALX148 in cynomolgus
monkeys. (A) Serum concentration of ALX148 and (B) occupancy of CD47 by ALX148 at theindicated time points in monkeys administered 30 mg/kg (black lines) or 10 mg/kg (greylines) ALX148. Curves for individual monkeys are shown. Vertical dashed lines indicate infusion of monkeys on day 1.
(TIF)
S1 Table. Apparent affinity of ALX148 for human FcRn receptor. The apparent affinity of
ALX148 for human FcRn receptor was determined using SPR as described in Materials andMethods. ALX222, which has a wildtype human IgG1 Fc domain was used as a positive control. The binding affinity of ALX148 for human FcRn is comparable to that of ALX222.
(DOCX)

Acknowledgments
We thank J. Cyster, A. Olson, D. Fontaine, M. Chang, S. Randolph, M. Warner and H. Sternfor discussion, critical reading of the manuscript, and/or technical support.

Author Contributions
Conceptualization: Steven E. Kauder, Tracy C. Kuo, Janet Sim, Jaume Pons, Hong I. Wan.
Investigation: Steven E. Kauder, Tracy C. Kuo, Ons Harrabi, Amy Chen, Emma Sangalang,
Laura Doyle, Sony S. Rocha, Janet Sim, Jaume Pons.
Methodology: Steven E. Kauder, Tracy C. Kuo, Ons Harrabi, Amy Chen, Emma Sangalang,
Janet Sim.
Project administration: Steven E. Kauder, Tracy C. Kuo, Sangeetha Bollini, Bora Han.
Supervision: Steven E. Kauder, Tracy C. Kuo, Janet Sim, Jaume Pons, Hong I. Wan.

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ALX148 blocks CD47 with favorable safety profile and antitumor immunity

Validation: Steven E. Kauder, Tracy C. Kuo, Ons Harrabi, Amy Chen, Emma Sangalang,
Laura Doyle, Sony S. Rocha, Sangeetha Bollini, Bora Han, Janet Sim, Jaume Pons, Hong I.
Wan.
Visualization: Steven E. Kauder, Tracy C. Kuo, Ons Harrabi, Amy Chen, Sony S. Rocha, Bora
Han.
Writing – original draft: Steven E. Kauder, Tracy C. Kuo, Ons Harrabi, Bora Han.
Writing – review & editing: Steven E. Kauder, Tracy C. Kuo, Ons Harrabi, Bora Han, Janet
Sim, Jaume Pons, Hong I. Wan.

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