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doi.org/10.26434/chemrxiv.8187146.v2
Targeted Desialylation Overcomes Glyco-Immune Checkpoints andPotentiates the Anticancer Immune Response in VivoMelissa Gray, Michal A. Stanczak, Han Xiao, Johan F. A. Pijnenborg, Natália R. Mantuano, Stacy A. Malaker,Payton A. Weidenbacher, Caitlyn L. Miller, Julia T. Tanzo, Green Ahn, Elliot C. Woods, Heinz Läubli, CarolynBertozzi
Submitted date: 13/11/2019 • Posted date: 25/11/2019Licence: CC BY-NC-ND 4.0Citation information: Gray, Melissa; Stanczak, Michal A.; Xiao, Han; Pijnenborg, Johan F. A.; Mantuano,Natália R.; Malaker, Stacy A.; et al. (2019): Targeted Desialylation Overcomes Glyco-Immune Checkpointsand Potentiates the Anticancer Immune Response in Vivo. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.8187146.v2
Currently approved immune checkpoint inhibitor (ICI) therapies targeting the PD-1 and CTLA-4 receptorpathways are powerful treatment options for certain cancers; however, the majority of patients across cancertypes still fail to respond. Addressing alternative pathways that mediate immune suppression could enhanceICI efficacy. One such mechanism is the increase in sialic acid-containing proteins and lipids (sialoglycans) inmalignancy, which recently has been shown to inhibit immune cell activation through multiple mechanismsincluding Siglec receptor binding, and therefore represents a targetable glyco-immune checkpoint. Here, wereport the design of a trastuzumab- sialidase conjugate that potently and selectively strips diversesialoglycans from breast cancer cells in vivo. In a syngeneic orthotopic HER2+ breast cancer model, targeteddesialylation delayed tumor growth and enhanced immune cell infiltration and activation, leading to prolongedsurvival of mice with trastuzumab-resistant breast cancer. Thus, antibody-sialidase conjugates represent apromising modality for cancer immune therapy.
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1
Targeted desialylation overcomes glyco-immune checkpoints and potentiates the anticancer immune response in vivo Melissa A. Gray1, Michal A. Stanczak2,3, Han Xiao1, Johan F. A. Pijnenborg1, Natália R. Mantuano2,3, Stacy A. Malaker1, Payton A. Weidenbacher1, Caitlyn L. Miller4, Julia T. Tanzo1, Green Ahn1, Elliot C. Woods1, Heinz Läubli2,3, Carolyn R. Bertozzi1,5* 1Department of Chemistry, Stanford University, Stanford, California, 94305, USA. 2Cancer Immunology Laboratory, Department of Biomedicine, University Hospital, Basel, Switzerland. 3Division of Oncology, Department of Internal Medicine, University Hospital, Basel, Switzerland. 4Department of Bioengineering, Stanford University, Stanford, California, 94305, USA. 5Howard Hughes Medical Institute, Stanford University, Stanford, California, 94305 USA. *Correspondence to: C.R.B. ([email protected]).
Abstract: Currently approved immune checkpoint inhibitor (ICI) therapies targeting the PD-1 and
CTLA-4 receptor pathways are powerful treatment options for certain cancers; however,
the majority of patients across cancer types still fail to respond. Addressing alternative
pathways that mediate immune suppression could enhance ICI efficacy. One such
mechanism is an upregulation of sialoglycans in malignancy, which has been recently
shown to inhibit immune cell activation through multiple mechanisms including Siglec
receptor binding, and therefore represents a targetable glyco-immune checkpoint.
Here, we report the design of a trastuzumab-sialidase conjugate that potently and
selectively strips diverse sialoglycans from breast cancer cells in vivo. In a syngeneic
orthotopic HER2+ breast cancer model, targeted desialylation delayed tumor growth and
enhanced immune cell infiltration and activation, leading to prolonged survival of mice
with trastuzumab-resistant breast cancer. Thus, antibody-sialidase conjugates
represent a promising modality for cancer immune therapy.
2
Introduction Immune checkpoint inhibitor (ICI) therapies have revolutionized treatment of certain
cancers. For example, blocking antibodies against PD-1, PD-L1, and CTLA-4, have
eradicated metastatic tumors in some patients, leading to long-term survival1,2.
Although immune activation can be lifesaving, most patients do not respond or relapse
after an initial response, and the underlying mechanisms of primary and secondary
resistance are not well understood3. Additional immune modulators might be at play,
including alternative T cell checkpoints (e.g., TIM-3, LAG-3, and A2AR4–6), innate
immune receptors and ligands (e.g., CD47 and SIRPα7,8), and enzymes (e.g., IDO and
ADAR19,10). Several of these targets are under clinical evaluation, often in combination
with PD-1/PD-L1 blockade11.
While most ICIs target protein checkpoints11, cell-surface glycosylation has recently
garnered interest as a mediator of immune inhibition12. This is supported by several
decades of literature, which have identified altered glycosylation as a hallmark of
malignancy13,14. One example of a glycosylation pattern associated with cancer
transformation is an increase in sialic-acid containing proteins and lipids (sialoglycans),
a phenotype that intensifies with tumor progression and enhances tumor growth only in
the context of an intact immune system in mice15–17. Subsequent work has
demonstrated that sialoglycans suppress immune activation and act as glyco-immune
checkpoints through multiple mechanisms: blocking complement-dependent cytotoxicity
(CDC), inhibiting immune-mediated apoptosis, masking immune-activating ligands, and
directly binding the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors15,18–
20. In particular, the Siglec-sialoglycan axis of immune modulation is emerging as an
important mediator of sialic acid-induced immune suppression in the context of
cancer21.
The Siglec receptor family binds to a variety of sialoglycan structures and populates,
often in combination, every immune cell class22,23. Eight family members (Siglecs-3, 5,
6, 7, 8, 9, 10, and 11) have intracellular domains that bear homology to that of PD-124,
including an immunoreceptor tyrosine-based inhibition motif (ITIM) preceding a switch
3
motif (ITSM) (Fig. 1a,b Supplementary Fig. 1). These cytosolic ITIM/ITSM domains
recruit protein tyrosine phosphatases, ultimately resulting in inhibitory signaling and
immune cell suppression25,26. Thus, we and others speculated that Siglec engagement
of cell-surface sialoglycans has inhibitory consequences similar to PD-1 engagement of
PD-L127,28. In support of this hypothesis, Siglec-9 was recently shown to be
upregulated on tumor-infiltrating T cells and correlated with reduced survival of cancer
patients17. Reciprocally, genetic knockout or inhibition of tumor sialic acid synthesis and
reduced presence of Siglec ligands enhanced immune infiltration and reduced tumor
growth17,29. The sialoglycan axis may therefore be a major contributor to tumor immune
suppression and an attractive target for cancer immune therapy.
Fig. 1. Targeted desialylation of Siglec ligands with the antibody-enzyme conjugate T-Sia 2 as a
modality for immune therapy. a, PD-1 and b, Siglecs are receptors that suppress immune cell function
upon ligand binding. Engagement of PD-1 and Siglec receptors leads to recruitment of SHP
phosphatases to the cytosolic ITIM/ITSM domains and inhibits immune cells, APC = antigen-presenting
cell. c, PD-L1 checkpoint inhibitor therapy uses antibodies to bind PD-L1 and block extracellular
interactions to PD-1, inhibiting SHP recruitment and enhancing the immune response to cancer. d,
Targeted desialylation with an antibody-sialidase conjugate catalytically removes a chemically diverse
Catalytic
domain
a
e
bPD-1 signaling
Cancer cell or APC
Immune cell
PD-1
PD-L1
SHP
1/2
ITSM
ITIM
Checkpoint inhibitor
ITSM
ITIM
PD-1
PD-L1
Cancer cell or APC
Immune cell
Targeted desialylation
ITSM
ITIM
Cleaved
glycans
Siglec
Cancer cell or APC
Immune cell
Siglec signaling
Siglec
Cancer cell or APC
Immune cell
SHP
1/2
ITSM
ITIM
Sialylated
proteins
& lipids
Lectin
binding
domain
T-Sia 1 T-Sia 2
f
c d
4
group of Siglec ligands and prevents SHP recruitment to the Siglec ITIM/ITSM domains. e,
Representation of previously reported T-Sia 1, in which trastuzumab was conjugated to two molecules of
V. cholerae sialidase. f, Illustration of T-Sia 2, where trastuzumab is linked to an average of one S.
typhimurium sialidase. Trastuzumab is represented by mouse IgG1 PDB: 1IGY, V. cholerae sialidase:
1W0P, S. typhimurium sialidase: 3SIL.
Sialoglycan ligands exhibit significant chemical heterogeneity and are fused to a wide
range of cell-surface protein and lipid scaffolds. Development of ligand-sequestering
antibodies (analogous to PD-L1 blockade as illustrated in Fig. 1c) is therefore
challenging. Thus, we previously conceived of a therapeutic modality comprising a
sialic acid-cleaving enzyme fused to a tumor-targeting antibody (Fig. 1d,e) to
catalytically deplete sialoglycans in a tumor-specific manner30.
Here, we advance this strategy to full proof-of-concept by demonstrating that selective
removal of sialoglycans from cancer cells using antibody-sialidase conjugates can
improve the antitumor immune response. These efforts necessitated rational design and
screening for optimal sialidase activity to yield Trastuzumab-sialidase conjugate 2 (T-
Sia 2) (Fig. 1f), which exhibits a low off-target activity and high chemical stability needed
for in vivo use. In a syngeneic orthotopic HER2+ breast cancer model, targeting glyco-
immune checkpoints with T-Sia 2 delayed tumor growth and enhanced immune
infiltration, leading to prolonged survival of mice with trastuzumab-resistant breast
cancer.
Results Minimizing off-target sialidase activity We previously reported on an antibody-sialidase molecule, T-Sia 1 (Fig. 1e),
constructed by conjugation of Vibrio cholerae (VC) sialidase to each trastuzumab heavy
chain (chemical conjugation strategy described in Supplementary Fig. 2)30. Although T-
Sia 1 efficiently cleaved sialoglycans from HER2+ cells at low doses, the conjugate had
considerable trastuzumab-independent activity as well30, which we ascribed to the low
apparent KM value of VC sialidase with polyvalent substrates (including cell surfaces)31.
5
This attribute reflects the engagement of VC sialidase’s two lectin domains (Fig. 1e),
which enable cell surface binding independent of antibody targeting31.
Fig. 2. Salmonella typhimurium sialidase cleaves sialic acid and enhances NK-mediated ADCC of
HER2+ cells with reduced off-target desialylation of HER2- cells. a, MDA-MB-468 cells were treated
with sialidases at various concentrations and stained with SNA-FITC probe. Flow cytometry gating
quantified the fraction of SNA+ (sialylated) cells and the results were fit to a four-parameter variable slope.
b, Representative flow cytometry dot plots of HER2+ (SK-BR-3) and HER2- (MDA-MB-468) cells treated
with T-Sia 1 or trastuzumab-ST sialidase (tras-ST). c, Fraction of sialylated (SNA+) cells quantified by
flow cytometry gating (as shown in b) after treatment with various concentrations of antibody sialidase
conjugates and fit to a variable four-parameter slope. d, Breast cancer cell lines were treated with 10 nM
trastuzumab-ST sialidase (tras-ST) or trastuzumab alone (tras). IL-2-activated human NK cells were
added at a ratio of 8:1 and NK cell-mediated ADCC was quantified by LDH assay after 4 h. P-values of
multiple two tailed t tests are shown with an asterisk indicating significance using the Holm-Sidak multiple
comparisons correction at α=0.05. Graphs a, c, and d, represent Mean ± SD of n=3 experimental
replicates.
