Letters
https://doi.org/10.1038/s41587-019-0135-x
A coiled-coil masking domain for selective
activation of therapeutic antibodies
Vivian H. Trang1, Xinqun Zhang1, Roma C. Yumul2, Weiping Zeng2, Ivan J. Stone2, Serena W. Wo1,
Melissa M. Dominguez1, Julia H. Cochran1, Jessica K. Simmons2, Maureen C. Ryan3, Robert P. Lyon1,
Peter D. Senter4 and Matthew R. Levengood 1*
The use of monoclonal antibodies in cancer therapy is limited
by their cross-reactivity to healthy tissue. Tumor targeting
has been improved by generating masked antibodies that are
selectively activated in the tumor microenvironment, but each
such antibody necessitates a custom design. Here, we present
a generalizable approach for masking the binding domains of
antibodies with a heterodimeric coiled-coil domain that sterically occludes the complementarity-determining regions.
On exposure to tumor-associated proteases, such as matrix
metalloproteinases 2 and 9, the coiled-coil peptides are
cleaved and antigen binding is restored. We test multiple
coiled-coil formats and show that the optimized masking
domain is broadly applicable to antibodies of interest. Our
approach prevents anti-CD3-associated cytokine release in
mice and substantially improves circulation half-life by protecting the antibody from an antigen sink. When applied to
antibody–drug conjugates, our masked antibodies are preferentially unmasked at the tumor site and have increased antitumor efficacy compared with unmasked antibodies in mouse
models of cancer.
Antibody therapeutics can be associated with substantial side
effects when target antigens are not exclusively expressed on the
tissue of interest1,2. Targets with abundant expression in diseased
tissues but low normal tissue expression have generally proven
difficult to identify, prompting the development of strategies that
harness other differences between tissues. One recent example has
been the development of antibody prodrugs, in which a masking
group is fused to the antibody to restrict binding in healthy tissues
while leveraging disease-associated proteases to remove the mask in
diseased environments3. This approach requires a custom mask for
each antibody4–11, which limits rapid application and leads to uncertainties regarding expression, pharmacokinetics, immunogenicity,
activation and stability. Here, we set out to design a more generalizable approach to antibody masking that could potentially address
these limitations.
The N-terminal residues of the light and heavy chains of a typical IgG are separated by approximately 30–35 Å and flank the complementarity-determining regions (CDRs) that form the paratope
responsible for imparting antigen specificity (Fig. 1a). We reasoned
that fusion of a peptide domain with propensity to form robust
secondary structure could sterically block the CDRs from binding
antigen. We focused on leucine zipper coiled-coils because they are
highly tunable peptide domains that have been used extensively for
antibody-engineering approaches, and some of these domains are
derived from native human proteins (Supplementary Table 1)12. As
an initial test of this strategy, different classes of coiled-coils were
evaluated, including parallel heterodimeric13,14 coils with low (CC1)
and high (CC2) inter-coil affinities and disulfide-linked covalent
coils (CC3)15, as well as antiparallel heterodimeric (CC4)16 and helixturn-helix homodimeric (CC5)17 pairs (Fig. 1b, Supplementary
Table 1 and Supplementary Fig. 1). These peptide domains were
genetically fused to the N termini of the CD19-binding antibody
hBU12 (ref. 18) through protease-cleavable linkers. The linker
sequences chosen (PLG-LAG19 and IPVS-LRSG20) are substrate
sequences for multiple matrix metalloproteinases (MMPs) such as
MMP-2 and MMP-9 that are upregulated in a variety of cancers21,22.
Binding of the coiled-coil hBU12 fusions was assessed on CD19expressing Raji cells by flow cytometry via competition with Alexa
Fluor 488-labeled hBU12, and it was found that the constructs had
decreased affinity compared with unlabeled hBU12 (Fig. 1c). The
effects for the antiparallel and micromolar-affinity parallel coils
were relatively modest, decreasing the affinity by 38- and 83-fold,
respectively, whereas the nanomolar-affinity parallel (CC2A),
covalent and helix-turn-helix domains reduced binding by over
300-fold. These results suggest that inter-coil affinity is an important factor for blocking binding. An intriguing application for the
masking concept is for antibody–drug conjugates (ADCs) that can
deliver potent cytotoxic small molecules to antigen-expressing
cells. While the covalent parallel heterodimeric coiled-coil CC3
demonstrated excellent blocking activity, the N-terminal disulfide
is incompatible for masked ADCs that employ endogenous disulfides for drug attachment. For this reason, a high-affinity parallel
heterodimeric coiled-coil was designed that was devoid of cysteine residues and the resulting fusion demonstrated marked loss in
binding (750-fold) compared with the parental monoclonal antibody (CC2B; see Fig. 1d). This confirmed that the N-terminal
disulfide was not required for efficient blocking. To test whether
cleavage of the CC2B mask could restore antibody binding, CC2BhBU12 was incubated with purified human MMP-2, resulting in
efficient site-specific removal of the mask, which was confirmed by
reverse-phase liquid chromatography–mass spectrometry (LC–MS)
(Supplementary Fig. 2). Binding of the precleaved antibody was
restored to that of unmasked hBU12, as assessed by saturation
binding on Raji cells (Fig. 1d).
To better understand how the CC2B mask impacts antibody
binding, the kinetics of binding for CC2B-hBU12 were assessed
by biolayer interferometry using recombinant CD19. As has been
reported with other masking modalities5–7, the decreased affinity of
Department of Protein Sciences, Seattle Genetics Inc., Bothell, WA, USA. 2Department of Nonclinical Science, Seattle Genetics Inc., Bothell, WA, USA.
Department of Antibody Discovery, Seattle Genetics Inc., Bothell, WA, USA. 4Department of Chemistry, Seattle Genetics Inc., Bothell, WA, USA.
