Received: 10 December 2019

Revised: 13 June 2020

Accepted: 30 June 2020

DOI: 10.1111/trf.16009

ORIGINAL RESEARCH

Assessing and mitigating the interference of ALX148,
a novel CD47 blocking agent, in pretransfusioncompatibility testing
Tae Yeul Kim1
Hong I. Wan4

| Mi Sook Yoon2 | Hein Hustinx3 |Janet Sim4 || Hyungsuk Kim2

1

Department of Laboratory Medicine andGenetics, Samsung Medical Center, Seoul,
South Korea

2

Department of Laboratory Medicine,
Seoul National University Hospital, Seoul,
South Korea

3Interregional Blood Transfusion SRC
Ltd., Berne, Switzerland

4

ALX Oncology, Burlingame, California

Correspondence
Hyungsuk Kim, Department ofLaboratory Medicine, Seoul National
University Hospital, 101 Daehak-ro,
Jongno-gu, Seoul 03080, South Korea.
Email: hyungsuk.kim79@gmail.com
Funding information
ALX148, soluble CD47, and high-affinity
SIRP monomers were provided by ALX
Oncology.

Background
ALX148, a novel CD47 blocking agent, is in clinical development for the treatment of advanced solid tumors and lymphoma. Because CD47 is highly
expressed on red blood cells (RBCs), its therapeutic blockade can potentiallyinterfere with pretransfusion compatibility testing. This study describes theinterference of ALX148 in pretransfusion compatibility testing and evaluates
the methods used for mitigating such interference.
Study Design and Methods: Routine serologic tests were performed on six
samples from four patients treated with ALX148. Antibody screening tests
were performed on ALX148-spiked plasma, and RBC testing including antigen
typing was performed on ALX148-coated RBCs. Soluble CD47 or high-affinity
signal regulatory protein α (SIRPα) monomers were used to remove the falsepositive reactivity of ALX148-spiked plasma with or without anti-E.
Results: ALX148 caused false-positive reactivity in antibody screening using
indirect antiglobulin testing (IAT) and two-stage papain testing. However, falsepositive reactivity was not observed at the immediate spin (IS), room temperature(RT), and 37°C phases. Direct antiglobulin testing, autologous controls, and eluates showed positive results. ALX148 did not affect blood group antigen typing
performed at the IS or RT phases. The use of 50- to 100-fold molar excess of soluble CD47 or 300-fold molar excess of high-affinity SIRPα monomers removed
false-positive reactivity in IAT without affecting anti-E detection.
Conclusion: ALX148 generates false-positive reactivity in IAT, interferingwith pretransfusion compatibility testing. The use of soluble CD47 or highaffinity SIRPα monomers can resolve the interference without possibly missing
clinically significant alloantibodies.
KEYWORDS
ALX148, CD47 blocking agents, high-affinity SIRPα monomers, Hu5F9-G4, interference,
pretransfusion compatibility testing, soluble CD47

Abbreviations: IS, immediate spin; RT, room temperature; SIRPα, signal regulatory protein α; SNUH, Seoul National University Hospital.
Transfusion. 2020;1–9.

wileyonlinelibrary.com/journal/trf© 2020 AABB1

2

The use of immune checkpoint inhibitors has recently
emerged as one of the most promising approaches for cancer treatment.1 Inhibitors targeting immune checkpoint
molecules, such as cytotoxic T-lymphocyte–associated protein 4 and programmed cell death-1/programmed deathligand 1, have already been used in clinical practice for thetreatment of a broad range of cancers.2 The signal regulatory protein α (SIRPα)-CD47 pathway, an immune checkpoint in macrophages, is also drawing considerable
attention as a potential therapeutic target.3 As a marker ofself, CD47 binds to SIRPα on the surface of macrophages
to deliver an inhibitory “donʼt eat me” signal.4 Cancer cells
are thought to evade macrophage surveillance by up -regulating CD47 expression.5 Several monoclonal antibodies(MoAbs) and SIRPα-Fc fusion proteins targeting CD47 arebeing investigated in clinical trials for the treatment of solidand hematologic cancers.2
CD47 is expressed on a broad range of cell types including red blood cells (RBCs).6 Unintended binding of CD47
blocking agents to RBCs has the potential to interfere withpretransfusion compatibility testing, similar to what has
been seen with CD38 blocking agents that also bind to
RBCs.7–9 Recently, the interference of a humanizedanti-CD47 MoAb of the IgG4 isotype (Hu5F9-G4) in
pretransfusion compatibility testing has been described.10,11
Multiple rounds of adsorption with papain-treated allogeneic RBCs and/or indirect antiglobulin testing (IAT) using
Gamma-clone anti-IgG (Immucor, Norcross, GA) without
IgG4 reactivity resolved the Hu5F9-G4 interference.10 The
interference of SIRPα-Fc fusion proteins in pretransfusioncompatibility testing also needs to be characterized,
because their RBC-binding profiles could differ from thoseof anti-CD47 MoAbs.
ALX148 is a fusion protein containing a high-affinity
engineered D1 domain of SIRPα genetically linked to an
inactive human IgG1 Fc domain. Here, we describe theinterference of ALX148 in pretransfusion compatibility
testing and evaluate the various methods for mitigatingsuch interference.