Accordingly, our first goal was to identify a more suitable sialidase from a repertoire that
lacks such lectin domains. We expressed six recombinant bacterial and human
sialidases in E. coli (Supplementary Fig. 3a). The sialidases were screened for in vitro
6 nM 120 nM
T-Sia 1
0 nM
Alexa Fluor
647-HER2
Tras ST
b
c
a
d
HER2 protein expression
Tras
Tras ST
60
40
20
00
0.5
1
p=0.42
p=0.004 p=1.9 x10
p=0.048
-5
p=4.2 x10-5
p=3.9 x10-6
SKBR3 361 ZR751 BT20 231 468
HER2
HER2
HER2
HER2
+ +
- -
+
% C
yto
toxic
ity
1
0.5
0
[Sialidase] (nM)
VC sialidase
ST sialidase
Fra
ctio
n o
f S
NA
c
ells
-1 0 1 2 3 410 10 10 10 10 10
-1 0 1 2 310 10 10 10 10
-210
[Conjugate] (nM)
* **
*
+F
ractio
n o
f S
NA
c
ells
FIT
C-S
NA
10
10
10
10 10 10 10
6
4
2
3 5 71
105
103
6
activity and for their ability to enhance natural killer (NK) cell-mediated antibody-
dependent cellular cytotoxicity (ADCC) against BT-20 breast cancer cells
(Supplementary Fig. 3b,c). The Salmonella typhimurium (ST) sialidase NanH was
selected for its relatively high KM value (mM range) against polyvalent targets31–33,
enhancement of NK cell-mediated ADCC, and stability (Supplementary Fig. 3b-d). To
ensure that ST sialidase would cleave Siglec-binding sialoglycans on breast cancer
cells, we performed flow cytometry assays using Siglec-9- and -7-Fc fusion proteins as
probes. Treatment of nine different breast cancer cell lines with ST sialidase reduced
Siglec-9- and -7-Fc binding signal by >96% and >50%, respectively (Supplementary
Fig. 3e-g). As postulated, ST sialidase on its own was less efficient than VC sialidase at
removing sialoglycans from the cell surface (EC50 > 10-fold higher) (Fig. 2a).
To determine whether ST sialidase could be rendered selectively active by cell-surface
targeting, we conjugated the enzyme to trastuzumab using a similar strategy as
previously reported30, but in this case the ST sialidase was modified at a recombinantly
inserted Cys residue (Supplementary Fig. 4a,b and 5a). The enzymatic activity of the
sialidase, as measured with a fluorogenic substrate, was retained after conjugation to
trastuzumab (Supplementary Fig. 5b). We next compared the activity and selectivity of
the trastuzumab-ST sialidase conjugate to T-Sia 1 in a co-culture assay comprising
HER2+ SK-BR-3 and HER2- MDA-MB-468 cells. Using the lectin Sambucus nigra
agglutinin (SNA) as a probe for cell-surface sialoglycans, both conjugates desialylated
HER2+ target cells at concentrations near trastuzumab’s reported KD of 5 nM (Fig. 2b)34
by flow cytometry. However, T-Sia 1 caused substantial off-target desialylation of MDA-
MB-468 cells with an EC50 of 38 nM, whereas the trastuzumab-ST sialidase completely
abrogated off-target reactivity at that concentration; its EC50 ~ 1 µM (Fig. 2c and
Supplementary Fig. 5c). Furthermore, the ST sialidase conjugate retained the ability to
enhance NK cell-mediated ADCC of cells expressing medium and low levels of HER2
(Fig. 2d and Supplementary Fig. 6a,b). As expected, the highly trastuzumab-sensitive
HER2-high SK-BR-3 cell line delivers a sufficiently strong activating signal to NK cells
via FcγRIII and removal of the inhibitory Siglec signal provides no added benefit30. In
summary, antibody conjugation to a sialidase with low intrinsic binding ability retains on-
7
target desialylation activity while increasing the therapeutic window from 60-fold (T-Sia
1) to 2000-fold.
Optimization of the chemical stability of T-Sia 2 To enhance the stability of our conjugates and enable in vivo examination, several
advances to the construct design were required. Specifically, the oxime bond used in T-
Sia 1 is prone to hydrolysis in biological settings (Supplementary Fig. 2)35,36. We
previously developed the Pictet-Spengler ligation, that forms a stable C-C adduct with
SMARTag (aldehyde)-modified proteins35,37. More recently, a related process termed
the hydrazino-iso-Pictet Spengler (HIPS) reaction was developed38. An antibody-HIPS-
maytansinoid conjugate demonstrated a long serum half-life and had the highest
tolerated dose of any maytansinoid antibody-drug conjugate reported in monkeys39,
enabling advancement into human clinical studies40. Based on these precedents, we
synthesized a HIPS-azide linker (1) (Supplementary Fig. 7) and conjugated this to
SMARTag-labeled trastuzumab with full conversion detected by mass spectrometry (Fig
3a and Supplementary Fig. 8a-c). We confirmed the enhanced stability of trastuzumab
conjugated to HIPS over oxime in human plasma and on live cells (Supplementary Fig.
S9a-e).
To improve the uniformity of the trastuzumab-ST conjugate, we selectively and stably
alkylated the engineered C-terminal Cys residue of ST sialidase with an α-
chloroacetamide-DBCO linker (2) (Fig. 3a and Supplementary Fig. 10) under mild
reducing conditions (Supplementary Fig. 11a). We confirmed that the engineered Cys
residue was uniquely modified, with no off-target reactivity towards the four endogenous
ST sialidase Cys residues (Supplementary Fig. 11b-d). Finally, trastuzumab-azide and
ST sialidase-DBCO were coupled by copper-free click chemistry41 to produce T-Sia 2
(Fig. 3a).
To determine the optimal enzyme/antibody ratio (EAR) of the conjugate, we isolated T-
Sia 2 fractions with EARs of ~1 and ~2 by size exclusion chromatography
(Supplementary Fig. 12a). In NK cell-mediated ADCC assays, T-Sia 2 with an EAR ~1
8
outperformed EAR ~2 by a small but significant margin (1.13-fold increase in
cytotoxicity, p=0.035) (Supplementary Fig. 12b-c). This improvement may result from
enhanced FcγRIII binding to a less sterically hindered epitope. Accordingly, we
optimized our conjugation procedure to increase the proportion of T-Sia 2 with an EAR
~ 1. We characterized the conjugate by SDS-PAGE (Fig. 3b) and mass spectrometry
(Supplementary Fig. 13a) and determined an EAR = 0.9 by HPLC analysis (Fig. 3c and
Supplementary Fig. 13b). We confirmed that T-Sia 2 binds its antigen HER2 similarly to
trastuzumab (Supplementary Fig. 13c), and also enhanced NK cell- and γd T cell-
mediated ADCC compared to trastuzumab (Supplementary Fig. 14a-c).
Fig. 3. Synthesis of T-Sia 2. a, T-Sia 2 scheme: (I) Trastuzumab with heavy-chain formylglycine
residues was reacted with 20 equiv. HIPS-azide 1 in citrate buffer pH 5.5. (II) ST sialidase was site-
selectively reacted with 20 equiv. α-chloroacetamide-DBCO 2 under mildly reducing conditions. (III)
Sialidase-DBCO and trastuzumab-azide were coupled via copper-free click chemistry to form T-Sia 2. b,
SDS-PAGE with non-reducing buffer (left) and reducing buffer (right) of sialidase-DBCO, trastuzumab-
HIPS-azide, and T-Sia 2. c, Representative HPLC trace of T-Sia 2. Abbreviations: LC = antibody light
chain, HC = antibody heavy chain, HC-Sia = antibody heavy chain conjugated to ST sialidase.
T-Sia 2 delays tumor growth in the trastuzumab-resistant syngeneic EMT6 breast cancer model
NN
NHN
OO N
NN
N
O
NH
O
OHN
OS
3
4
NN
NN3
T-Si
a 2
Tras
STT-Si
a 2
Tras
ST
a bHIPS-azide α-chloroacetamide-DBCO
I II
III
98198
624938
28
98198
624938
28
c
4 6 8 10 12 14 16 18Time (min)
LCHC HC-Sia
Non-reduced Reduced
OH
OH
NN
NN3
SO
NH
O
O
HN
O
N4
N
O
NH
O
OHN
OCl
4
NN
NHN
OO N3
3
HN
N
NHN
OO
N33
HC
HCSia
LC
1 2
9
To test the efficacy of T-Sia 2 in vivo, we selected the syngeneic orthotopic mouse
EMT6 mammary carcinoma model42, in which the EMT6 cell line was engineered to
express HER2 yet remained resistant to trastuzumab monotherapy in mice43. In vitro,
T-Sia 2 cleaved sialoglycans from HER2+ EMT6 cells, causing a decrease in binding to
both human and mouse Siglec-Fc fusion probes (Supplementary Fig. 15), and
enhanced human NK cell-mediated ADCC (Fig. 4a).
Fig. 4. T-Sia 2 delays tumor growth in a
HER2+ EMT6 syngeneic mouse breast cancer model that is resistant to
trastuzumab alone. a, HER2+ EMT6 cells
were treated with PBS, 10 nM trastuzumab
(Tras), or 10 nM T-Sia 2. Human NK cells
were added at a ratio of 3:1 and NK cell-
mediated ADCC was quantified by flow
cytometry after 4 h and subtracted from PBS
controls. A paired t test is shown for three
biological replicates using different NK cell
donors. b, HER2+ EMT6 cells were injected
into the mammary fat pad of Balb/c mice at day
0. At day 8, mice were injected
intraperitoneally (IP) with PBS (n=5),
trastuzumab (15 mg/kg, n=6), or T-Sia 2 (10
mg/kg or 15 mg/kg, n=6). Tumor volume was
measured over time. c, Individual growth
curves of the HER2+ EMT6 tumors in mice. d,
Mean + SEM tumor volume over time for
animals described in b, RM two-way ANOVA comparing trastuzumab to T-Sia 2 (10 mg/kg). e, Lectin
stain with PNA to detect exposed galactose of extracted tumor cells from mice in b. Geometric mean
(gMFI) ± SD PNA/ConA, n=4 (PBS and tras) and n=9 (T-Sia 2, both doses), ordinary one-way ANOVA
with Tukey’s multiple comparison.