*e-mail: mlevengood@seagen.com
1
3
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761
Letters
NaTure BiOTeCHnOlOgy
a
b
Light chain
N terminus
Coil masking
domain
hBU12
Micromolar parallel (CC1)
Nanomolar parallel (CC2A)
Covalent parallel (CC3)
Antiparallel (CC4)
Helix-turn-helix (CC5)
50
100
10,000
Geometric mean (MFI)
Maximum binding (%)
100
1
s-s
Antiparallel
Helix-turn-helix
heterodimeric (CC4) homodimeric (CC5)
d
Antibody binding to CD19+
Raji cells
0
0.01
Covalent
(CC3)
s-s
Antibody
c
High affinity
(CC2)
Cleavable
linker
32 Å
Heavy chain
N terminus
Parallel heterodimeric
Low affinity
(CC1)
250,000
Antibody binding to CD19+
Raji cells
hBU12
200,000
CC2B-IPVSLRSG
-hBU12
Cleaved
150,000
100,000
50,000
0
0.01
1
100
10,000
Concentration (nM)
Competitor (nM)
Fig. 1 | Steric blockade of antibody CDRs as a modular antibody masking system. a, Structure of an antibody Fab domain depicting the CDRs (blue)
spanning approximately 32 Å between the N termini of the heavy and light chain (rituximab, PDB 4KAQ). b, Schematic depicting a coiled-coil masking
domain fused to an antibody via cleavable linkers (left) and coiled-coils with various orientations and inter-coil interactions (right). c, Binding of coiled-coil
masked anti-CD19 hBU12 antibodies was assessed using flow cytometry via competition against Alexa Fluor 488-labeled hBU12 on CD19+ Raji cells. Data
represent the mean. The sequences for each coiled-coil domain are shown in Supplementary Fig. 1. d, The binding of CC2B-hBU12 compared with hBU12
was assessed via saturation binding on CD19+ Raji cells. For the cleaved antibody, the CC domain was removed using recombinant MMP-2 as described in
the Methods. For c and d, the data shown are mean values of independent duplicate cell samples, and two independent experiments were conducted with
similar results.
CC2B-hBU12 to CD19 is driven largely by a slower rate of association of the antibody, whereas the dissociation rate is only modestly
affected (Supplementary Fig. 3). One distinguishing aspect of this
technology is that the CC2B peptides are fused to all four antibody N termini (two light chain, two heavy chain). To understand
whether this could impact efficient protease activation, we designed
‘half-cleavable’ monoclonal antibodies that contained an MMP-2cleavable sequence on only one chain (either light or heavy) and
a site that is not cleaved by MMP-2 (LALGPG19) on the opposing
chain. These monoclonal antibodies were treated with purified
MMP-2 until either the light chain or heavy chain had been cleaved,
and the resulting products were evaluated for binding by flow
cytometry on CD19+ Raji cells (Supplementary Fig. 4). Two cleavage events (out of a possible four) were sufficient to restore hBU12
binding to within approximately 10-fold of the parent antibody,
whereas the intact masked monoclonal antibodies had over 900fold reduced affinity. Therefore, fusion of a coiled-coil peptide to
a single antibody chain provides approximately 1% of the blocking
ability of a coiled-coil mask that bridges the heavy and light chains.
Further, these results demonstrate that limited mask proteolysis can
have a dramatic impact on CC2B-mAb binding.
The CC2B domain was then applied to several other monoclonal
antibodies, including rituximab (anti-CD20)23, trastuzumab (antiHER2)24, h15H3 (anti-αVβ6)25 and 145-2C11 (anti-mouse CD3)26,
and binding for each antibody was tested on antigen-positive
cells by flow cytometry. For each of the CC2B-masked antibodies,
minimal binding was observed at concentrations as high as 2 μM,
representing decreases in affinity of at least 80-fold for rituximab,
470-fold for trastuzumab, 290-fold for 145-2C11 and 1,000-fold
for h15H3 (Fig. 2a and Supplementary Table 2). For each of these
antibodies, the mask could be efficiently removed through incubation with purified MMP-2, and cleavage restored binding to within
762
1.7-fold of the parent antibody. These results demonstrate that, in
contrast to previously described methods4–10, the CC2B domain is
modular and is a general solution for antibody masking. With this
approach, there is no need for extensive screening to identify suitable masking domains, and the extent of masking achieved with the
CC2B domain meets or exceeds those obtained using individualized
antigen-blocking technologies4–10. The CC2B mask was applied to
antibodies with a wide range of affinities (0.4 nM for h15H3 compared with 25 nM for rituximab) and receptor densities (100,000
copies per cell for CD19 on Raji cells compared with 1.3 million
copies per cell for HER2 on SK-BR-3 cells). An additional benefit
is that the masking domain has no intrinsic affinity for the antibody paratope, minimizing the possibility that the mask will remain
bound once the linker has been cleaved27.
We next examined the effects of the coiled-coil mask on monoclonal antibody internalization, a key factor that drives ADC activity. A quenched fluorescence assay was utilized to track hBU12
internalization into CD19+ Ramos cells over the course of 14 h,
showing that CC2B-hBU12 had decreased internalization compared with hBU12, both in terms of the rate and extent of internalization (Fig. 2b). The impact of masking on in vitro cytotoxicity
was then evaluated utilizing four-load ADCs bearing an auristatinbased drug-linker, mc-MMAF28. The unmasked hBU12 and
MMP-activated masked ADCs both had half-maximum inhibitory concentration (IC50) values of 2.5 ng ml−1 on Ramos cells after
96 h continuous incubation, whereas the masked ADC was much
less active, with an IC50 of approximately 1.5 μg ml−1 (Fig. 2c).
To further examine the biological consequences of masking, we
assessed the complement-dependent cytotoxicity (CDC) activity
of rituximab and CC2B-rituximab. Rituximab (4 μg ml−1) elicited
approximately 60% maximal cell lysis on CD20+ Raji cells in the
presence of human serum, whereas no lysis was observed with
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Letters
50,000
CC2B-hBU12-mcMMAF(4)
Cleaved
4
6
8
Time (h)
10 12 14
1,
00
0
10
,0
00
2
10
0
0
0
0.