1 | MATERIALS AND METHODS
1.1 | Serologic testing of patient samples
The institutional review board of Seoul National University
Hospital (SNUH) approved the retrospective chart review forthis study and the in vitro experiments using ALX148-spiked
plasma. Blood samples were collected as part of routine carefrom four patients enrolled in a phase I clinical trial of
ALX148 conducted at SNUH (NCT03013218). A baseline
type and screen was performed before initial ALX148 infusion. After ALX148 administration, serologic tests were

KIM ET AL.

conducted on patient samples as per the standard protocol of
the SNUH blood bank. ABO forward grouping was performed using the tube method with anti-A and anti-B
reagents (SIHDIA, Shinyang Diagnostics, Siheung, Korea).
When a weak ABO subgroup was suspected, the RBCs wereadditionally typed using the tube method with anti-A1 andanti-H lectins (Lorne Laboratories, Reading, UK). ABO
reverse grouping was performed using the tube method with
A1 and B cells (HEMATEST, Diagast, Loos, France). The D
phenotype was determined using the immediate spin(IS) tube method with two anti-D reagents (TOTEM, humanmonoclonal IgM/IgG blend, Diagast; SIHDIA, human monoclonal IgM/IgG blend, Shinyang Diagnostics). Antibody
screening was carried out using the tube method with a twocell panel (Selectogen I-II, Ortho-Clinical Diagnostics, Raritan, NJ) at room temperature (RT), 37°C, and albumin-IAT
phases with polyspecific antihuman globulin reagents (antiIgG and anti-C3d, Diagast). Antibody screening was also
conducted using gel IAT (ID-Card LISS/antiglobulin test,
BIO-RAD, Cressier, Switzerland) with a two-cell panel (IDDiaCell I-II, BIO-RAD). Antibody identification was performed using gel IAT with an 11-cell panel (ID-DiaPanel,
BIO-RAD). Direct antiglobulin testing (DAT) was performed
using the gel method (ID-Card LISS/antiglobulin test). If thepolyspecific DAT was positive, the patient RBCs were further
tested using the gel method with monospecific anti-IgG andanti-C3d (DC-Screening II, BIO-RAD).
Samples from two patients (Samples 3-2 and 4-2) weresent to a reference laboratory (Interregional Blood Transfusion SRC Ltd., Berne, Switzerland) for further serologic testing. Polyspecific DAT was performed using the gel method(ID-Card LISS/antiglobulin test). Monospecific DAT was
performed using ID-Card antiglobulin test anti-IgG (BIORAD) and the neutral gel card (BIO-RAD) with anti-C3d(Dako, Hamburg, Germany). Eluates were prepared usingacid elution (BAG-Elutions-Kit, BAG Health Care, Lich,Germany) and tested using gel IAT (ID-Card LISS/antiglobulin test). In-house RBC panels were used for antibody
screening and identification. Antibody screening was performed using the gel method (ID-Card LISS/antiglobulintest) and the tube method. The tube method was performedat the IS and low-ionic-strength solution (LISS)–IAT phases
using two types of anti-IgG reagents: anti-IgG with reactivity
against all human IgG subclasses (Sanquin Reagents,
Amsterdam, the Netherlands) and anti-IgG without IgG4
reactivity (Gamma-clone). Additionally, 0.2 M dithiothreitol(DTT)-treated screening RBCs were used for tube LISS-IATwith Gamma-clone anti-IgG. Antibody identification was
performed using gel IAT. Antibody identification was also
performed using a papain-pretreated RBC panel and theneutral gel card. Antibody titration and allogeneic adsorption were carried out only in Sample 3-2. Patient plasma
serially diluted with phosphate-buffered saline (PBS) at

KIM ET AL.