Next we performed in vivo experiments as depicted in Fig. 4b. HER2+ EMT6 cells were
injected into the mammary fat pads of mice followed by intraperitoneal (IP) treatment
a b
d e
Days after tumor inoculation
PBS
Tras
0 10 205 15 25
0
1.5
1.0
0.5
HER2 EMT6
orthotopic
injection
+
Conjugate
IP injections
T-Sia 2 (15 mg/kg)
T-Sia 2 (10 mg/kg)
Days
Endpoint:
tumor volume
p < 0.0001
p < 0.0001
Tras T-Sia 2
% C
yto
toxic
ity
100
80
0
20
40
60
p = 0.013
p <
0.0
001
8 1713100
Tum
or
volu
me (
cm
)
3
0.8
PN
A/C
onA
ratio (
gM
FI)
Tras T-Sia
2
PBS
0.2
0.4
0.6
c
Days after tumor inoculation
Tum
or
volu
me (
cm
)
3
0 10 20 30 40
0
0.3
1.8
1.5
1.2
0.9
0.6
10
with PBS, trastuzumab, or T-Sia 2. After 28 days, all mice in the PBS or trastuzumab
treated groups had reached a tumor burden requiring sacrifice (Supplementary Fig.
16a). In contrast, T-Sia 2 at both doses extended mouse survival to 40 days and
demonstrated significant tumor growth delay compared to trastuzumab alone (Fig. 4c,d
and Supplementary Fig. 16a). No significant difference in tumor growth was observed
between the two doses of T-Sia 2, and although the 10 mg/kg dose of T-Sia 2 was well
tolerated, the 15 mg/kg dose resulted in a minimal weight loss in mice compared to
untreated controls at day 24 (Supplementary Fig. 16b). Lectin staining of tumor
suspensions with Peanut Agglutinin (PNA), which binds to galactose residues exposed
upon sialidase treatment, showed increased labeling in the T-Sia 2-treated tumors
compared to the PBS- or trastuzumab-treated mice 2-4 weeks after the final conjugate
injections (Fig. 4e). These data indicate that T-Sia 2 desialylated the tumor
microenvironment (TME) and delayed EMT6 tumor growth in mice.
To determine where T-Sia preferentially accumulates in mice, we modified the
conjugate with IRDye 800CW NHS ester and imaged mice bearing subcutaneous EMT6
tumors treated with 500 pmol conjugate (~4 mg/kg), 100 pmol, and 20 pmol, and PBS
control. After 48 hours, fluorescent dye clearly localized to the tumors on the left flank
of the mice at the highest dose (Supplementary Fig. 17a). After 4 days, mice were
sacrificed to further analyze T-Sia localization in individual organs. Both the 500 and
100 pmol doses of 800-labeled T-Sia 2 significantly accumulated in tumors, however the
higher dose of 500 also had significant fluorescent accumulation in the liver and
kidneys, likely involved in antibody and fluorophore clearance (Supplementary Fig. 17b-
d). Interestingly, all doses exhibited significant desialylation of the tumors analyzed by
flow cytometry after 4 days by MALII and PNA lectin staining (Supplementary Fig. 17e).
Sialidase activity, HER2 targeting, and FcγR binding contribute to T-Sia 2 activity in mice
11
Fig. 5. HER2-targeting and FcγR engagement are important mechanisms of T-Sia 2 therapy. a,
Schematic of the antibody-sialidase conjugates. b, NK cell-mediated ADCC of ZR-75-1 cancer cells
treated with antibody conjugates, (n=3), E/T =3, detecting percent cytotoxicity by LDH release after 8 h.
c, HER2+ EMT6 cells (1x106) were injected into the mammary fat pad of Balb/c mice. On day 8, mice
were injected intraperitoneally with PBS (n=6), or 10 mg/kg trastuzumab (tras) (n=6), T-Sia-LOF (n=6),
Isotype-Sia (n=6), T-FcX-Sia (n=7), or T-Sia 2 (n=7). d, Individual tumor growth curves for mice in c. e,
Mean + SEM of tumor growth from mice in c; RM two-way ANOVA of treatments compared to T-Sia 2. f,
Kaplan-Meier plot of the time-to-sacrifice of mice in c; with Log-rank Mantel-Cox tests compared to T-Sia
2. g, Total tumor-infiltrated leukocyte count normalized to tumor weight. h, Ratio of CD8+ T cells to Treg
cells. i, %CD69+ of NK cells. Leukocyte analysis quantified from the two independent mouse experiments
(Figs 4 & 5); ordinary one-way ANOVA with Dunnet’s multiple comparisons to T-Sia 2, n=8-15
mice/group, mean ± SD.
% C
ytot
oxic
ity
a
Tum
or v
olum
e (c
m )3
1.61.20.80.40.0
Time (days after tumor injection)0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
TrasPBS control T-Sia-LOF
T-FcX-SiaT-Sia 2 Isotype-Sia
b
c d e
0
20
10
0 10 20 30 40Time (days after tumor injection)
50 600
20
40
60
80
100
Perc
ent s
urviv
al*
f
8
16
1311
0
ConjugateIP
injections
Endpoint: tumor volume
HER2 EMT6 orthotopic injection
0 10 200
0.4
0.8
1.2
PBSTrasT-Sia-LOFT-Sia 2 Isotype-SiaT-FcX-Sia
Tum
or v
olum
e (c
m )3
Sialidase activity
T-Sia 2 IsotypeSia
T-SiaLOF
T-FcXSia
+ + -HER2 targeting
FcγR binding
++ +-+ -+ [Antibody] (pM)
10 101010100 4321
g
25
15
5
Days
1.61.20.80.40.0
Tras
+p =
0.0003<0.0001<0.0001
0.0280.0008
+
PBSTras
T-Sia 2
T-Sia-LOFIsotype-SiaT-FcX-Sia
0.00020.00050.00030.0260.89p =
CD45
ce
lls/m
g (x
10 )5
15
0
5
10
20
2
4
CD8
/ T
reg
ratio
0
6
8p = 0.098
p = 0.0071p = 0.0033p = 0.0006 h
PBSTrasT-Sia 2
+
+ +
Time (days after tumor injection)
12
To better understand the mechanisms underlying T-Sia 2’s antitumor activity, we
synthesized three control molecules using the same conjugation strategy as with T-Sia
2 (Fig. 5a). The first was T-Sia 2 in which the sialidase’s catalytic nucleophile was
mutated (YàA), causing loss of function (T-Sia-LOF) (Supplementary Fig. 18a-c). The
second was an isotype control human IgG1-sialidase conjugate with the antibody
motavizumab that targets the respiratory syncytial virus, which has 87% identity to
trastuzumab (Isotype-Sia) (Supplementary Fig. 19a-f)44,45. The third was a variant
possessing several point mutations (ELLGàPVA-) in the FcγR binding domain of
trastuzumab, which abolishes most effector FcγR and CDC interactions on immune
cells yet maintains the majority of the neonatal Fc receptor (FcRn) binding and therefore
native antibody recycling (T-FcX-Sia) (Supplementary Fig. 20a-f)46,47. NK cell-mediated
ADCC assays with several HER2+ cell lines demonstrated that T-Sia 2 was superior in
eliciting cell death whereas T-Sia-LOF had comparable activity to trastuzumab alone
(Fig. 5b and Supplementary Fig. 21). As expected, Isotype-Sia was ineffective in
stimulating ADCC against HER2+ cells and T-FcX-Sia had greatly diminished FcγRIII-
mediated ADCC activity compared to T-Sia 2 in vitro.
The antibody-sialidase conjugates were then injected intraperitoneally into Balb/c mice
growing HER2+ EMT6 tumor cells in their mammary fat pads (scheme in Fig. 5c). For
PBS-, trastuzumab-, and T-Sia 2-treated mice, the tumor growth curves were consistent
with the previous mouse experiment: tumors in PBS- and trastuzumab-treated mice
grew quickly, while tumors in T-Sia 2-treated mice exhibited delayed growth (Fig. 5d
and Supplementary Fig. 22a). T-Sia-LOF-treated mice had tumor growth that was
indistinguishable from trastuzumab- and PBS-treated mice, indicating that sialidase
activity was necessary for therapeutic effect (Fig. 5d,e and Supplementary Fig. 22b). T-
Sia 2-treated mice showed an early delay in tumor growth that was not evident in T-
FcX-Sia-treated mice (Fig. 5e and Supplementary Fig. 22b); however, there was no
significant difference in time to sacrifice between the two treatment groups (Fig. 5f).
These data suggest that FcγR binding or CDC function may be more important in the
early phases of the antitumor response, and that T-Sia 2 is potentiating both FcγR-
dependent and -independent immune responses.
13
We observed a delay in tumor growth in mice treated with the Isotype-Sia construct
when compared to untreated, trastuzumab, or T-Sia-LOF controls (Fig. 5e). It is
possible that systemic exposure to sialidase activity potentiates immune cell reactivity
against the tumor through a more general loss of immunomodulatory sialoglycans.
Alternatively, the dose may be high enough to permit some desialylation of the tumor
(Supplementary Fig. 22c). Nonetheless, targeting the sialidase activity to the tumors
through conjugation to the trastuzumab antibody enhanced the therapy significantly; T-
Sia 2 was more effective at prolonging the time to sacrifice than Isotype-Sia (Fig. 5f)
and Isotype-Sia-treated tumors grew more rapidly than the HER2-targeted conjugate
(Fig. 5e and Supplementary Fig. 22b).
To further investigate effects of sialidase exposure, we analyzed blood cell counts
pursuant to administration of the above antibody-sialidase conjugates. Forty-eight
hours after the first injection of conjugates, red and white blood cell counts were
comparable to PBS-treated controls (Supplementary Fig. 22d,e) whereas the platelet
count decreased to an average of 157 platelets/nL blood in the mice treated with any
construct containing active sialidase (Supplementary Fig. 22f), likely mediated by the
asialoglycoprotein receptor in the liver, which binds to exposed galactose upon
desialylation48,49. Thrombocytopenia is an unfortunate but common symptom of cancer
treatment and some platelet loss may be a favorable prognostic biomarker of ICI
therapy50. None of the sialidase conjugates instigated weight loss or signs of ill health
in treated mice (Supplementary Fig. 22g).
T-Sia 2-treatment enhanced activated immune cell infiltration Tumors of untreated, trastuzumab, and T-Sia 2-treated mice were collected upon
sacrifice and the infiltrating immune cells were extracted and analyzed by flow
cytometry. The results from the combined mouse experiments showed an increase in
total tumor leukocytes in the T-Sia 2-treated mice compared to untreated- and
trastuzumab-treated mice (Fig. 5g). We also observed a general increase of tumor-
infiltrating lymphocytes (TILs) (Supplementary Fig. 23). This is encouraging as studies
14
have shown that high TILs following treatment of breast cancer patients are a favorable
prognostic marker for overall and metastasis-free survival51,52. Additionally, T-Sia 2
treatment augmented the ratio of CD8+ T cells to Treg cells (Fig. 5h), another indicator of
improved patient prognosis in breast cancer53.