1
0
hBU12-mcMMAF(4)
50
Rituximab CDC
on CD20+ cells
80
60
40
20
0
C
C
2B
Concentration (ng ml–1)
1,000
co
nt
ro
l
100,000
d
Cytotoxicity of ADCs
on CD19+ Ramos cells
100
10
le
av
ed
150,000
0.1
Concentration (nM)
yp
e
CC2B-IPVSLRSGhBU12
Survival (%)
Signal intensity
per cell per well
c
hBU12
200,000
0
0.001
Concentration (nM)
Antibody internalization
on CD19+ Raji cells
250,000
10,000
ot
b
100
10,000
ab
Concentration (nM)
1
Cleaved
Is
0
0.0001 0.01
10,000
CC2B-IPVSLRSGanti-mouse CD3
20,000
itu
xi
m
100
10,000
Anti-mouse CD3
R
1
Cleaved
Maximum lysis (%)
0
0.0001 0.01
20,000
30,000
SG
50,000
CC2B-IPVSLRSGtrastuzumab
C
100,000
Cleaved
Trastuzumab
LR
CC2B-IPVSLRSGrituximab
VS
150,000
30,000
Antibody binding to CD3+
HT-2 cells
-IP
Rituximab
1
200,000
Antibody binding to HER2+
SK-BR-3 cells
Geometric mean (MFI)
Antibody binding to CD20+
Raji cells
10
Geometric mean (MFI)
a
Geometric mean (MFI)
NaTure BiOTeCHnOlOgy
Fig. 2 | Masking and reactivation of CC2B-masked antibodies. a, The binding of CC2B-masked rituximab (anti-CD20), trastuzumab (anti-HER2) and
145-2C11 (anti-mouse CD3) was assessed on CD20-, HER2- and mouse CD3-expressing cells, respectively. Cleaved comparators were generated using
recombinant MMP-2 before the binding study. Data represent the mean values of two independent cell samples and are representative of two independent
experiments. b, Internalization of anti-CD19 antibodies CC2B-hBU12 and hBU12 on CD19+ Raji cells was compared using a quenched-fluorophore pair
(Alexa Fluor 488 and QSY9). On catabolism of the internalized antibody, a fluorescent signal is released and measured using a high-content fluorescence
imager. Data represent the mean of two independent cell samples and are representative of two independent experiments. c, hBU12-mc-MMAF(4)
ADCs were assessed for cell killing after 96-h incubation at 37 °C on CD19+ Ramos cells. Cytotoxicity was assessed using CellTiter-Glo (Promega). Data
represent the mean ± s.d. of four independent cell samples and are representative of two independent experiments. d, CDC was assessed for masked and
unmasked rituximab using CD20+ Raji cells. Cells were incubated with 4 μg ml−1 antibody for 2 h at 37 °C in the presence of human serum and assessed
for cell lysis using Sytox Green (ThermoFisher). Data represent the mean of two individual cell samples and are representative of two independent
experiments. MFI, mean fluorescence intensity.
CC2B-rituximab or an isotype control antibody (Fig. 2d).
Re-activated CC2B-rituximab had activity comparable to the
unmasked antibody. These results demonstrate that the mask is
able to conditionally prevent binding and biological function, and
that removal of the mask restores activity.
Anti-CD3 antibodies have been shown to induce rapid and profound on-target toxicity in both humans and mice29–31. We utilized
an anti-mouse CD3 mAb (145-2C11) to test whether the coiledcoil domain could prevent systemic CD3 binding on lymphocytes
and the subsequent production of pro-inflammatory cytokines
in vivo. As shown in Fig. 2a, the anti-mouse CD3 antibody is effectively masked with the CC2B domain, as no binding was observed
at concentrations up to 2 μM on CD3+ HT2 cells. Additionally, the
unmasked antibody is able to induce mouse T-cell proliferation when
immobilized on a culture dish at a concentration of 500 ng ml−1,
whereas no proliferative effect was observed when cells were treated
with the same concentration of a masked antibody (Supplementary
Fig. 5). After confirming that masking impaired in vitro function,
we compared the pharmacokinetics and cytokine production of
masked and unmasked antibodies in immunocompetent BALB/c
mice. Following intravenous administration at a dose of 0.5 mg kg−1,
the parental anti-mouse CD3 antibody was undetectable in plasma
2 d after dosing, suggesting the presence of an antigen sink due to
abundant CD3 expression (Fig. 3a). In contrast, the masked antibody displayed the same pharmacokinetic profile as a non-binding
isotype control antibody, consistent with the mitigation of systemic
CD3 binding. We next probed the consequences of masking on the
production of inflammatory cytokines such as interferon-γ (IFN-γ)
and interleukin-2 (IL-2). As expected, the parental antibody elicited
a large spike in inflammatory cytokines 4 h post-dose, in contrast
to the masked version (Fig. 3b). These results demonstrate that the
mask protects the antibody from an antigen sink and unwanted systemic CD3 engagement, which both enhances circulation half-life
and mitigates on-target effects.
To evaluate whether mask activation could occur in tumors, we
tested the activity of anti-human CD19-targeted ADCs in a human
CD19+ Ramos non-Hodgkin lymphoma xenograft tumor model.
Before initiating these studies, we confirmed the presence of protease activity in the Ramos model using in situ zymography with
fluorescently labeled antibodies (Supplementary Fig. 6)4. We then
dosed anti-CD19 or control IgG ADCs at 6 mg kg−1 in severe combined immunodeficient (SCID) mice bearing 200 mm3 subcutaneous tumors. Both CC2B-hBU12-mc-MMAF(4) and an unmasked
ADC had similar activities while a CC2B-hBU12 ADC containing the inefficiently cleaved sequence (LALGPG) was less active
(Fig. 3c). As both ADCs had approximately 600-fold reductions in
cytotoxicity experiments in vitro, the results demonstrate cleavage
sequence-dependent anti-tumor activity.
We tested the consequences of masking on the pharmacokinetics and efficacy of an ADC that cross-reacts with mouse antigen.
h15H3 is an antibody directed against the αVβ6 integrin complex
that binds to both human and mouse antigens with similar affinities25. This integrin complex is expressed in a variety of tumors but is
also expressed on skin in mice32. Binding of the masked antibody to
human integrin αVβ6-transfected human embryonic kidney (HEK)
cells was impaired by greater than 1,000-fold (Supplementary Fig. 7),
as was the cytotoxic activity of glucuronide-cleavable MMAE33
ADCs on αVβ6+ BxPC3 cells (Supplementary Fig. 8). Binding and
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Letters
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b
4,000
CC3-IPVSLRSGanti-mouse CD3
3,000
Isotype
control
2,000
10
15
Time (d)
CC3-IPVSLRSGanti-mouse CD3
3,000
0
Isotype
control
2,000
Single dose
6 mg kg–1 at day 10
300
45
5
4,000
Isotype control
35
40
0
Anti-mouse CD3
Untreated
600
25
30
0.01
IFN-ϒ
5,000
CC2B-LALGPG-hBU12
900
0
Animals dosed
at 0.5 mg kg–1
CC2B-PLGLAG-hBU12
1,200
15
20
0
4
8
12
16
20
24
Time (h)
Isotype control
LLQ (0.08 µg ml–1)
hBU12
1,500
0
CC3-IPVSLRSG
-anti-mouse CD3
0.1
ADC activity
in Ramos xenograft
1,000
Anti-mouse CD3
1
Anti-mouse CD3
5
10
Concentration
(pg ml–1)
10
Concentration
(pg ml–1)
Total Ab (µg ml–1)
Pharmacokinetics of antibodies
in BALB/c mice
c
IL-2
5,000
Median tumor
volume (mm3)
a
Time (d)
1,000
0
4
8
12
16
20
24
0
Time (h)
1,000
h15H3
10
CC2B-PLGLAG-h15H3
CC2B-LALGPG-h15H3
1
Animals dosed
at 1 mg kg–1
0.1
–1
LLQ (0.015 µg ml )
0.01
0
5
10
15
h15H3
800
CC2B-PLGLAG-h15H3
600
Untreated
400
Single dose
3 mg kg–1 at day 6
200
0
0
10 20 30 40 50 60 70
Time (d)
Tumor
20
Plasma
Liver
15
10
5
0
PL
Time (d)
Activation of CC2B-h15H3 ADCs
in HPAF-II xenograft
L
LA AG
LG
PG
PL
G
LA LAG
LG
P
PL G
G
L
LA AG
LG
PG
100
f
ADC activity
in BxPC3 xenografts
G
Pharmacokinetics of ADCs
in nude mice
Cleaved heavy chain (%)
e
Mean tumor
volume (mm3)
Total Ab (µg ml–1)
d
Fig. 3 | In vivo assessment of masked antibodies and ADCs. a, The pharmacokinetics of 3H-labeled CC3-masked and unmasked anti-mouse CD3
antibodies were compared in BALB/c mice following 1 intravenous (i.v.) dose of 0.5 mg kg−1 (n = 3 mice per time point; data represent the mean ± s.d.).