pH 7.0 to 7.2 was tested against a pool of R1R1, R2R2, and rr
RBCs using tube LISS-IAT with Gamma-clone anti-IgG.
Patient plasma underwent five rounds of allogeneic
adsorption using ZZAP-treated phenotype-matched RBCs
(R1r, K–). Equal volumes of ZZAP-treated RBCs, patientplasma, and LISS were mixed and incubated at 37°C for
15 minutes with periodic agitation. The mixture wascentrifuged for 5 minutes, and the adsorbed plasma/
LISS mixture was harvested. After four additionalrounds of adsorption, the adsorbed plasma was tested
using tube LISS-IAT with Sanquin anti-IgG or Gammaclone anti-IgG. The agglutination strength was graded
as 0, 0.5+, 1+, 2+, 3+, or 4+.

1.2 | Serologic testing of ALX148-spiked
plasma and ALX148-coated RBCs
Discarded plasma samples (group AB with a negative antibody screen or with anti-E) were used for these experiments. Plasma with a negative antibody screen was pooled
and spiked with ALX148 at final concentrations of 0.1, 1.0,
10.0, 100.0, and 500.0 μg/mL. The highest concentration(500.0 μg/mL) was expected to be above the plasma concentrations reported in patients receiving ALX148 at
10 mg/kg weekly.12 To describe ALX148 interference in
ABO, D, and extended blood group antigen typing, in vivo
binding of ALX148 to RBCs was mimicked in vitro by
incubating test RBCs (group O) with 500.0 μg/mL
ALX148-spiked plasma (group AB) at 37°C for 15 minutes,
followed by three washes with saline. The test RBCs used
for this experiment were Selectogen I-II and two D– panel
cells from Resolve Panel A (Ortho-Clinical Diagnostics).
After incubation with ALX148, DAT was performed using
the tube method with Diagast anti-IgG to evaluate whether
the test RBCs were actually coated with ALX148. ABO forward typing was performed on the ALX148-coated RBCs
using the tube method with SIHDIA anti-A and anti-B
reagents. ABO reverse typing was performed on 500.0 μg/
mL ALX148-spiked plasma using the tube method with
HEMATEST A1 and B cells. ABO forward and reverse typing
was also performed using the gel method (DiaClon ABO/D+ Reverse Grouping, BIO-RAD). The ALX148-coated RBCs
were tested for the D phenotype using the tube method with
anti-D TOTEM and SIHDIA anti-D at the IS and IAT phases.
Before and after ALX148 coating, extended blood group antigen typing was conducted using the tube method with
anti-C, -E, -c, -e, -K, -Jkb, -N, and -P1 reagents at the IS phase;anti-Lea and -Leb reagents at the RT phase; and anti-Fya,-Fyb, -S, and -s reagents (Diagast) at the IAT phase.
To describe ALX148 interference in antibody screening, ALX148-spiked plasma was tested using gel IAT(ID-Card LISS/antiglobulin test) with ID-DiaCell I-II and3

the tube method with Selectogen I-II at the IS, RT, 37°C,
and IAT phases. Tube IAT was performed using Diagast
anti-IgG without enhancement media or with polyethylene glycol (PEG), LISS, or albumin enhancement.

1.3 | PEG adsorption
To reduce the effects of ALX148 on IAT, six rounds of
PEG adsorption were performed on ALX148-spiked
plasma using allogeneic R1R1, R2R2, and rr RBCs
according to the method described in the AABB Technical
Manual.13 Briefly, equal volumes of untreated RBCs,
500.0 μg/mL ALX148-spiked plasma, and PEG weremixed and incubated at 37°C for 15 minutes with periodic agitation. After 5 minutes of centrifugation, theadsorbed plasma/PEG mixture was harvested. Five additional rounds of adsorption were carried out, and theadsorbed plasma was tested using tube IAT with Diagast
anti-IgG. As a dilution control, equal volumes of
500.0 μg/mL ALX148-spiked plasma and PEG weremixed, incubated at 37°C for 15 minutes in the absenceof adsorbing RBCs, and tested in the same manner.