Other immune marker changes in the T-Sia 2-treated tumors included an increase in
MHCII+ tumor associated macrophages (TAMs) and a decrease in CD206+ TAMs
(Supplementary Fig. 23), indicating a potential switch to a more inflammatory
macrophage polarization54. Additionally, T-Sia 2-treated tumors demonstrated an
increase in activated (CD69+, Fig. 6c,d) and cytotoxic (granzyme b+, Fig. 6e,f) CD8+ T
and NK cells55,56. Analysis of other immune cell populations showed trends suggesting
enhanced infiltration of dendritic- and B-cells (Supplementary Fig. 23). In summary, a
more infiltrated and activated tumor immune microenvironment could be observed with
activation of both innate and adaptive immune cells.
T-Sia 2 does not delay tumor growth in a Siglec-E knockout mouse tumor model Recent evidence suggests that Siglec-E (a Siglec-7/-9 homolog) is the major Siglec
present on the tumor infiltrating T cells of several cancer models including the EMT6
tumor model in mice17. We therefore hypothesized that Siglec-E might be a major
contributor to the immunosuppressive phenotype in these tumors and that destroying
Siglec-E ligands is an important mechanism of action of T-Sia 2 in mice. As there are
no known fully antagonistic blocking antibodies for Siglec-E, we decided to test our T-
Sia 2 conjugate in mice lacking inhibitory Siglec-E (Sig-E-/-), a mouse model with the
C57BL/6 background developed by the Crocker lab57.
15
Fig. 6. T-Sia 2 therapeutic effect is dependent on
functional Siglec-E in mice. a, HER2+ B16D5 cells
(5x105) were injected subcutaneously into either b, wt
C57BL/6 mice or c, Sig-E-/- C57BL/6 mice. On day 7, mice
were injected intraperitoneally with T-Sia 2 or the inactive
T-Sia-LOF (n=6 mice per group). Tumor size was plotted
for all surviving mice until a group reached n<4 surviving
mice. d, wt and Sig-E-/- mouse tumor volumes were plotted
and analyzed by unpaired t test on day 17, the final day
with n=6 surviving mice for each group.
To do this, we used HER2+ B16D5 melanoma
tumors, which expressed Siglec ligands that
were cleavable by ST sialidase (Supplementary
Fig. 24), and injected them subcutaneously into
the flanks of either wt or Sig-E-/- C57BL/6 mice,
after 7 days, mice were treated with T-Sia 2 or
the T-Sia-LOF control conjugate, and tumor
growth was assessed over time (Fig. 6a). As
expected, the T-Sia 2 molecule delayed tumor
growth in wt C57BL/6 mice compared to T-Sia-
LOF (Fig. 6b). Interestingly, in Sig-E-/- mice
there was no benefit of T-Sia 2-treatment over
the loss-of-function sialidase conjugate (Fig. 6c,d), indicating that the benefit of targeted
sialidase activity in vivo is dependent on functional Siglec-E in this mouse model.
Discussion
The correlation of hypersialylation with cancer progression was first reported in the
1960s58. In the 1970s, researchers explored the effects of sialidase treatment for
cancer therapy, but without the framework for a cogent mechanistic hypothesis and with
mixed results likely due to rapid recovery of cell-surface sialic acid58. The development
of modern techniques for bioconjugation, as well as recent breakthroughs in our
wt mice
0 5 10 15 20 25 300
0.25
0.5
0.75
1.0
1.25
1.5
Days after B16D5 HER2 injection
Tum
or s
ize (c
m3 )
T-Sia-LOFT-Sia 2
Treatments
Sig-E + T-Sia-LOFSig-E + T-Sia 2
Treatments
HER2 B16D5subcutaneous
injection
+
ConjugateIP injections
Days
Endpoint: tumor volume
7 131190
a
b
c
0 5 10 15 20 25 300
0.25
0.5
0.75
1.0
1.25
1.5
Days after B16D5 HER2 injection
Tum
or s
ize (c
m3 )
dp = 0.0028
Tum
or s
ize (c
m3 )
0.5
1.0
1.5
0Sig-E mice
p = 0.35
-/-
T-Sia-LOFT-Sia
-/--/-
16
understanding of the role of sialoglycans in numerous pathways of immune suppression
prompted us to explore whether tumor-targeted sialidase enzymes could now be
effectively harnessed for cancer therapy. These efforts yielded T-Sia 2, a trastuzumab-
S. typhimurium sialidase conjugate that significantly delayed tumor growth in a
trastuzumab-resistant cancer model. The use of two bioorthogonal chemistry tools – a
HIPS reaction with aldehyde-tagged antibodies and copper-free click chemistry –
enabled modular assembly of conjugates using common functionalized antibody and
enzyme moieties. Key factors in the performance of T-Sia 2 were its target specificity
and plasma stability imparted by antibody conjugation, which facilitated desialylation of
the tumor microenvironment that was measurable 2-4 weeks after conjugate injection.
T-Sia 2-treatment enhanced the CD8+ T cell/ Treg ratio in the tumors and showed an
increase in activated and cytotoxic NK and CD8+ T cells. These results are consistent
with literature that implicates NK cells15,16 and CD8+ T cells29 as key mediators of the
immune response against hyposialylated tumor cells. Much of T-Sia 2’s anticancer
activity appears to be mediated by the removal of ligands for immune-suppressing
mouse Siglec-E receptor. Several human Siglecs have now been implicated as
immune suppressors in the TME, including Siglecs 7, 9, and 1517,27,59,60, but the
importance of their relative contributions may vary from tumor to tumor. Although the
molecular details of their specific biological ligands remain an active area of research,
these Siglec family members all require terminal sialoglycans for ligand recognition, and
the chemical diversity and complexity of these sialic acids has made it challenging to
generate targeted therapeutics with broadly neutralizing activity. The antibody-sialidase
conjugates exemplified herein can destroy such sialoglycans in a Siglec-agnostic
manner. Further study of Siglec-sialoglycan biology in a range of different cancer
subtypes will be essential to elucidate the precise indications where sialidase
conjugates like T-Sia 2 may be most effective. Significantly, the modular chemical
synthesis of T-Sia 2 can be readily adapted to other FDA-approved therapeutic
monoclonal antibodies, allowing for simple and rapid targeting of sialidase enzymes to a
wide range of tumor types.
17
While this study demonstrates the efficacy of T-Sia 2 as a monotherapy, there is
significant future scope for combining antibody-sialidase molecules that target glyco-
immune checkpoints with more traditional ICI therapies or antibody-drug conjugates
(ADCs). Siglec receptors are expressed across a broad range of innate and adaptive
immune cells and Siglec-9+ CD8+ TILs co-express immune checkpoint inhibitors17,
indicating that T-Sia 2 may synergize with immune checkpoint agents. Recent evidence
also suggests that removal of surface sialic acid increases the internalization rate of
ADCs, indicating that targeted desialylation could enhance their cytotoxic effects61. We
anticipate that glycan-editing antibody therapies will prove a potent tool amongst the
wider arsenal of new anticancer therapies moving towards clinical translation.
Methods Statistical Analysis All statistical analyses were performed using GraphPad Prism 6. P-values are reported
for t-tests. For multiple t tests, an asterisk is used to indicate statistical significance
after correction for multiple comparisons using the Holm-Sidak method with α=0.05. For
one-way ANOVA, p-values of the ANOVA are reported if p>0.05; if p<0.05, post-hoc
statistics are reported (Tukey’s or Dunnet’s) as multiplicity-adjusted p-values. The p-
values of ordinary and RM two-way ANOVA analyses are reported on the figure or in
the figure legend; in Supplementary Fig. 8 and 16, post-hoc analyses were performed
after two-way ANOVA with Sidak’s and Dunnet’s multiple comparisons, respectively,
and are reported in the figures. For mouse survival curve analysis, a log-rank (Mantel-
Cox) test was used to compare between treatment groups.
General synthetic chemistry procedures All chemical reagents were purchased in reagent grade from commercial suppliers
including Sigma-Aldrich, Thermo Fisher Scientific, TCI, and BroadPharm and used
without purification. Unless stated otherwise, all chemical reactions were performed in
standard, oven-dried glassware fitted with a rubber septa under an inert atmosphere of
nitrogen using anhydrous solvents. Stainless steel syringes were used to transfer air-
and moisture-sensitive liquids. Anhydrous solvents (acetonitrile (ACN), diethyl ether,
18
dichloromethane, N,N-dimethylformamide, and tetrahydrofuran) were prepared by
passing the solvent through an activated alumina column. See supplemental methods
for detailed synthetic procedures and characterization of compounds.
Human cell lines Cell media, PBS, DPBS, and serum were purchased from Corning Mediatech unless
otherwise specified. SK-BR-3, ZR-75-1, and HCC-1954 were purchased from the
American Type Culture Collection (ATCC) and cultured in filtered RPMI media 10%
heat-inactivated FBS and no added antibiotics. BT-20, MDA-MB-468, MDA-MB-361,
MDA-MB-231, MCF-7, and MDA-MB-453 were purchased from ATCC and cultured in
DMEM media containing 10% heat-inactivated FBS and no added antibiotics. The
HEK-Blue cell line for endotoxin detection was thawed into growth medium (DMEM +
10% HI FBS + Penicillin-Streptomycin + 1X Normocin) and cultures were maintained in
growth medium + 1X HEK-Blue selection. Cultures were grown in T25 and T75 flasks
(Thermo Fisher) and maintained at 37 °C with 5% CO2. All cultures tested negative for
mycoplasma infection by the Lonza MycoAlert Mycoplasma Detection Assay.
Mouse cell lines EMT6 cells expressing the HER2 protein were a generous gift from the Zippelius lab
(University of Basel, Switzerland) and were cultured in Waymouth’s MB 752/1 medium
(Thermo Fisher) + 15% heat-inactivated FBS without antibiotic. B16D5 melanoma
tumors expressing HER2 were cultured in DMEM supplemented with 10% FBS, 1%
gluatamine, 1% sodium pyruvate, and 1% amino acids.
Human NK and T cell isolation procedure LRS chambers were obtained from healthy anonymous blood bank donors and isolated
using Ficoll-Paque (GE Healthcare Life Sciences, GE-17-1440-02) density gradient
separation. Cells were cultured in X-VIVO 15 media (Lonza, 04-418Q) supplemented
with 5% heat-inactivated human male AB serum (Sigma Aldrich, H4522). For some
experiments, recombinant carrier-free IL-2 (Biolegend, 1:2000 dilution, 589104) was
added to further activate NK cells overnight. After 12-24 h, NK cells were isolated from
19
PBMCs by immunomagnetic negative selection using the EasySep™ Human NK Cell
Enrichment Kit (STEMCELL Technologies Catalog # 19055) according to the
manufacturer’s protocol. Human γδ T cell were isolated from PBMCs by
immunomagnetic negative selection using the EasySep™ Human Gamma/Delta T Cell
Isolation Kit (STEMCELL Technologies, 19255) according to the manufacturer’s
protocol.
Protein gels and protein concentration Protein concentration was determined from the absorbance at 280 nm using
the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific) divided by the molar
extinction coefficient calculated from protein sequence using ExPASy server (provided
by the Swiss Institute of Bioinformatics)62. SDS-PAGE protein gels were run on 18 well
10% Criterion XT Bis-Tris Protein Gels (Bio-Rad, 3450112), 180 V, 40 min – 1 h.