b, The levels of IL-2 and IFN-γ were quantified over the course of 24 h from mice treated with 1 mg kg−1 CC3-masked or unmasked anti-mouse CD3
antibodies (n = 3 mice per time point; data represent the mean ± s.d.). c, The efficacy of anti-human CD19 hBU12-mc-MMAF(4) ADCs was assessed in
a human CD19+ Ramos xenograft in SCID mice. Mice were administered 1 intraperitoneal (i.p.) dose of 6 mg kg−1 once tumors reached 200 mm3. Data
represent the median value of n = 5 mice. d, The pharmacokinetics of 3H-labeled anti-human/mouse αVβ6 CC2B-h15H3-gluc-MMAE(8) and h15H3-glucMMAE(8) ADCs were compared in nude mice after a single i.v. dose of 1 mg kg−1 (n = 3 mice per time point; data represent the mean ± s.d.). e, The efficacy
of anti-human/mouse αVβ6 h15H3-gluc-MMAE(8) ADCs was assessed in a human αVβ6+ BxPC3 xenograft. Mice were administered 1 3 mg kg−1 i.p. dose
once tumors reached 100 mm3. Data represent the mean value of n = 5 mice. f, CC2B-mask cleavage was assessed in tumor, plasma and liver samples
of HPAF-II tumor-bearing mice at 4 d post-dose. Shown is the percentage of cleaved heavy chain, as determined by western blot analysis of antibodies
purified from each tissue. Data represent the mean ± s.d. (n = 3 mice for PLGLAG and n = 2 mice for LALGPG). The difference in cleaved antibody for
PLGLAG and LALGPG linkers in the tumor was statistically significant (P = 0.045, two-tailed unpaired t-test). For a–f, each experiment was conducted
once but included control test articles that provided similar response in at least one additional independent experiment. LLQ, lower limit of quantitation.
activity could be restored on mask cleavage. As with the anti-CD3
mAb, the unmasked αVβ6-binding ADC cleared quickly from circulation in mice due to the presence of an antigen sink, whereas
the masked forms of the ADC had prolonged circulation half-lives
(Fig. 3d). To assess the impacts of improved pharmacokinetics on
efficacy, we tested ADC activity in three different cell line xenograft
tumors that express αVβ6 integrin. In two of these models (BxPC3
and Detroit 562), increased circulation of the masked ADC led to
improved efficacy in comparison with the unmasked h15H3 ADC
(Fig. 3e and Supplementary Fig. 9).
The masked ADC bearing a cleavable PLGLAG linker and the
parent h15H3 ADC had comparable efficacy in an HPAF-II pancreatic carcinoma xenograft model, but a masked ADC bearing
the scrambled LALGPG linker had only minimal effects on tumor
growth (Supplementary Fig. 10). We used western blot analysis to
establish ratios of masked and unmasked ADCs in the tumor 4 d
after administration (Fig. 3f and Supplementary Fig. 11). There was
a statistically significant (P = 0.045) 7-fold increase in cleaved ADC
for the PLGLAG linker compared with the scrambled sequence,
suggesting that in vivo efficacy (Supplementary Fig. 10) correlates with intratumoral activation. A similar activation trend was
observed using in situ zymography (Supplementary Fig. 12). While
764
the in vivo conversion does not take into account the portion of
activated ADC that bound antigen and internalized inside target
cells, even low-level conversion can lead to pronounced increases
in tumor antigen binding (Supplementary Fig. 13). Finally, we
showed (Fig. 3f and Supplementary Fig. 11) that there was no
circulating unmasked ADC resulting from cleavage of the linkers from the protease-sensitive or scrambled ADC constructs. To
exclude the possibility that the unmasked ADC might be taken up
by antigen-positive cells, we injected both tumor-bearing and naïve
mice with a non-binding CC3-masked control antibody. In both
cases, the cleaved antibodies in circulation were barely detectable
(Supplementary Fig. 14). These results are consistent with systemic
stability and selective intratumoral activation.
We have described a modular approach for conditional antibody
masking, in which parallel heterodimeric coiled-coils with high
inter-coil affinities reversibly impair antibody binding and biological function. Unlike designer antibody masks that can require extensive screening to identify an antibody that is efficiently masked, this
approach is general and the same mask can be readily applied to
an array of antibodies of interest. The pharmacological benefits
of antibody masking via coiled-coils include improved antibody
pharmacokinetics and mitigation of unwanted side effects, such as
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Letters
NaTure BiOTeCHnOlOgy
cytokine release, that arise from systemic target engagement. With
ADCs, the inclusion of disease-specific protease-cleavable linkers
between the mask and antibody can impart improved efficacy of
masked antibody therapeutics compared with unmasked controls
due to avoidance of an antigen sink. This masking approach may
facilitate the targeting of antigens that are inaccessible to conventional approaches due to undesirable normal tissue expression. The
system described here should enable the rapid identification and
optimization of antibodies for therapeutic applications.
Online content
Any methods, additional references, Nature Research reporting
summaries, source data, statements of code and data availability and
associated accession codes are available at https://doi.org/10.1038/
s41587-019-0135-x.