1.4 | Mitigation using soluble CD47
or high-affinity SIRPα monomers
Mitigation studies were performed utilizing soluble
human CD47 or high-affinity SIRPα monomers. Soluble
CD47 and high-affinity SIRPα monomer production
methods are detailed in Supplementary Methods (available as supporting information in the online version ofthis paper). First, we prevented the binding of ALX148
to RBCs by neutralizing the ALX148 in plasma.
ALX148-spiked plasma samples were incubated with
10-, 30-, and 50-fold molar excess of soluble CD47 at RTfor 30 minutes. Antibody screening using gel IAT with
ID-DiaCell I-II was performed on the ALX148-spiked
plasma preincubated with soluble CD47. When positive
reactivity in the antibody screening remained, an additional neutralization step using 100-fold molar excess ofsoluble CD47 was performed. Second, we inhibited thebinding of ALX148 to RBCs by masking the drug bindingsite on the RBCs. Screening cells (ID-DiaCell I-II) wereincubated with 10-, 50-, 100-, and 300-fold molar excess ofhigh-affinity SIRPα monomers at RT for 30 minutes undercontinuous shaking at 600 rpm. Antibody screening using
gel IAT with the preincubated screening cells was performed on ALX148-spiked plasma. We finally demonstrated
the utility of the above described mitigation strategies
in ALX148-spiked plasma containing anti-E. Antibody
screening using gel IAT with a three-cell panel (ID-DiaCell

4-2b

Abbreviations: Auto, autologous control; ID, identification; mf, mixed-field agglutination; NT, not tested; Poly, polyspecific; PR, panreactive; QW, every week.
a
The patientʼs RBCs showed 4+ reactivity with anti-A, 3+mf reactivity with anti-B, no reactivity with anti-A1, and 2+ reactivity with anti-H. The patientʼs plasma exhibited no reactivity with
A1 and B cells.
bThese samples were sent to Interregional Blood Transfusion SRC Ltd. for further serologic testing.
c
The results were same for Sanquin anti-IgG and Gamma-clone anti-IgG.

PR (3+)
Poly (4+), IgG (4+), C3d (0) 2+
PR (3+)
PR (2+)c
PR (3+)

NT
Poly (4+), IgG (4+), C3d (0) 2+
PR (3+)
PR (2+)
4

3-2b

4-1

10 mg/kg ALX148 QW Group O, D+
with pembrolizumab

PR (3+)

PR (3+)
Poly (4+), IgG (4+), C3d (0) 2+
PR (3+)
PR (2+)c
PR (3+)

NT
Poly (4+), IgG (4+), C3d (0) 2+
PR (2+)
3-1
3

10 mg/kg ALX148 QW
with rituximab

Group AB (A2B3a), D+ PR (3+)

PR (3+)

Poly (3+), IgG (3+), C3d (0) 2+
PR (3+)
PR (2+)
PR (3+)
Group O, D+
2
2

10 mg/kg ALX148 QW
with rituximab

Poly (4+), IgG (4+), C3d (0) 2+
PR (3+)
PR (2+)
PR (3+)
10 mg/kg ALX148 QW Group AB, D+
with pembrolizumab
1Tube IAT

Antibody screening

Gel IATABO/D

Treatment regimens and serologic test results of four
patients treated with ALX148 are summarized in Table 1.
ALX148 interference in ABO forward typing was evaluated in group O RBCs from Patients 2 and 4. ALX148
interference in ABO reverse typing was investigated ingroup AB plasma from Patients 1 and 3. In all patients,
ABO forward and reverse typing gave the same results
before and after ALX148 administration. All samples
showed a negative antibody screen before the beginningof ALX148 treatment. After ALX148 administration, allsamples showed panreactivity in both gel and tube IAT(Table 1) but nonreactive results at other phases (IS, RT,
and 37°C) of the tube method. Of note, panreactivity (2+)
was observed in tube IAT with Gamma-clone anti-IgG aswell as with Sanquin anti-IgG (Table 1). In Samples 3-2

Patient Sample ALX148 regimen

2.1 | Interference in ALX148-treatedpatient samples and mitigation methods

Treatment regimens and serologic test results of four patients treated with ALX148

2 | R E SUL T S

TABLE 1

Flow cytometry was performed to assess ALX148 binding totest RBCs and ability of soluble CD47 or high-affinity SIRPαmonomers to reduce such binding. The following three mixtures were prepared: 25 μL of ALX148-spiked plasma (0.1,
1.0, 10.0, 100.0, and 500.0 μg/mL ALX148) or normal plasma
with a negative antibody screen was added to 50 μL of test
RBCs (ID-DiaCell I); 25 μL of ALX148-spiked plasma(500.0 μg/mL) preincubated with 10-, 30-, and 50-fold molar
excess of soluble CD47 was added to 50 μL test RBCs; and25 μL of ALX148-spiked plasma (500.0 μg/mL) was added to
50 μL test RBCs preincubated with 10-, 50-, 100-, and300-fold molar excess of high-affinity SIRPα monomers. The
mixtures were incubated at 37°C for 15 minutes, washedfour times with PBS, incubated at 4°C for 30 minutes with
0.5 μL of FITC-conjugated F(abʼ)2 fragment goat anti-human
IgG (Jackson ImmunoResearch, West Grove, PA), andwashed twice with PBS. A total of 20,000 events per sample
were acquired on a flow cytometer (FACSCanto II, BD Biosciences, San Jose, CA), and RBCs were gated by forward
and side scatter parameters.