Running buffer (2x) for protein gels was made in-house and contained: 4% sodium
dodecyl sulfate (Millipore Sigma L3771), 0.004% bromphenol blue (Sigma-Aldrich
B0126), 20% glycerol (Chem-Impex 00599), and 120 mM Tris HCl (Sigma-Aldrich
T5941), pH 6.8. For reduced gels, SDS page buffer additionally contained 10% 2-
mercaptoethanol, and proteins in 1x running buffer were heated in buffer to 95 °C for 5
min before loading onto gels. Protein gels were stained with AcquaStain Protein Gel
Stain (Bulldog Bio AS001000) and imaged on a LI-COR Odyssey CLx imaging system.
Protein sequences are reported in the supplementary methods
General DNA procedures and instrumentation DNA gBlocks were ordered from Integrated DNA Technologies (IDT). DNA primer
sequences were ordered from IDT or Elim Biopharmaceuticals. Plasmids were
sequenced by ELIM Biopharmaceuticals and analyzed using SnapGene 3.3.3. PCR
was performed in the C100 Touch Thermal Cycler from Bio-Rad. Unless otherwise
specified, PCR amplification was performed using the CloneAmp HiFi PCR premix
(Takara) with the following conditions.
20
Amplified DNA was purified by agarose gel: 1% agarose (Thermo Fisher 16500500) in
1x TAE buffer (Bio-Rad 1610773), containing 1x SYBR Safe DNA Gel Stain (Thermo
Fisher S33102) on a Bio-Rad PowerPac HC electrophoresis power supply at 120V, 40
min. DNA inserts were incorporated into plasmids using In-Fusion HD Enzyme premix
according to the manufacturer’s protocol (Takara) and transformed into Stellar
Competent Cells (Takara), an E. coli HST08 strain. DNA sequences are reported in the
supplementary methods.
Resuspending cells prior to flow cytometry or ADCC (Procedure #1) In all cases, breast cancer cells were split/processed 2-3 days prior to an assay, plated
on a T75 culture flask (Thermo Fisher), and allowed to grow to 60-90% confluence. To
lift cells for an assay, cells were first washed 1x with room temperature PBS –Ca –Mg,
(10mL), then 7 mL cell dissociation buffer pre-warmed to 37 °C was added and cells
were incubated at 37 °C with 5%CO2 until just lifted off the plate (5-20 min). Cell flasks
were rinsed vigorously with 7 mL normal growth media, transferred to 15 mL falcon
tubes, and pelleted by centrifugation at 300 x g for 5 min. Cells were resuspended in 2-5
mL assay media, counted with a Countess II FL Automated Cell Counter (Thermo
Fisher Scientific), and then diluted in assay media to desired concentration for assay.
General procedure for desialylation of cells (Procedure #2)
PCR conditions Amount to add Final concentration
2x HiFi Polymerase 12.5 µL 1x
Primer 1 5 pmol 0.2 µM
Primer 2 5 pmol 0.2 µM
Template DNA 10-100 pg
Water Add to 25 µL
95 °C 10 s
55 °C 10 s
72 °C 40 s (repeat steps 1-3, 29x)
72 °C 5 min
21
Cells were lifted as described in Procedure #1 and diluted to 1x106 cells/mL in normal
growth media. 200 µL of cells were added to V bottom 96-well plates (for mixed cell
assays, 100 µL MDA-MB-468 and 100 µL of SK-BR-3 were added to make 200 µL final
well volumes). Individual wells were treated with various concentrations of sialidase,
conjugates, or equivalent volume PBS. Cells were incubated with constructs for 1 h at
37 °C, 5% CO2. Following this, cells were pelleted in the plates by centrifugation at 300
x g for 5 min. Supernatant was removed and replaced with 200 µL PBS; this was
repeated a total of three times to complete washing of the cells prior to staining.
SNA staining procedure Following desialylation at various sialidase/T-Sia concentrations as described in
Procedure 2, cells were resuspended in PBS+0.5%BSA containing Alexa Fluor® 647
anti-human Her2 antibody and (FITC)-labeled Sambucus nigra lectin and incubated for
30 min at 4 °C in the dark. Cells were then washed 3x in PBS+0.5% BSA, resuspended
in PBS+0.5% BSA, and analyzed by flow cytometry on a BD Accuri C6 Plus Flow
Cytometer (BD Biosciences). Flow cytometry data was analyzed using FlowJo v. 10.0
software (Tree Star) and gated to distinguish HER2+ and HER2- cells as well as to
quantify SNA+ and SNA- cells. For the flow plot in Fig. 2b, >16,000 cells are shown for
each antibody treatment condition. For the analysis in Fig. 2a,c, & Supplementary Fig.
5 >5,000 cells are reported for each treatment.
Procedure for Siglec-Fc staining of cancer cells Breast cancer cell lines were treated as described in Procedure #1, followed by
treatment with 2 µM ST sialidase for 1 h (Procedure #2). After washing, cells were
resuspended in PBS+0.5% BSA containing Siglec-7, -9, or F-Fc that had pre-incubated
for 30 min at 4 °C with Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG, or Siglec-E-
mFc pre-incubated for 30 min at 4 °C with Alexa Fluor® 647 AffiniPure Goat Anti-Mouse
IgG. Stain was incubated with the cells for 30 min at 4 °C, followed by 3x washes and
resuspension in PBS+0.5% BSA and analysis by flow cytometry (BD Accuri C6 Plus or
LSR II). Gating was performed using FlowJo software to eliminate debris (FSC/SSC)
22
and analyze single cells (FSC-A/FSC-H), at least 2,000 cells are reported for each
replicate and treatment, except when otherwise noted as in Supplementary Fig. 17.
Determining KD of T-Sia 2 HER2+ HCC-1954 cells were lifted (Procedure #1), resuspended to 1×106 cells/mL in
PBS + 0.5% BSA, and 180 µL were distributed into wells of a 96-well V-bottom plate
(Corning). Various concentrations (20 nM - 0.01 nM) of trastuzumab or T-Sia 2 were
added to the cells in equal volumes and incubated with cells for 30 min, 4 °C. Following
this, cells were washed 3x in PBS + 0.5% BSA, pelleting by centrifugation at 300 x g for
5 min between washes. Cells were resuspended in Alexa Fluor® 488 AffiniPure Goat
Anti-Human IgG in PBS + 0.5% BSA for 15 min at 4 °C. Cells were further washed 2x
and resuspended in PBS + 0.5% BSA and fluorescence was analyzed by flow cytometry
(BD Accuri C6 Plus). Gating was performed using FlowJo v. 10.0 software (Tree Star)
to eliminate debris and isolate single cells. MFI (median fluorescence intensity) of the
cell populations were subtracted from control cells treated with secondary antibody only,
and normalized to the maximum MFI population from each experimental replicate.
Values were fit to a one site – total binding curve using GraphPad Prism 6, which
calculated the KD values as the antibody concentration needed to achieve a half-
maximum binding.
General procedure for activity assay of sialidases using 4-MUNANA. Sialidases were diluted (10 pM – 1 µM) in DPBS containing Ca2+ and Mg2+ in a 96-well
clear bottom black microplate (Corning 3904). Immediately before beginning the read,
2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (“4-MUNANA”, Sigma Aldrich,
M8639) was added to the sialidase solutions solution to a 1 mM final concentration, with
a final solution volume of 100 µL. Coumarin release was measured by fluorescence
(excitation 365 nm; emission 450 nm) in the SpectraMax i3x plate reader at 37 °C.
Background fluorescence was subtracted from control wells lacking enzyme, and
fluorescence was compared to a standard curve of 4-methylumbelliferone (0-150 µM)
(Sigma Aldrich, M1381) to calculate the amount of hydrolyzed substrate. Specific
23
activity (reported µmol substrate converted × min-1 per mg enzyme) was calculated from
the rate of hydrolysis in the initial linear range and mg of enzyme.
General procedure for an LDH ADCC assay After lifting the cells (Procedure #1, above) Cells were resuspended in 2-5 mL phenol-
red-free RPMI + 1% heat-inactivated FBS, counted with a Countess II FL Automated
Cell Counter (Thermo Fisher Scientific), and then diluted in media to desired
concentration. 1x104 cells were plated in 96-well V-bottom plates (Corning), and
trastuzumab, sialidase, conjugates, or PBS were added to a final volume of 100 µL and
treatments were pre-incubated with target breast cancer cells for 30 min. Next, 100 µL
of effector cells in phenol-red free RPMI + 1% heat-inactivated FBS were added at E/T
ratios ranging from 0-15 for the various assays. The assay plate was incubated at 37
°C + 5% CO2 for 4-8 h. Cells were pelleted by centrifugation at 500 x g, 5 min, and 50
µL of supernatant was transferred to a 96 well flat-bottom microplate to perform the LDH
Cytotoxicity Assay Kit (Pierce, 88953) according to the manufacturers protocol.
Absorbance was measured with the SpectraMax i3x plate reader at 490 nm and 680
nm, final cytotoxicity was calculated according to the assay kit manufacturer’s protocol.
NK cell-mediated ADCC by flow cytometry against EMT6 target cells Cells were lifted according to Procedure #1. Cells were resuspended in serum-free
media containing 5 µM CellTracker™ Green CMFDA Dye (Thermo Fisher Scientific,
C7025) and incubated at 37 °C for 30 min in 5%CO2. Cells were recollected by
centrifugation at 300 x g for 5 min and resuspended in 2-5 mL normal growth media,
counted with a Countess II FL Automated Cell Counter (Thermo Fisher Scientific), and
then diluted in media to desired concentration. Then, 2x104 cells/mL cells were pre-
treated with trastuzumab, PBS, or T-Sia 2 for 30 min at a final volume of 100 µL and
human NK cells (without IL-2 pre-treatment) were added at an effector:target ratio of
3:1. After 4 h, SYTOX™ Red Dead Cell Stain (Thermo Fisher Scientific, S34859) was
added at 5 nM final concentration to the cell mixture, which was analyzed on the BD
Accuri C6+ flow cytometer (BD Biosciences). Using FlowJo v. 10.0 software (Tree
Star), cells positive for CellTracker green in the FL1 channel were selected and gated
24
for live/dead by the red FL4 channel. Data are presented with PBS-treated cells
subtracted from the trastuzumab and T-Sia 2 treated mixtures (Fig. 4A)
Cloning and expression of proteins (antibodies, FGE, and sialidases) Cloning, sequences, and expression of proteins are described in the supplemental
methods.
Representative Procedure for Antibody cysteine à fGly conversion tbFGE in TEAM buffer (25 mM triethanolamine, 50 mM NaCl, pH 9) (101.4 µL, final 0.9
µM, 0.1 equiv.) was added to Motavizumab in TEAM buffer (3.7 mL, final 9 µM, 1
equiv.) and CuSO4 (5 mM stock in MQ water) was immediately added to 5 µM final
concentration. 2-Mercaptoethanol was added for a final concentration of 2 mM.