Received: 21 August 2018; Accepted: 16 April 2019;
Published online: 27 May 2019
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Acknowledgements
We thank our Seattle Genetics colleagues L. Benoit, D. Meyer and J. Mitchell for help
with antibody–drug conjugation and C. Yu for help with internalization experiments.
Author contributions
V.H.T., M.R.L. and P.D.S. participated in writing, reviewing and editing of the manuscript.
V.H.T. and M.R.L. participated in the planning, initiation, data generation and analysis
of biological experiments. V.H.T., X.Z. and M.R.L. performed cell-based assays. V.H.T.
performed zymography experiments. S.W.W. and M.M.D performed biophysical
characterization. J.H.C. conducted analysis of pharmacokinetics experiments. R.C.Y.,
W.Z., J.K.S. and I.J.S. performed in vivo anti-tumor activity and pharmacodynamics
experiments. M.C.R., R.P.L. and P.D.S. provided project oversight and review.
Competing interests
All of the authors were employees and shareholders of Seattle Genetics at the time of
these studies.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41587-019-0135-x.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to M.R.L.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature America, Inc. 2019
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Methods
All chemicals were purchased from Sigma Aldrich unless otherwise noted. All
proteases were purchased from R&D Systems with the exception of human
MMP-2 (Sino Biological). Cancer cell lines were obtained from the American Type
Culture Collection. Cell culture media and media components were purchased
from Gibco unless otherwise stated. Auristatin-based drug-linkers, including
maleimidocaproyl-MMAF maleimide (mc-MMAF) and MDpr-glucuronideMMAE-maleimide (gluc-MMAE), were prepared as previously described28,33.
Antibody production. Antibodies were expressed via transient transfection of
either HEK (Gibco) or Chinese hamster ovary (Gibco) cells and were purified
using MabSelect SuRe Protein A resin (GE Healthcare Life Sciences). Further
purification using size-exclusion chromatography was performed when necessary,
using a Superdex S200 column (GE Healthcare Life Sciences).
General procedure for mass spectrometric analysis of antibodies or ADCs.
Reverse-phase LC–MS data were acquired on a Waters Xevo GS-S QTOF coupled
to a Waters Acquity H-Class UPLC system. Samples were reduced with 10 mM
dithiothreitol for 10 min at 37 °C and then separated over an analytical reversedphase column (Agilent Technologies, PLRP-S, 300 Å, 2.1 mm × 50 mm, 3 μm) at
80 °C. Samples were eluted using a linear gradient of 0.01% trifluoroacetic acid
(TFA) in acetonitrile from 25% to 65% in 0.05% aqueous TFA over 5 min, followed
by isocratic 65% 0.01% TFA in acetonitrile for 0.5 min at a flow rate of 1.0 ml min−1.
Mass spectrometry data were acquired in ESI+ mode using a mass range of
500–4,000 m/z and were deconvoluted using MaxEnt1 to determine masses of the
resulting antibodies or conjugates.
Fluorescent labeling of antibodies. Antibodies were fluorescently labeled using
N-hydroxysuccinimide ester-activated Alexa Fluor 488 or Alexa Fluor 647
(Life Technologies) following the vendor protocol. Briefly, antibody (1 molar
equivalent) was incubated with fluorophore (4–6 molar equivalents) at pH 8
at room temperature for 1 h. Fluorescent antibody was purified using a NAP-5
column (GE Healthcare Life Sciences) and fluorophore loading was quantified
using a UV-Vis spectrophotometer (Agilent). Typical fluorophore loadings were
2–4 fluorophores per antibody.
For quenched fluorescent antibodies used in internalization experiments,
antibodies containing an engineered cysteine at S239C of the heavy chain
were used. To enable preferential conjugation to the S239C site, the entire
antibody was first reduced using an excess of Tris(2-carboxyethyl)-phosphine
(TCEP, 20 molar equivalents, pH 8, 37 °C, 90 min) in the presence of 1 mM
diethylenetriaminepentaacetic acid (DTPA). Excess TCEP and DTPA were
removed via buffer exchange using Amicon 30-kDa molecular weight cutoff
centrifugal filters (EMD Millipore) into PBS containing 2 mM EDTA. The
disulfides were then re-oxidized using dehydroascorbic acid (20 molar equivalents,
pH 7.4, room temperature, 45 min, 2 additions). Excess dehydroascorbic acid
was removed by buffer exchange in Amicon 30-kDa molecular weight cutoff
centrifugal filters. QSY9-maleimide (2.5 molar equivalents, ThermoFisher) was
added to the re-oxidized antibody (1 molar equivalent) containing free thiols at
S239C. The reaction was incubated for 15 min at room temperature, then purified
using a NAP-5 column. Drug loading was determined by LC–MS. To conjugate
Alexa Fluor 488 at the interchain disulfides, the QSY9-labeled antibody was again
reduced using TCEP (10 molar equivalents, 37 °C, 90 min) in the presence of
1 mM DTPA, then buffer exchanged to remove excess TCEP and DTPA. The fully
reduced antibody containing 8 free Cys residues was then incubated with Alexa
Fluor 488-maleimide (12 molar equivalents, pH 7.4, Life Technologies). The fully
conjugated quenched fluorescent antibody was buffer exchanged and concentrated
into PBS as previously described.
Preparation of masked ADCs. For mixed 4-load mc-MMAF ADCs, antibody
interchain disulfides were partially reduced using TCEP (~2.5 molar equivalents,
pH 8, 37 °C, 90 min) in the presence of 1 mM DTPA until a mean of 4 reduced
thiols were present. Excess TCEP and DTPA were removed via buffer exchange
with Amicon centrifugal filters into PBS containing 2 mM EDTA. Reduced
antibodies were conjugated for 15 min at room temperature with a 1.5-fold molar
excess per thiol of mc-MMAF from dimethylsulfoxide stock solutions. The
conjugation reaction was purified using a NAP-5 column and buffer exchanged
into PBS as described above. Drug loading was determined by LC–MS. For
8-loaded MDpr-glucuronide-MMAE (gluc-MMAE) ADCs, conjugates were
prepared in a similar fashion, except 12 molar equivalents of TCEP were used for
the reduction step to generate an antibody containing 8 free thiols. Purification and
quantification of drug loading were performed using the same methods.
Masked antibody proteolysis. MMPs were activated via incubation with 1.25 mM
4-aminophenyl mercuric acetate at 37 °C for 1 h (MMP-2 and MMP-7), 2 h
(MMP-13) or overnight (MMP-9).