Antibody ID gel IAT Gel DAT

1.5 | Flow cytometry analysis

1

Auto tube IAT Elution gel IAT

I-II-III) was performed on prespiked plasma (containing
anti-E only) to measure the original anti-E reactivity.
Anti-E titration was not performed in the prespiked plasma.
Before and after soluble CD47 (50-fold molar excess) andhigh-affinity SIRPα monomers (300-fold molar excess) wereused, antibody screening was performed on 500.0 μg/mL
ALX148-spiked plasma containing anti-E.

NT

KIM ET AL.

NT

4

KIM ET AL.

5

and 4-2, antibody screening using tube IAT with DTTtreated RBCs exhibited panreactivity (2+). Antibody identification using the neutral gel card with papain-treated
RBCs showed panreactivity (4+). The ALX148 plasma
titer was higher than 4096 in Sample 3-2. Despite fiverounds of adsorption using ZZAP-treated allogeneic RBCs
on plasma from Sample 3-2, 2+ reactivity remained intube IAT with Sanquin and Gamma-clone anti-IgG.

2.2 | The effects of ALX148 on ABO, D,
and extended blood group antigen typing
Test RBCs (group O) coated in vitro with ALX148 showedstrong reactivity (4+) in DAT but exhibited no reactivity
with anti-A and anti-B reagents in ABO forward typing
using the tube and gel methods. A quantity of 500.0 μg/mL
ALX148-spiked plasma (group AB) exhibited no reactivity
with A1 and B cells in ABO reverse typing using the tube
and gel methods. ALX148-coated D– RBCs displayed noreactivity with anti-D reagents at the IS phase. In all
extended blood group antigens tested at the IS or RT
phases, antigen-negative RBCs coated with ALX148 showedno reactivity with the corresponding antigen typing reagent.
When extended blood group antigen typing was performedon antigen-positive RBCs, no notable differences in agglutination strengths between pre- and post-ALX148 coatingwere observed. However, the 4+ DAT caused false-positive
reactivity in all antigens tested at the IAT phase.
TABLE 2CellsaI

ALX148 (μg/mL)
0.1

1.0

II
I

10.0

II
I

100.0

II
I

ALX148-spiked plasma exhibited panreactivity in both
gel and tube IAT (Table 2). The strengths of agglutinationin tube IAT were same (2+) regardless of the presence ofan enhancement medium. However, agglutination was
not observed in any concentration of ALX148-spiked
plasma at the IS, RT, and 37°C phases. When
ALX148-spiked plasma was incubated with test RBCs,
the cell surface binding of ALX148 was confirmed using
flow cytometry analysis (Figure 1). Binding of 0.1 μg/mL
ALX148 to test RBCs was much weaker than that of
ALX148 at higher concentrations (Figure 1), but it wasstrong enough to induce 2+ reactivity in both gel andtube IAT (Table 2). Even after six rounds of PEG adsorption using untreated allogeneic RBCs, the adsorbed
plasma showed 2+ reactivity in tube IAT. The dilution
control of PEG adsorption also exhibited 2+ reactivity intube IAT. In contrast, ALX148-induced false-positive
reactivity was completely mitigated using 50- to 100-foldmolar excess of soluble CD47 or 300-fold molar excess ofhigh-affinity SIRPα monomers (Table 2). The use of mitigating agents at insufficient concentrations exhibited apartial reduction of the false-positive reactivity in a
gradient-dependent manner. Flow cytometry analysis
revealed that the use of soluble CD47 or high-affinity
SIRPα monomers reduced the binding of ALX148 to test
RBCs (Figure 1). The effects of ALX148 on IAT wereMitigation of interference in ALX148-spiked plasma with or without anti-E

III

2.3 | Interference in ALX148-spiked
plasma and mitigation methods

500.0

Tube IAT

Gel IAT

Gel IAT (soluble CD47)

Gel IAT (high-affinity SIRPα monomer)