Conversion occurred at 37 °C, 400 rpm for 16 h and was analyzed by ESI-TOF mass
spectrometry. Detection of and quantification of fGly conversion is difficult because the
shift is small (-17 Da) and can exist as a hydrate very close to the original antibody
molecular weight (-1 Da). Antibodies were re-purified and concentrated by protein A
chromatography as described above and proceeded onto the next step.
Representative procedure for HIPS-azide addition to antibody Trastuzumab fGly was buffer exchanged by PD-10 column (GE Life Sciences,
17085101) into 50 mM sodium citrate and 50 mM NaCl, pH 5.5. A HIPS-azide aliquoted
solid stock was removed from -80 °C, dissolved in DMSO to 36 mM, and added (11.987
µmol, 20 equiv., 333 µL) to trastuzumab fGly in citrate buffer (600.6 nmol, 1 equiv.,
7.947 mL). Mixture was shaken at 230 rpm, 37 °C overnight, followed by PD-10 buffer
exchange into PBS. Only HIPS converted trastuzumab was detected, with a 96%
recovery of protein. Methods for copper-click labeling of fluorophore onto HIPS-azide
labeled antibodies and subsequent stability assays are further described in the
Supplementary methods.
Procedure for α-chloroacetamide labeling of ST sialidase
25
ST sialidase (1.04 µmol, 1 equiv., 16 mL) in 50 mM ammonium bicarbonate buffer pH
8.3 was incubated in the dark with TCEP (2 mM final concentration, 66 µL addition),
while shaking at 300 rpm for 10 min. α-chloroacetamide-DBCO was dissolved into
DMSO to 50 mM and added to the ST sialidase solution (20.88 µmol, 20 equiv., 417.6
µL), and the mixture was shaken in the dark at 300 rpm overnight at room temperature
in a 50 mL falcon tube. Reaction completion was determined by desalting an aliquot
(Zeba spin desalting columns, 7k MWCO, Thermo), and performing UV-vis
measurements at 280 nm and 309 nm to check for %DBCO conjugated. Once fully
conjugated, purification was performed using a HiLoad 26/600 Superdex 75 pg on the
ÄKTA pure chromatography system in PBS at 4 °C, followed by 0.2 µm syringe filtration
to avoid contamination.
Maleimide-PEG4-DBCO labeling of ST sialidase To ST sialidase (618 nmol, 30 mL, 1 equiv.) in PBS, TCEP was added (30 µmol, 60 µL)
(Fisher Scientific, T2556) and the mixture was rotated at 4 °C for 30 min protected from
light. Maleimide-PEG4-DBCO (12.36 µmol, 618 µL, 20 equiv.) (Click Chemistry Tools,
A108P) in DMSO was added and the reaction was mixed at 600 rpm for 2.5 days at 4
°C. Purification was performed using a HiLoad 26/600 Superdex 75 pg on the ÄKTA
pure chromatography system in PBS.
Representative synthesis of antibody-sialidase conjugate T-FcX-HIPS-azide (63.3 nmol, 1 equiv. 11.9 mL) and ST sialidase (316.9 nmol, 5 equiv.,
16 mL), both in PBS buffer, were mixed together and concentrated to ~25 mg/mL using
10,000 MWCO Amicon spin filters. The final mixture (~0.92 mL) was incubated at 25 °C
in the dark with shaking at 500 rpm for 3 days. The reaction was monitored by SDS-
PAGE and purified by size exclusion chromatography using a Superdex 200 increase
10/300 column on the ÄKTA pure chromatography system in PBS (GE Healthcare Life
Sciences) to remove unconjugated and aggregated protein, followed by protein A
chromatography to re-concentrate and further purify the antibody sialidase conjugates.
Final conjugates were buffer exchanged to PBS buffer with PD-10 columns and 0.2 µm
syringe filtered for sterility.
26
Endotoxin detection The HEK-Blue LPS Detection Kit (Invivogen, rep-lps2) was used according to the
manufacturer’s protocol. Briefly, serial dilutions of antibody-enzyme conjugates were
incubated with HEK-Blue cells both alone and spiked with 0.1 EU/mL Endotoxin
standard to verify that no inhibition occurred. After 24 h, an aliquot of the cell media
was incubated with QUANTI-Blue reagent (Invivogen) and absorbance was read on a
SpectraMax i3x Multi-Mode microplate reader and compared to a standard curve. For
all in vitro cell experiments and mouse experiments, endotoxin of the antibody-enzyme
conjugates was determined to be <1 EU/mg.
RP-HPLC Antibody conjugates (1 mg/mL) were buffer exchanged into 100 mM ammonium
bicarbonate + 8 M urea in water (pH 8.3). Stock DTT was added to 5 mM final
concentration and antibody was heated to 56 °C, with shaking at 700 rpm for 25-45 min
in an Eppendorf thermomixer. The sample was allowed to cool, spun down briefly in
tabletop centrifuge, and 14 mM iodoacetamide was added from a freshly prepared 500
mM stock in water; sample was protected from light and incubated at 25 °C with shaking
at 700 rpm for 30 min. The reaction was quenched by addition of another 5 mM DTT
and incubated again in the dark, at RT, 700 rpm, 15 min. Reversed-phase high
performance liquid chromatography was performed on an 1100/1200 series instrument
(Agilent Technologies) connected in-line to a UV-vis spectrophotometer. A total of 10
µg protein was injected onto a Zorbax 3.5 µm 300SB 300Å C8 2.1 x 50 mm column
(Agilent). RP-HPLC was performed at 0.9 mL/min at 60 °C using 0.1% trifluoroacetic
acid (TFA) in water (mobile phase A) and 0.1% TFA in acetonitrile (mobile phase B,
MPB). The 28 minute method consisted of a 4 min isocratic hold at 28% MPB, a linear
gradient for 6 min to 34% MBP, an isocratic hold at 34% MBP for 1.5 min to increase
the separation between HC and HC-Sia, and a 6.5 min linear gradient to 42% MBP,
followed by a 5 min wash using 100% MBP, and a 5 min re-equilibration at 28% MBP.
Antibody/Enzyme ratio (EAR) was calculated by integrating the area-under-the-curve of
the light chain and heavy chain peaks and calculating molar ratios of the antibody
27
chains using the protein extinction coefficients at A280. When HC and HC-Sia were not
perfectly separated, as in the case of motavizumab, the equation of [LC] = [HC] + [HC-
Sia] and the known extinction coefficients of each of the three chains was used to
determine the EAR.
Mass spectrometry of full-length proteins Protein samples (30 µL, ~10 µM) in PBS or 50 mM Ammonium bicarbonate buffer were
treated with 1 µL PNGaseF (New England Biolabs, P0704S) overnight at 37 °C.
Following this, the samples were analyzed by ESI-LC/MS on an Agilent 1260 HPLC and
Bruker MicroTOF-Q II time-of-flight mass spectrometer. The column was a Waters
BioResolve RP mAb Polyphenyl 450A 2.7u 100 x 2.1 mm maintained at 50 °C; the flow
rate was 0.3 mL/min, and the injection volume was 5 µL. Solvent A was 0.095% formic
acid and 0.05% TFA in water, solvent B was 0.1% formic acid in acetonitrile. The
gradient began with 5% B held for 1.5 min then ramped to 35% B at 2 min, 46% B at 10
min, and 95% B at 11 min held for 1 minute. Data was collected in full scan MS mode
with a mass range of 400-4000 Da with a Collision RF setting of 800 V. The protocol for
trypsin digest and mass spectrometry of digested proteins is detailed in the
Supplemental methods
T-Sia 2 treatment of EMT6 tumors in mice All mouse experiments were approved by the local Ethical Committee (Basel Stadt).
BALB/c mice were obtained from Janvier Labs (France) and bred in-house at the
University Hospital Basel, Switzerland. Animals were housed under specific pathogen-
free conditions.
For tumor growth experiments, 7-9 week old females were used. EMT6-HER2 cells
were injected into the right mammary gland of female BALB/c mice (1x106 cells in 40 µL
of PBS). Tumor size and health score, as well as weight, were measured and monitored
three times weekly. Perpendicular tumor diameters were measured by caliper and
tumor volume calculated according to the following formula: tumor volume (mm3) =
(d2*D)/2, where d and D are the shortest and longest diameters of the tumor (in
28
millimeters), respectively. Mice were sacrificed once tumor size reached 1000-500 mm3.
Animals developing ulcerated tumors were removed from the further analysis. All
treatments were given intraperitoneally. A total of four doses were administered every
second to third day once the tumor size reached approx. 100 mm3.
Sig-E-/- mouse experiments Sig-E-/- C57BL/6 mice were provided by Paul Crocker, College of Life Sciences,
University of Dundee, Dundee, UK57 and bred in-house at the University Hospital Basel,
Switzerland. Animals were housed under specific pathogen-free conditions. Briefly,
500,000 B16D5 cells expressing the HER2 protein were resuspended in PBS and
injected subcutaneously into the flanks of C57BL/6 mice (wt or Sig-E-/-). After 7 days,
mice were injected IP with 10 mg/kg T-Sia 2, T-Sia-LOF, or PBS 4x over 2 weeks.
Tumor size was measured as described above every 2-4 days until tumors reached
1500 mm3.
Leukocyte analysis For analysis of tumor-infiltrating immune cells, resected tumors were mechanically
dissociated and digested with a mixture of Accutase, collagenase IV, hyaluronidase and
DNAse type IV. Samples were filtered through a 70 µm mesh and tumor-infiltrating
lymphocytes were enriched by density centrifugation using Histopaque-1119 (Sigma).
Samples were frozen (90% FCS, 10% DMSO) and stored in liquid nitrogen until further
analysis.
Multicolor flow cytometry was performed on single cell suspensions. Samples were
incubated with fixable live/dead dye and Fc receptor block followed by staining with
primary antibodies. Stained samples were fixed with IC fixation buffer (eBioscience)
until time of analysis. For intracellular staining, samples were permeabilized after
fixation. All tumor samples were analyzed by flow cytometry using a Fortessa LSR II
flow cytometer (BD Biosciences, USA) and cells analyzed after serial duplet exclusion
and live/dead discrimination using FlowJo v. 10.0 software (Tree Star).
Lectin staining of mouse tumor digests
29
Lectin staining was performed using biotinylated PNA, SNA, MALII, and ConA obtained
from Vector Laboratories (USA). Single cell suspensions of tumor digests were
incubated with lectins at 10 µg/ml and detected in a second step using Streptavidin-PE-
Cy7 or Streptavidin-AF647, both for 20 minutes at 4 °C. Sialylation was assessed by
flow cytometry using a CytoFLEX (Beckman Coulter) cytometer or an LSR II and
quantified after live/dead and duplet exclusion using the geometric mean of PNA
staining. ConA was used as a sialic acid-independent control.
T-Sia 2 imaging in mice T-Sia 2 was labeled with 3x excess IRDye® 800CW NHS ester at pH 8.3 in PBS for 1 h.