For all cleaved antibodies used in binding or cytotoxicity assays, masked
antibody (~50 µg) was incubated at 37 °C for 2–16 h with activated human MMP-2
(100 enzyme units, where 1 unit is the amount of MMP-2 that hydrolyzes 1 μg of
fluorogenic substrate peptide per minute at 37 °C, pH 7.5). The antibody was then
NaTure BiOTeCHnOlOgy
purified using MabSelect SuRe Protein A resin (GE Healthcare Life Sciences).
Briefly, antibody was bound to the resin at room temperature for at least 2 h with
constant mixing. The resin was then washed with PBS (5 column volumes) and
antibody was eluted with 50 mM glycine pH 3.0. The eluent was then neutralized
to pH 7.4 and buffer exchanged into PBS using dilution and concentration with
Amicon 30-kDa molecular weight cutoff filters.
Competition binding experiments. To evaluate cell binding of masked antibodies,
2 × 105 Raji cells were mixed with fluorescently labeled parent antibody (2 nM)
mixed with serial dilutions of competitor (masked antibody) in staining buffer
(PBS, 2% fetal bovine serum (FBS), 0.2% NaN3). Samples were incubated on ice for
1 h and washed twice with ice-cold staining buffer. Labeled cells were examined by
flow cytometry on a BD LSRII gated to exclude non-viable cells and analyzed using
FloJo software. The IC50 was calculated using GraphPad Prism 6.
Saturation binding experiments. Binding of antibody or ADC to cell-surface
antigen was assessed by flow cytometry on CD19+ Ramos, CD20+ Raji,
HER2+ SKBR3, mCD3+ HT2 or αVβ6+ HPAF-II and HEK cells. Cells (2 × 105)
were combined with a serial dilution of indicated antibody in staining buffer (PBS,
2% FBS, 0.2% NaN3) in a total volume of 100 µl. The cells were incubated on ice for
1 h and washed twice with ice-cold staining buffer. Cells were resuspended with
anti-human IgG-AF647 (Jackson ImmunoResearch, 200-fold dilution in staining
buffer) on ice for 1 h. Cells were washed twice with ice-cold staining buffer and
resuspended in staining buffer. Labeled cells were examined by flow cytometry on
an Attune NxT cytometer (Life Technologies) gated to exclude non-viable cells and
analyzed using FloJo software. The dissociation constant (Kd) was calculated using
nonlinear regression analysis in GraphPad Prism 6.
Biolayer interferometry. Recombinant human CD19-human Fc fusion protein
was produced in Chinese hamster ovary cells and labeled with biotin following the
SureLINK chromophoric biotin labeling kit (Sera Care). Briefly, protein (1 molar
equivalent) was incubated with biotin-N-hydroxysuccinimide ester (4 molar
equivalents) at pH 8 at room temperature for 1 h. Biotinylated protein was purified
using a NAP-5 column and biotin loading was measured using a UV-Vis (Agilent).
A typical extent of biotinylation was 1–2 biotins per CD19.
All kinetic measurements were assessed using the OctetRed384 (Pall Forte Bio)
at room temperature. The biotinylated CD19 was bound to SAX streptavidin
biosensors (Pall Forte Bio) at 7 µg ml−1 in kinetics buffer (0.1% BSA, 0.02%
Tween-20, PBS) for 300 s. After equilibration, hBU12 or CC2B-IPVSLRSG-hBU12
was allowed to associate for 600 s and dissociation was measured for 2,000–6,000 s.
Kinetic parameters were determined using the Octet analysis software using a 1:1
global fit binding model.
Viability assay. Cell viability assays were performed using CellTiter-Glo
(Promega). Ramos and BxPC3 cells (2,000 cells per well) were seeded into 96-well
clear-bottom culture plates. ADC dilutions were added to each well (1,000 ng ml−1
to 0 ng ml−1) and the samples were incubated for 96 h at 37 °C. Luminescence was
measured using an EnVision Multilabel Plate Reader (Perkin Elmer). The IC50
value was determined in quadruplicate and is defined as the concentration that
results in half-maximal growth inhibition over the course of the titration curve.
The data were fit using GraphPad Prism 6 and an IC50 was used to compare the
change in cytotoxicity of parent ADC against masked ADC.
CDC assay. In a 96-well plate (Costar), 100,000 Raji cells were plated in 200 μl
RPMI 1640 medium containing 5 µM Sytox Green (Invitrogen), 10% normal
human serum (Complement Technology) and titrated rituximab, CC2BIPVSLRSG-rituximab, cleaved CC2B-IPVSLRSG-rituximab or control antibody
(hIgG1k). A solution of 1% Triton-100 was used as a positive lysis control. Samples
were incubated for 2 h at 37 °C and read with the EnVision Multilabel Plate Reader
(Perkin Elmer) using 504/523-nm excitation and emission filters. Each experiment
was performed in triplicate.
T-cell proliferation assay. T cells isolated from BALB/c mouse spleens were
labeled with carboxyfluorescein succinimidyl ester (BD Horizon) by incubating
cells in prewarmed PBS and carboxyfluorescein succinimidyl ester (1 µM) at 37 °C
for 15 min. The labeling reaction was quenched by addition of FBS (Gibco) and
cells were washed and resuspended in murine lymphocyte medium (RPMI 1640,
10% FBS, 10 mM HEPES, 4 mM GlutaMAX, 50 µM beta mercapto-ethanol and
1X penicillin/streptomycin).
T-cell stimulation was assessed using a CSFE dilution assay. Either 145-2C11
or CC2B-145-2C11 antibody was coated on a U-bottom clear 96-well plate (50 μl
per well, 500 ng ml−1) along with 8 µg of anti-mouse CD28 antibody. The plate was
incubated overnight at 37 °C. After 16–24 h, the supernatant was removed through
aspiration. In each well, 5 × 106 cells were added in a volume of 200 µl and allowed to
incubate at 37 °C. On day 4, the T cells were monitored via flow cytometry using an
Attune NxT cytometer (Life Technologies). Data were analyzed using FloJo software.
Quantification of cytokines using multiplex immunoassay. (Protocol was
adapted from MCYTOMAG-70K manufacturer protocol from Luminex Corp.)
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Letters
NaTure BiOTeCHnOlOgy
Plasma samples obtained from mice dosed with antibodies were analyzed using
a mouse cytokine/chemokine magnetic bead panel (EMD Millipore). In a 96-well
plate (Costar), 25 μl plasma was combined with antibody-immobilized beads
and allowed to incubate with agitation overnight at 4 °C. The plate was washed
twice and 25 μl detection antibody was added into each well. The plate was sealed,
agitated at room temperature for 1 h and then 25 μl Streptavidin-Phycoerythrin
was added to each well. The plate was agitated at room temperature for 30 min and
washed twice. The luminal signal was detected using the MAGPIX Luminex plate
reader (Luminex Corp.).