50×

100×

100×

300×

2+

2+

0.5+

0

1+

0

2+

2+

0.5+

0

1+

0

2+

2+

0.5+

0

1+

0

2+

2+

0.5+

0

1+

0

NT

1+

0

1+

0

1+

0

1+

0

1+

0

0.5+

0

NT

0

2+

3+

0

2+

3+

0

2+

3+

0

2+

3+

0

NT

2+

3+

0

2+

3+

0

NT

3+

0

II

3+

1+

1+

III

3+

0

0

II
I

b

500.0 (with anti-E )NT

NT

Abbreviation: NT, not tested.
a
The Rh phenotypes of RBCs used for antibody screening tests were as follows. Screening cell I, R1R1; screening cell II, R2R2; screening cellIII, R1R1.
b
Anti-E in prespiked plasma showed 1+ reactivity with screening cell II (R2R2) in gel IAT.

6

KIM ET AL.

FIGURE 1

The binding of ALX148 to test RBCs and its reduction using soluble CD47 or high-affinity SIRPα monomers. In thehistograms, the x-axis represents fluorescent intensity, while the y-axis represents the number of cells. A, Flow cytometry shows that
ALX148 binds to test RBCs (black and gray indicate binding of normal plasma and ALX148-spiked plasma, respectively). B and C, Flowcytometry reveals that using, B, soluble CD47 or, C, high-affinity SIRPα monomers diminishes ALX148 binding to test RBCs (black, gray,and stripe indicate binding of normal plasma, ALX148-spiked plasma, and ALX148-spiked plasma after using the mitigation measure,respectively)

selectively eliminated by using soluble CD47 or highaffinity SIRPα monomers, allowing accurate identification of the underlying anti-E (Table 2).

3 | DISCUSSION
ALX148 is currently being evaluated in a clinical trial asa monotherapy or in combination with pembrolizumab,
trastuzumab, or rituximab for the treatment of advanced
solid tumors and lymphoma (NCT03013218).12 However,anti-IgG used in routine serologic testing can interact
with the Fc domain of ALX148 bound to the RBC membrane, thereby having the potential to induce RBC agglutination in IAT. Interference with pretransfusioncompatibility testing can potentially complicate the safe

and timely provision of blood products. Thus, there is anurgent need for assessing the potential of a new drug tointerfere with pretransfusion compatibility testing during
the early phases of drug development and introducing
effective strategies to circumvent such interference.14
Spontaneous RBC agglutination might produce falsepositive reactivity in blood group antigen typing. While
RBCs from patients treated with Hu5F9-G4 showed weak
spontaneous agglutination,10 human RBCs incubatedwith varying concentrations of ALX148 in vitro did not
agglutinate spontaneously.15 Hence, we presumed that the
impact of ALX148 on blood group antigen typing performed at the IS or RT phases would be minimal, if any atall. Indeed, our study involving samples from four patientstreated with ALX148 as well as artificial samples(ALX148-coated RBCs and ALX148-spiked plasma

KIM ET AL.

prepared in vitro) demonstrates that ALX148 does notinterfere with blood group antigen typing performed at the
IS or RT phases. To provide extended antigen-matched
RBC units, it would be prudent to obtain a baseline
ABO, D, and extended blood group antigen profile before
initiating ALX148 therapy. After ALX148 therapy has
begun, along with genotyping, phenotyping with antigen
typing reagents used at the IS or RT phases can also be performed as long as the patient has not been recently
received a transfusion.
As seen with CD38 blocking agents, including
daratumumab,7–9 CD47 blocking agents generate panreactivity in IAT, interfering with pretransfusion compatibility testing.10,11,16,17 While plasma from patientstreated with daratumumab typically shows weak reactivity in IAT,18 plasma from patients treated with
Hu5F9-G4 or ALX148 exhibits strong reactivity in
IAT.10,16,17 Similarly, in our study, strong reactivity (3+)
in gel IAT was observed in the plasma from all four
patients treated with ALX148. Retesting patient samples
that initially exhibit daratumumab interference in gel
IAT or solid-phase testing with the tube method can partially obviate the need for routine DTT treatment,
because the tube method may not be able to detect theeffects of daratumumab on IAT in some cases.19 However, our study and that of Velliquette et al.10 reveal thatthe panreactivity in IAT caused by ALX148 or Hu5F9-G4
cannot be prevented using the tube method. Experiments
using ALX148-spiked plasma suggest that even very low
concentrations of ALX148 in patient plasma can bind totest RBCs strongly, generating 2+ reactivity in both gel
and tube IAT. Thus, panreactivity in IAT might persist
for a period of time after the last ALX148 infusion. The
greater reactivity of CD47 blocking agents compared tothat of CD38 blocking agents is probably due to the factthat CD47 is expressed at higher levels than CD38 in the
RBC membrane.10 It should be noted that the reactivityof CD47 blocking agents may vary with the Rh phenotype of RBCs used for pretransfusion compatibility testing, because CD47 expression levels differ among
different Rh phenotypes.10 In this study, when antibody
screening and identification tests were performed on
plasma samples from patients treated with ALX148, thereactivity with all reagent RBCs, including rr RBCs whichhave the highest CD47 expression, was consistent instrength and phase regardless of their Rh phenotypes.
Velliquette et al. reported that the use of D– – and Rhnull
RBCs with markedly decreased CD47 expression failed tosignificantly reduce false-positive reactivity induced by
ALX148.16 The potential influence of Rh phenotypes on
the mitigation strategies used in this study (soluble CD47
and high-affinity SIRPα monomers) requires further
investigation as we utilized only R1R1 and R2R2 cells.