After buffer exchanging to PBS, fluorophore labeling was quantified by nanodrop UV
measurements at 280 nm and 774 nm. T-Sia was found to have 1.7 fluorescent
molecules for every T-Sia 2 conjugate, which was then diluted to a 1:1
fluorophore:conjugate ratio using unlabeled T-Sia 2. Balb/c mice were then injected
subcutaneously with 1x106 cells in 100 µL PBS, after 5 days, mice were injected IP with
fluorophore-labeled T-Sia 2. Live mice were anesthetized with isoflurane and imaged at
2 and 4 days on an IVIS Lumina instrument (ex. 700 nm, em: 790 nm, 2s exposure
time), then sacrificed and organs and tumors were imaged ex vivo on the IVIS Lumina.
Tumors were then resuspended and stained for lectin binding as described above.
Blood counts Blood was collected from the tail vein of PBS, trastuzumab and T-Sia 2 treated mice
(Fig. 5) into EDTA-coated Microtainers (BD Biosciences) 48h after the first dose of
treatment. Generally, 40-60 µL of blood were diluted to a total volume of 240 µL using
0.9% NaCl. Complete blood counts were measured on the ADVIA120 Hematology
Analyzer using the Multispecies Version 5.9.0-MS software (Bayer) and adjusted to the
respective dilution factor.
30
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Acknowledgements We thank Drs. Steven Banik, CJ Cambier, and Simon Wisnovsky for their critical reading of this manuscript. We thank Theresa McLaughlin and the Stanford University Mass Spectrometry (SUMS) facility for performing intact protein characterization and HRMS analysis. We are grateful to the Zippelius lab for kindly providing the HER2+ EMT6 cell line, Dr. Eric R. Vimr for the gift of the plasmid pCVD364, Dr. David Rabuka (Catalent Pharma Solutions) for kindly providing humanized trastuzumab with an aldehyde tag and Dr. Mason Appel for generating the pET28-MBP-tev-tb-FGE plasmid used to make SMARTag antibodies. We thank Dr. Søren Christensen for the AU54pET9d* plasmid and Dr. Jennifer Kohler for the pGEX-Neu2 construct. Funding This work was supported in part by the Goldschmidt-Jacobson Foundation (to H.L), the Promedica Foundation (to M.A.S) and a Swiss National Science Foundation grant (SNSF Nr. 310030_184720/1), as well as a grant from the National Institutes of Health to C.R.B (NIH CA227942). M.A.G. was supported by the National Science Foundation Graduate Research Fellowship (NSF GRFP) and the Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program. N.R.M. was supported by
34
the Swiss Government Excellence Scholarship for Foreign Scholars and Artists (FCS) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico. S.A.M. was supported by an NIH F32 Postdoctoral Fellowship. J.T.T. received support from the Stanford Undergraduate Summer Research Program in Chemistry (Funding: Stanford VPUE/UAR). G.A. was supported by the NSF GRFP. P.A.W. was supported by the NSF GRFP and the Stanford ChEM-H Program. E.C.W. was supported by US National Institutes of Health Predoctoral Fellowship F31CA200544. Author contributions M.A.G., H.X., E.C.W, and C.R.B. conceived the project. M.A.G., M.S., H.X., J.F.A.P., N.R.M., S.A.M., J.T., C.L.M. G.A., and P.A.W., carried out experiments and interpreted data. M.A.G and C.R.B wrote the manuscript with input from all authors. H.L. and C.R.B. provided supervision. Competing interests M.A.G, H.X., E.C.W., and C.R.B., are inventors of the patent filed by Stanford University (international publication number WO2018006034A1) titled “Conjugates for targeted cell-surface editing” published on January 4, 2018 and licensed by Palleon Pharmaceuticals on 06/27/2017. C.R.B. is a co-founder and Scientific Advisory Board member of Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent) and InterVenn Biosciences, and a member of the Board of Directors of Eli Lilly & Company. H.L. received research and traveling support from Bristol-Myers Squibb. H.L. received traveling support from Merck Sharp Dome and Roche. H.L. is a member of the Scientific Advisory Board of Palleon Pharmaceuticals. Data and materials availability All data are available in the main text or the supplementary materials. List of Supplementary materials Supplementary Figures 1-24 Supplementary Methods NMR Spectra Supplementary References
download fileview on ChemRxivT2Manuscript_Gray-2019-11-11.pdf (1.60 MiB)
1
Supplementary Information
Targeted desialylation overcomes glyco-immune checkpoints and potentiates the anticancer immune response in vivo
Melissa A. Gray, Michal A. Stanczak, Han Xiao, Johan F. A. Pijnenborg, Natália R.
Mantuano, Stacy A. Malaker, Payton A. Weidenbacher, Caitlyn L. Miller, Julia T. Tanzo, Green Ahn, Elliot C. Woods, Heinz Läubli, Carolyn R. Bertozzi
Correspondence to: [email protected]
This PDF file includes:
Supplementary Figures 1-24 Supplementary Methods NMR spectra Supplementary References
2
Supplementary Figure 1. The PD-1 receptor and eight members of the human Siglec family have a homologous ITIM-ITSM cytosolic sequence motif and are expressed on a variety of immune cells. Both PD-1 and Siglecs contain an N-terminal binding domain to their ligands (PD-L1/2 and glycans, respectively), a helical single pass through the cell membrane, and an 82-126 amino acid cytosolic C-terminus containing an ITIM motif, a 12-18 amino acid spacer, and an ITSM or ITSM-like motif. Siglecs are expressed on a variety of immune cells; cell types that are Siglec+ or have Siglec+ subsets in humans are indicated above for each Siglec receptor. T = T cell, B = B cell, MDSCs = myeloid-derived suppressor cells, My = Myeloid-precursor, Mon = Monocyte, Mi = Microglia, Neu = Neutrophil, Mac = Macrophage, Troph = Trophoblast, NK = Natural killer cell, Eos = Eosinophil, Bas = Basophil, DC = Dendritic cell, Mast = Mast cell. Figure adapted from Angata et al63 and Riley24, with added cell expression information from multiple sources64-69.
Extracellular Cytosolic
VDYGEL TEYATI T, B, MDSCs
Neu, Mon, Mac, B, DC, Bas, Mast
NK, Neu, Mon, Mac, T, DC
ITIM Motif ITSM Motif
B, Troph, Bas, Mast
NK, Mon, Eos, T, DC, Mac
Eos, Bas, Mast, Mac
B, DC, T, Mon, DC, Eos, NK
Mac, Mi, Mon
LHYAVL TEYSEISiglec 6
IQYAPL NEYSEISiglec 7
LHYATL SEYSEISiglec 8
LQYASL TEYSEISiglec 9
LDYINV LHYATL ADYAEVSiglec 10
LHYASL TEYSEISiglec 11
PD-1
Cell Expression
LHYASL TEYSEISiglec 5
LHYASL TEYSEVSiglec 3
V/I/LxYxxL/V TxYxxI/V
Mon My, Mi, DC, Mac, Bas, Mast, Neu
18
amino
acid spacer
12
17
18
14
17
17
18
18
3
Supplementary Fig. 2. Chemistry of T-Sia version 1 linker previously reported30. T-Sia 1 was constructed by chemical conjugation of VC sialidase to a site near the C-terminus of each trastuzumab heavy chain30. SMARTag technology37 was employed to introduce site-specifically an aldehyde group onto trastuzumab’s heavy chains, which was then conjugated via oxime formation to an azide-terminated PEG linker. In parallel, a cyclooctyne group was incorporated onto VC sialidase through non-specific acylation of the enzyme’s lysine residues. The two proteins were finally joined by copper-free click chemistry41.
T-Sia version 1
Acid-sensitive linkerHeterogeneous conjugation sites
KM sizeSialidase:
HN
OONNN
ONH
OON
H
O
NH
O
23
4
Supplementary Fig. 3. ST sialidase is a small, stable sialidase that enhances NK cell-mediated ADCC towards breast cancer cells and efficiently cleaves Siglec-7 and -9 ligands at high concentrations. a, PAGE-SDS reducing gel showing relative purity of six recombinantly expressed and purified sialidases: Neu2, Neu3, Vibrio cholerae sialidase, Salmonella typhimurium NanH, Clostridium perfringens NanH, Arthrobacter ureafaciens NanH. b, Molecular weights and specific activities (in µmol substrate converted /min /mg enzyme) of sialidases determined by in vitro activity assays with the fluorogenic substrate 4-MUNANA (averaged from n=3 experimental replicates). c, NK cell-mediated ADCC assay against target BT-20 breast cancer cells treated with trastuzumab (Tras) and concentrated sialidases (Mean ± SD, n=3 experimental replicates). NK cells were IL-2 activated, and incubated with BT-20 target cells at an E/T = 4, %cytotoxicity was calculated from detecting the LDH release after 4 h. d, Significant activity of ST sialidase is maintained even after 2 years storage at 4 °C. In vitro activity assay with the fluorogenic substrate 4-MUNANA of freshly expressed sialidase compared with sialidase stored in PBS at 4 °C for 2 years reveals that the majority of ST sialidase activity is preserved (Mean ± SD, n=3 experimental replicates). e-g, Siglec ligand depletion by ST is effective across many breast cancer cell lines. ST sialidase (2 µM) was incubated with nine different breast cancer cell lines for 1 h and the removal of Siglec ligands were assessed by staining with Siglec-9-Fc (e) or Siglec-7-Fc (f) and anti-human-488 secondary antibody (control was treated with PBS followed by anti-human-488 secondary). Representative images shown of n=2 experiments,
PBS
ST
Control
PBS
ST
Control
PBS
ST
Control
a
% C
yto
toxic
ity
Tras
Sialidase
- + + + + + + +
- - Neu2 Neu3 VC ST AU CP
60
40
20
0
98
198
62
49
38
28
17
14
6
bSialidase MW Activity (U/mg)*
H. sapiens Neu2
H. sapiens Neu3
V. cholerae nanH
S. typhumirium nanH
C. perfringens nanH
A. ureafaciens sialidase
44
50
83
46
44
52
0.3 0.1
0.1 0.1 (N/A)
10.5 0.8
114 5
41 3
3.1 0.4
c
d
BT-20
MDA-MB-231
MDA-MB-468
SK-BR-3
HCC-1954 ZR-75-1
MCF-7 MDA-MB-453
MDA-MB-361
PBS
ST
Control
Alexa Fluor 488-Siglec-9-Fc
0
20
80
60
40
100
% S
igle
c-F
c f
luo
resce
nce
aft
er
ST
cle
ava
ge
7 9 7 9
Median
fluor. (au)
Mean
fluor. (au)
Siglec-Fc
PBS
ST
Control
PBS
ST
Control
e
BT-20
MDA-MB-231
MDA-MB-468
SK-BR-3
HCC-1954 ZR-75-1
MCF-7 MDA-MB-453
MDA-MB-361
f
g
0 10 20 300
20
40
60
new ST Sia
old ST Sia
Time (min)
Pro
du
ct
rele
ase
d (μ
M)
61 5%
of original
ST activity
10 10 10 102 4 6 8
10 10 10 102 4 6 8
Alexa Fluor 488-Siglec-7-Fc
5
each containing >2,000 cells. The mean and median (± SD) percent decrease in Siglec ligand-binding fluorescent signal upon ST treatment of the nine cell lines are quantified in g.