Quantification of cleaved antibody from xenograft studies. Frozen tissues
obtained from mice dosed with antibodies were homogenized into powder on dry
ice by physical disruption. The homogenized tissue was then resuspended into
T-PER homogenization solution (Thermo Scientific) containing protease inhibitor
cocktail using a ratio of tissue homogenate to protease inhibitor based on T-PER
product instructions. Human IgG-specific affinity resin, IgSelect (GE Healthcare
Life Sciences), was used to capture antibodies from the tissue homogenates by
incubation at 4 °C overnight. The antibodies were eluted from the resin using
50 mM glycine buffer, pH 3, and concentrated using an Amicon Ultra 30-kDa
spin filter to a volume of ~30 μl. Purified antibodies were separated on a 12% Tris
glycine gel (ThermoFisher) under reduced denaturing conditions, then transferred
onto nitrocellulose membranes. The masked and cleaved antibody signals were
detected using an HRP-conjugated anti-human IgG (H + L) antibody (Jackson
ImmunoResearch). The blots were scanned using an Amersham Imager 600
(GE Healthcare Life Sciences) and band intensities were quantified with ImageJ
software (NIH). The percentage of cleaved antibody signal was calculated as
cleaved signal divided by total signal of cleaved and uncleaved antibodies.
Assessment of antibody internalization. Ninety-six-well plates (as described
earlier) were coated with poly-l-lysine by adding 150 μl poly-l-lysine solution
(450 µl poly-l-lysine with 3 ml water) into each well. Plates were incubated at room
temperature for 5 min. The coating solution was removed via aspiration and the
plate rinsed with PBS two times. The plates were allowed to dry overnight in a
sterile environment.
Raji cells were labeled with Hoescht 33342 at 0.1–0.5 µg ml−1 final
concentrations at 37 °C for 20 min. The cells were washed twice with RPMI 1640
medium and plated at a density of 100,000 cells per well in a total volume of 200 µl.
Quenched hBU12 or CC2B-IPVSLRSG-hBU12 was added to each well to a final
concentration of 2 µg ml−1 in a total volume of 300 μl. The plate was analyzed using
an IN Cell Analyzer 2200 (GE Healthcare) at 37 °C over the course of 14 h. The
data were analyzed using IN Cell Analyzer Workstation v.3.7.3(x64), where the
fluorescence intensity of each cell was quantified over the course of the experiment.
Assessment of protease activity via in situ zymography. For hBU12 antibodies
directly labeled with Alexa Fluor 647, fresh-frozen Ramos tumors were
cryosectioned (5 μm) and mounted onto positively charged slides (Leica). The
tumor slices were incubated in PBS at room temperature for 5 min and blocked
with 2% human serum in PBS at room temperature for 30 min. A hydrophobic
square was drawn around the tumor using a hydrophobic pen (ThermoFisher).
The tumor slice was incubated with antibody (1 µg ml−1 in staining buffer: 25 mM
Tris, 150 mM NaCl, 0.05% Tween-20, 100 μM ZnCl, 5 mM CaCl2, pH 7.4) at room
temperature for 2 h. Antibody was aspirated off and slides were washed three times
with TBS-T buffer (25 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.4) with
gentle agitation for 5 min. Slides were washed one time with PBS and mounted
with ProLong Diamond Antifade Mountant (ThermoFisher). For protease
inhibitor control experiments, slides were pretreated with protease inhibitor
cocktail III (1:100, EMD Millipore) and 50 µM Galardin (EMD Millipore) before
addition of labeled antibodies.
For unlabeled h15H3 antibody staining, fresh-frozen HPAF-II tumors were
cryosectioned (5 μm) and mounted onto positively charged slides (Leica). The
tumor slices were incubated with PBS as described above and blocked with 5% goat
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serum in PBS at room temperature for 30 min. A hydrophobic square was drawn
around the tumor and antibody (1 µg ml−1 in staining buffer with 1 mM MnCl2) was
added. The slide was incubated with antibody at room temperature for 1 h before
the slide was washed as described above. Anti-human AF594 secondary antibody
(1:1,000, Invitrogen) was added and allowed to incubate at room temperature for
1 h. Antibody was removed and slides were washed with TBS-T buffer with gentle
agitation at room temperature for 5 min. This process was repeated three times and
then slides were washed with PBS and mounted as described above.
All slides were analyzed using an Olympus IX83 inverted fluorescence
microscope with ×10 or ×20 objective and then analyzed using Olympus
CellSens Software.
In vivo experiments. All animal handling and experimentation were performed
under Seattle Genetics Institutional Animal Care and Use Committee guidelines.
Seattle Genetics is fully accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care International.
Pharmacokinetics of masked antibodies. Pharmacokinetics experiments were
performed using radiolabeled antibodies.
Antibodies (1 mg) were incubated with 55 μCi N-succinimidyl propionate
[propionate-2,3-3H] (Moravek Biochemicals, 80 Ci mmol−1, 1 mCi ml−1, 9:1 hexane/
ethyl acetate solution) at room temperature for 2 h at pH 8.0. The mixture was
centrifuged at 4,000g for 5 min and the lower aqueous layer was removed. The
aqueous layer was buffer exchanged four times using Amicon Ultra-15 Centrifugal
Filter Units (Millipore, catalog No. UFC903024, 30 kDa molecular weight cutoff)
to remove excess radioactive material. The radiolabeled antibodies were filtered
through sterile 0.22-μm Ultrafree-MC Centrifugal Filter Units (Millipore) and
the final antibody or ADC concentration was measured spectrophotometrically.
The specific activity (μCi per mg) of each product was determined by liquid
scintillation counting.
The radiolabeled antibodies were injected at 0.5 mg kg−1 in BALB/c mice via
tail vein (3 animals per dose group, randomly assigned) or at 1 mg kg−1 in nude
mice via tail vein (3 animals per dose group, randomly assigned). The blood was
drawn into K2EDTA tubes via the saphenous vein at various time points and
processed to plasma. Plasma samples were added to Ecoscint-A liquid scintillation
cocktail (National Diagnostics), and the total radioactivity was measured via liquid
scintillation counting. The specific activity of the radiolabeled samples was used to
calculate the antibody concentration at each time point.