7

Patterns of interference in pretransfusion compatibility testing can differ among CD47 blocking agents
depending on their drug classes, IgG subclasses, andRBC-binding capacity. For example, Hu5F9-G4 interfereswith ABO typing and all phases of antibody screening,
including IS.10 However, as seen with daratumumab,18
ALX148 interferes with antibody screening at the IAT
phase but does not affect other phases of antibody screening as well as ABO typing. The comparison of interference in pretransfusion compatibility testing andmitigation strategies between Hu5F9-G4 and ALX148 isshown in Table 3. Although denaturing CD47 on test
RBCs may be a universal approach to overcome the interference of all CD47 blocking agents, CD47 is not denatured by DTT or proteolytic enzymes such as papain.10
Our study demonstrated that ALX148 reacted with 0.2 M
DTT-treated RBCs in tube IAT and two-stage papaintreated RBCs in the neutral gel card. Velliquette et al.16
also observed that ALX148 reacts with two-stage papaintreated RBCs at the IAT phase. Alternatively, adsorption
techniques can be viable options to negate the interference of CD47 blocking agents owing to the high expression of CD47 on adsorbing RBCs. Multiple rounds ofadsorption using papain-treated allogeneic RBCs (R1R1,
R2R2, and rr) successfully eradicated panreactivity ofpatient plasma induced by Hu5F9-G4.10 Our study
showed that five rounds of adsorption using ZZAPtreated allogeneic RBCs (R1r) failed to remove plasma
panreactivity in a patient treated with ALX148, and six
rounds of PEG adsorption using untreated allogeneic
RBCs (R1R1, R2R2, and rr) failed to eliminate thepanreactivity of 500.0 μg/mL ALX148-spiked plasma.
However, Velliquette et al.16 reported that plasma
panreactivity was eliminated by six rounds of adsorption
using papain-treated allogeneic RBCs (rr) in a patient
treated with ALX148, and the elimination might havebeen attributed to the short incubation time (10 minutes).
Although allogeneic adsorption may be effective in eliminating ALX148-induced panreactivity, it is laborious andtime-consuming for routine clinical use and carries therisk of adsorbing alloantibodies against high-frequency
antigens. The use of anti-IgG without IgG4 reactivity, such
as Gamma-clone anti-IgG, proves effective in nullifying
the effects of IgG4-based CD47 blocking agents on IAT.10
In contrast, this mitigation method does not work against
IgG1-based CD47 blocking agents, including ALX148.
The interference of CD47 blocking agents in
pretransfusion compatibility testing can be avoided byneutralizing the drug in plasma or masking the drug
binding site on RBCs. We demonstrated that the bindingof ALX148 to test RBCs could be selectively inhibitedusing an excess of soluble CD47 or high-affinity SIRPαmonomers. A disadvantage of these mitigation methods

8

KIM ET AL.

Comparison of interference in pretransfusion compatibility testing and mitigation strategies between Hu5F9-G410,11 and
ALX148 (our study)15-17

TABLE 3

Hu5F9-G4

ALX148Drug type

MoAb

Fusion protein

IgG subclass

IgG4

Engineered IgG1

Spontaneous agglutination

Observed

Not observed

ABO typing

Observed

Not observed

D and extended blood group antigen typing

Possible at IS and IAT phase

Observed at IAT phase

Antibody screening and identification

Observed at all phases

Observed at IAT phasea

DAT/autologous control

Negative or weakly positive

Positive

Eluate IAT

Positive

Positive

DTT or enzyme treatment

Not effective

Not effective

IAT using anti-IgG without IgG4 reactivity

Effective

Not effective

Multiple rounds of allogeneic adsorption

Effective

Variableb

Soluble CD47

Probably effective (not tested)

Effective

High-affinity SIRPα monomers

Probably effective (not tested)

Effective

Interference

Mitigation strategiesaExceptionally, plasma strongly reacted with papain-treated RBCs in the neutral gel card.
The effectiveness of allogeneic adsorption can vary depending on many factors, including the number of adsorption procedures, enzyme
treatment and Rh phenotype of adsorbing RBCs, plasma concentration of ALX148, enhancement medium, and incubation time.

bis that large volumes of soluble CD47 or high-affinity
SIRPα monomers are required to mitigate the interference induced by high concentrations of ALX148. In thisstudy, when 50-fold molar excess of soluble CD47 or
300-fold molar excess of high-affinity SIRPα monomers
were used to eradicate the false-positive reactivity of
500.0 μg/mL ALX148-spiked plasma, the total incubation
volume in gel IAT increased by almost 50% (solubleCD47) or 200% (high-affinity SIRPα monomers).
Although such increases did not affect anti-E detection inthis study, it is important to understand that adding soluble CD47 or high-affinity SIRPα monomers carries therisk of diluting out very weakly reactive clinically significant alloantibodies. One peculiar finding was that theminimum molar excess of soluble CD47 needed for neutralization was different at low and high concentrationsof ALX148 (100-fold vs 50-fold). Such different neutralization efficiencies may be explained by the collision theory, stating that as reactant concentration increases, thenumber of collisions between reactant molecules
increases, leading to a faster rate of reaction. The use ofsoluble CD47 or high-affinity SIRPα monomers could bea universal solution to the interference caused by any
CD47 blocking agent, although both methods need to bevalidated for each CD47 blocking agent. Another alternative is the use of pepsin- or papain-digested CD47 blocking agents lacking the Fc domain. This approach has
only been validated for daratumumab20,21 but could

potentially work for CD47 blocking agents. A panel ofantigen typed RBCs with decreased CD47 expression may
be useful to minimize the effects of CD47 blocking
agents. Unlike daratumumab interference, which can be
mitigated using a panel of antigen-typed cord RBCs withextremely low CD38 expression,22 the interference
induced by Hu5F9-G4 or ALX148 cannot be resolved
using RBCs with significantly decreased CD47 expression, such as D– – and Rhnull RBCs.10,16 Finally, the provision of phenotypically or genotypically matched RBC
units is a general solution to address drug interference inpretransfusion compatibility testing. The major limitations are that extended phenotype or genotype matching
is not available in many countries, and even if available,
clinically significant antibodies might be missed,
depending on the degree of matching.9
In summary, we demonstrated that ALX148 binds to test
RBCs, thereby interfering with the IAT phase of
pretransfusion compatibility testing, although it does not confound tests performed at other phases, including IS and
RT. Various methods were investigated to mitigate
ALX148-induced interference. The use of soluble CD47 or
high-affinity SIRPα monomers proves effective in resolving
the interference without possibly missing clinically significant
alloantibodies. Phenotyping of patient RBCs for extended
antigen-matched blood transfusion can be accomplishedusing antigen typing reagents at the IS or RT phases, evenafter ALX148 administration. The increasingly widespread

KIM ET AL.

use of MoAbs and fusion proteins with unintended binding to
RBCs poses a growing challenge to blood banks. International
efforts should be undertaken to investigate the potential ofeach drug to interfere with pretransfusion compatibility testing and establish a guideline to solve the problem.
A C K N O WL E D G M E N T
The authors thank Editage (www.editage.co.kr) for
English language editing.
CONFLICT OF INTEREST
Janet Sim and Hong I. Wan are employed by ALX Oncology. The other authors declare no potential conflict ofinterest.
ORCID
Tae Yeul Kim https://orcid.org/0000-0002-6405-5305
Hyungsuk Kim https://orcid.org/0000-0002-0574-9200
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SU PP O R TI N G I N F O RMA TI O N
Additional supporting information may be found online
in the Supporting Information section at the end of thisarticle.
How to cite this article: Kim TY, Yoon MS,
Hustinx H, Sim J, Wan HI, Kim H. Assessing andmitigating the interference of ALX148, a novel
CD47 blocking agent, in pretransfusioncompatibility testing. Transfusion. 2020;1–9.
https://doi.org/10.1111/trf.16009