In vivo efficacy studies. For therapy experiments, 5 × 106 cells were injected
subcutaneously into 5–8 female SCID mice (Harlan) for Ramos study or nude mice
(Envigo) for the BxPC3, Detroit 562 and HPAF-II studies. Mice were randomly
divided into study groups and dosed with test article via intraperitoneal injection
once the tumors reached approximately 100–200 mm3. Animals were euthanized
when tumor volumes reached 500–1,000 mm3. Tumor volume was calculated
with the formula: volume = ½ × length × width × width. Mice showing durable
regressions were terminated around day 40–66 after implant. In all xenograft
studies, no weight loss or treatment-related toxicities were observed for mice
treated with any of the test articles.
Statistics. Statistical significance was analyzed using two-tailed t-test, as indicated
in the figure legends. Further information can be found in the Nature Research
Reporting Summary.
Reporting Summary. Further information on research design is available in the
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The data that support the findings of this study are available within the paper and
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Mass spectrometry data was acquired using UNIFI software. Flow cytometry data was acquired using Attune NxT Software (version
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acquired using Envision Workstation software (version 1.13.3009.1401) Multiplex immunoassay data was collected using Luminex
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Antibodies used
The preparation of all therapeutic and surrogate antibodies is described in the Materials and Methods. Saturation binding
experiments with human antibodies used secondary antibodies from Jackson Immunoresearch (109-135-098, Allophycocyanin
Goat Anti-human IgG Fcg, 109-096-008, FITC F(ab')2 Fragment Goat Anti-Human IgG, or 109-116-170, R-Phycoerythrin F(Ab')2
Fragment Goat Anti-human IgG Fcg at a 1:200 dilution). For saturation binding studies involving mouse antibodies, an RPhycoerythrin F(ab')2 Fragment Goat Anti-mouse IgG Fcg antibody was used (Jackson Immunoresearch, 115-116-071, 1:200
dilution). For the T cell activation experiment, anti-mouse CD3e (eBioscience, 16-0031-85, lot# E06294-1646, 1 mg/mL) and
anti-mouse CD28 (Tonbo Biosciences, clone 37.51, 40-0281-U500, 2 mg/mL) was used. For the in situ zymography experiments,
a Goat Anti-human IgG (H+L), Alexa Fluor 594 conjugated antibody was used (Life Technologies, A11014 at a 1:1000 dilution). for
Western blot experiments, Peroxidase F(ab')2 Fragment Goat Anti-human IgG (H+L) (Jackson Immunoresearch 109-036-088,
1:10,000 dilution )
Validation
All primary aAll primary antibodies tested in the manuscript have been published or described previously and are referenced
accordingly. All commercially available antibodies were validated by the vendor. Links for each antibody used are:
Allophycocyanin Goat Anti-human IgG Fcg : https://www.jacksonimmuno.com/catalog/products/109-135-098
FITC F(ab')2 Fragment Goat Anti-Human IgG: https://www.jacksonimmuno.com/catalog/products/109-096-008
R-Phycoerythrin F(Ab')2 Fragment Goat Anti-human IgG Fcg: https://www.jacksonimmuno.com/catalog/products/109-116-170
R-Phycoerythrin F(ab')2 Fragment Goat Anti-mouse IgG Fcg: https://www.jacksonimmuno.com/catalog/products/115-116-071
anti-mouse CD3e: https://www.thermofisher.com/antibody/product/CD3e-Antibody-clone-145-2C11-Monoclonal/16-0031-85
anti-mouse CD28: https://www.tonbobio.com/antibodies-and-reagents/flow-cytometry-reagents/in-vivo-readytm-mousecd28-37-51.html
Goat Anti-human IgG (H+L), Alexa Fluor 594: https://www.thermofisher.com/antibody/product/Goat-anti-Human-IgG-H-L-CrossAdsorbed-Secondary-Antibody-Polyclonal/A-11014
All primary antibodies tested in the manuscript have been published or described previously and are referenced accordingly. All
commercially available antibodies were validated by the vendor. Links for each antibody used are:
Allophycocyanin Goat Anti-human IgG Fcg : https://www.jacksonimmuno.com/catalog/products/109-135-098
FITC F(ab')2 Fragment Goat Anti-Human IgG: https://www.jacksonimmuno.com/catalog/products/109-096-008
R-Phycoerythrin F(Ab')2 Fragment Goat Anti-human IgG Fcg: https://www.jacksonimmuno.com/catalog/products/109-116-170
R-Phycoerythrin F(ab')2 Fragment Goat Anti-mouse IgG Fcg: https://www.jacksonimmuno.com/catalog/products/115-116-071
anti-mouse CD3e: https://www.thermofisher.com/antibody/product/CD3e-Antibody-clone-145-2C11-Monoclonal/16-0031-85
anti-mouse CD28: https://www.tonbobio.com/antibodies-and-reagents/flow-cytometry-reagents/in-vivo-readytm-mousecd28-37-51.html
Goat Anti-human IgG (H+L), Alexa Fluor 594: https://www.thermofisher.com/antibody/product/Goat-anti-Human-IgG-H-L-CrossAdsorbed-Secondary-Antibody-Polyclonal/A-11014
Peroxidase F(ab')2 Fragment Goat Anti-human IgG (H+L): https://www.jacksonimmuno.com/catalog/
products/109-036-088accordingly.
nature research | reporting summary
Antibodies
Eukaryotic cell lines
Policy information about cell lines
Cell line source(s)
Raji (ATCC)
SKBR-3 (ATCC)
HT-2 (ATCC)
Ramos (ATCC)
HPAF-II (ATCC)
Detroit 562 (ATCC)
BxPC3 (ATCC)
Authentication
All cell lines were authenticated using hort tandem repeat (STR) profiling and interspecies contamination testing
Mycoplasma contamination
All cell lines tested negative for mycoplasma.
Commonly misidentified lines
No commonly misidentified cell lines were used in these studies.
(See ICLAC register)
Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
Female BALB/c, nude, and SCID mice were purchased from Harlan laboratories.
Wild animals
The study did not use wild animals.
Field-collected samples
The study did not use field-collected samples.
April 2018
Laboratory animals
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Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Methodology
Sample preparation
All experiments were performed using single-color flow cytometry with authenticated human cancer cell lines or transfected
cells
Instrument
Life Technologies Attune NxT
Software
Data collected using Attune NxT software and processed using FloJo (FloJo, Inc.)
Cell population abundance
N/A
Gating strategy
Nonviable cells were excluded based upon FSC/SSC gating
nature research | reporting summary
Flow Cytometry
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.
April 2018
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