6
Supplementary Fig. 4. Maleimide-PEG4-DBCO was site-specifically incorporated at the ST sialidase C-terminus in the synthesis of the first trastuzumab-ST conjugate. a, ST sialidase expressed with the amino acid sequence SLCTPSRGS at the C-terminus was incubated with 20 mM TCEP at 4 °C in the dark for 10 min, then 20 equiv. maleimide-PEG4-DBCO was added and the reaction was rotated overnight at 4 °C and purified by size exclusion chromatography. Expected m/z shift was observed by ESI-TOF MS for addition of 1 DBCO molecule; no peaks were observed for m/z corresponding to multiple maleimide-PEG4-DBCO additions. b, A control reaction performed with ST sialidase that lacked the C-terminal peptide tag on sialidase resulted in no observable addition of DBCO to endogenous cysteine residues by ESI-TOF-MS.
46671.3
2
4
6
0
845996.1
91992.6
10000 20000 30000 40000 50000 60000 70000 80000 90000 m/z10 20 30 40 50 60 70 80 90
Inte
nsity
(x10
)
20 40 60 80 1003
Expected: 45996.5Observed: 45996.1, 91992.6
Expected: 46671.8Observed: 46671.3
ST-sialidase ST-DBCO
LCTPSR
SHN
O NH
O
OO O
ONH
O
N
O
O
LCTPSR
N
O NH
O
OO O
O
NH
ON
O
O
S
A
m/z(10 )
N
O NH
O
OO O
ONH
O
N
O
O
no reaction
B
22554.0
45107.8
4020 60 80m/z (10 )3
ST-sialidase: 45107.5ST-DBCO: 45782.8Observed: 45107.8
4
0.51.0
1.5
2.0
0100
3m/z(10 )
100
Inte
nsity
(x10
)4
0.5
1.0
1.5
2.0
0
7
Supplementary Fig. 5. Characterization of the preliminary ST sialidase-trastuzumab conjugate including selective desialylation activity against a HER2+ breast cancer cell line. a, Left: PAGE-SDS of ST sialidase (ST), ST Sialidase modified with maleimide-PEG3-DBCO (ST-DBCO), trastuzumab-oxime-azide (tras-Az), and trastuzumab ST sialidase conjugate (tras-ST). Right: ESI-TOF mass spectra of the light and heavy chains of tras-ST demonstrating ST sialidase addition selectively to the heavy chain. Below: representation of the trastuzumab ST sialidase molecule. b, In vitro activity assay of trastuzumab ST sialidase conjugate (tras-ST) (50 pM) with an Antibody/Enzyme ratio =2, and ST sialidase (100 pM). Release of the fluorescent 4-methylumbellierone from the 4-MUNANA fluorogenic probe was detected by plate reader (Mean ± SD, n=3). c, Representative of n=3 flow cytometry plots of the averaged data shown in Fig. 2C. The SNA stain is somewhat toxic to cells as is apparent by depleted cell count ratio of the HER2- cell line when the HER2+ cell line is desialylated.
HN O O
NN N
N
O
NH
O
OHN
O
N
O
O
S
3
4
HN
O
a
98198
62493828
1714
6
ST
ST
-DB
CO
tras
-ST
tras
-ST
tras
-Az
tras
-Az
nonreduced reducedb
Time (min)
Pro
du
ct
form
ed
(μ
M)
0.0256 nM 0.32 nM 1.6 nM 8 nM 200 nM40 nM
1000 nMT-Sia 1
Tras ST sialidase
Conjugate concentration c
95957.3 96775.9
96919.0
97323.10
1
2
3
4
523439.2
0
5
10
15
23300 23500 m/z m/z96000 97000
Inte
nsit
y (
x10 )
(au
)2
Trastuzumab-ST sialidase conjugate
0 5 10 15 20 250
20
40
60
80
100 pM free ST sia50 pM tras-ST
Expected: 23442.1
Observed: 23439.2
Expected: 96927.3
Observed: 96919.0
101010101010
7
6
5
4
3
2
10 10101020 4 6 Alexa Fluor 647 - HER2
FITC
- SN
A
8
Supplementary Fig. 6. Trastuzumab-ST enhances NK cell-mediated ADCC against many HER2+ cell lines using human NK cells from multiple donors; this effect increases with increasing E/T ratios. a, Percent cytotoxicity from IL-2 activated NK cell-mediated ADCC against six target breast cancer cell lines at E/T = 2 and E/T = 4 (in addition to the E/T =8 displayed in Fig. 2D). Cells were treated with PBS, ST sialidase (20 nM), trastuzumab (10 nM), or trastuzumab-ST sialidase (trastuzumab-ST, 10 nM). N=3 experimental replicates of % cytotoxicity detected with the LDH release method after 4 h. b, IL-2 activated NK cell-mediated ADCC from two additional biological NK donors on various breast cancer cell lines, E/T =4, n=3 experimental replicates, detecting LDH release after 4 h, statistical analysis by multiple t tests; an asterisk indicates statistical significance after correction for multiple comparisons using the Holm-Sidak method with an alpha of 0.05.
Trastuzumab-STTrastuzumabST sialidasePBS
60
40
20
0
2 4 6 8
% C
yto
toxic
ity
E/T ratio
60
40
20
0
2 4 6 8
% C
yto
toxic
ity
E/T ratio
60
40
20
0
2 4 6 8
E/T ratio
60
40
20
0
2 4 6 8
E/T ratio
60
40
20
0
2 4 6 8
E/T ratio
60
40
20
0
2 4 6 8
E/T ratio
SK-BR-3 MDA-MB-361
MDA-MB-231
ZR-75-1
BT-20 MDA_MB-468
a
b NK donor B NK donor C
NK donor A
PBSTrastuzumabTrastuzumab-ST
SKBR3 361 BT20
% C
yto
toxic
ity
HCC-1954 ZR-75-1 231 468
0
10
20
30
0
10
20
30
40
50
HER2 protein expression HER2 protein expression
p = 0.093
p = 0.0018
p = 0.00098
p = 0.90
p = 0.0027
p = 0.026p = 0.17
*
*
*
9
Supplementary Fig. 7. Synthesis of HIPS-azide linker. Synthetic route to HIPS-azide (1) and associated reaction yields. Synthesis of 3-9 was performed as described previously36, with a modified purification of compound 9. Synthesis and purification of compounds 9, 10, and 1 are described further in the methods section.
NH
HN 2 HCl
NH
FmocN
NH
OH
NH
OTBS
N
OTBS
COOMe
N
OH
COOMe
N
O
COOMe
N
N
COOH
H
N
O
COOH
H
NFmoc
3
4 5
6 7
8 9
FmocClTEAACN37%
TBSClimidazole
DCMqu.
DBUmethyl acrylate
ACN72%
TBAFTHF66%
Dess-Martin periodinane,
pyridineDCM83%
LiOHwaterTHF80%
1,2 dichloroethane87%
Na(OAc)3BH
Azido-PEG3-amineCOMU
2,4,6-trimethylpyridine
DMA75%
N3 OHN
ON
NFmocN
10
piperidineDMA86%3
N3 OHN
ON
NHN
1
3 NH
FmocN
3
10
Supplementary Fig. 8. Synthesis and characterization of the site-specific modification of HIPS-azide to the trastuzumab formylglycine residue. a, Tras-HIPS-azide was synthesized by reacting HIPS-azide (1) in DMSO with SMARTag-labeled trastuzumab under acidic conditions in citrate buffer for 24 h at 37 °C shaking. b, ESI-TOF mass spectra of reduced antibody chains before and after HIPS conjugation. c, Peptide mass spectrum of the trastuzumab heavy-chain C-terminal trypsin digested peptide covalently modified with the HIPS linker; HIPS addition was not detected on other trastuzumab cysteine residues.
1Citrate buffer
pH 5.5
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z
25
50
75
100
Rela
tive
Abu
ndan
ce
b2
b3b3
-18
b4b4
-18
b5
b6
b7
b8
b9
b10
S L S L S P G S L C T P S R
14 13 12 11 10 9 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14
y12y1
0
y9
y7y5
y4y3
y8++
M
0
23440.0
50022.4
20000 30000 40000 50000 60000m/z
15
10
5
0
Inte
nsity
x10
(au
)4
23440.2
50465.9
5
10
15
20
25
010000 20000 30000 40000 50000 60000
m/z
ExpectedLC: 23443.1HC: 49963.9
Observed:LC: 23440.0HC: 49878.5, 50022.4
ExpectedLC: 23443.1HC: 50321.8, 50465.7
Observed:LC: 23440.2HC: 50321.7, 50465.9
a
b
c
HN
N
NHN
OO N3
3
3NN
NHN
OO N3
NN
NN3
O
H
O
H
11
Supplementary Fig. 9. The HIPS linker used to make T-Sia 2 is more stable in serum and on living cells than the oxime linker. a, Alexa Fluor 647 Alkyne was reacted with trastuzumab-azide antibodies using copper-click chemistry. Trastuzumab-oxime-AF647 contains the oxime bond used in T-Sia 1; Trastuzumab-HIPS-AF647 contains the new HIPS linker. b, Trastuzumab-HIPS-647 and trastuzumab-oxime-647 were incubated at 0.56 mg/mL in 80% human plasma at 37 °C for several days. To determine presence of remaining conjugate, antibody-fluorophores were bound to HCC-1954 HER2+ cells and fluorescence was monitored by flow cytometry and compared to a standard curve of trastuzumab-AF647. After two days, there was significantly less trastuzumab-oxime-647 than trastuzumab-HIPS-647. Ordinary two-way ANOVA (p = 0.036) with Sidak’s multiple comparisons reported below each time point (n=3 experimental replicates, Mean ± SD). c-d, To determine the conjugates’ stabilities on cell surfaces and in endocytic compartments, 50 nM Tras-HIPS-647 and tras-oxime-647 were incubated on the surface of adherent HCC-1954 cells for 1 hour, and then solution was removed and replaced with normal growth media (c) or media containing protease inhibitor (d). Fluorescence was monitored by IncuCyte and total integrated fluorescence was quantified at 2 h time points. In both instances, the HIPS linker outlasted the oxime linker, and when proteases were inhibited there was minimal degradation of trastuzumab-HIPS-AF647, while trastuzumab-oxime-647 lost fluorescence on cells. e, Conditions from (b) were used, except on the surface of cells that had been first fixed with 4% PFA to prevent cellular endocytosis and proteolysis. On fixed cells without active endocytosis, both chemistries gave similar fluorescence signals over time, as expected in normal media with 10% heat-inactivated fetal bovine serum. Values are from n=2 (c) or n=3 (d,e) experimental replicates; where experimental replicates are each an average of two technical well replicates.
HN O O N
NN3
NN
NHN
OO 3
NNN
HIPSOxime
100
50
00 21 3 4
Time (days)
% S
tabl
e co
njug
ate p=
0.036
a
b c
Inte
grat
ed fl
uore
scen
ce
inte
nsity
(x10
)
4
0 1 2 3 40
10
20
30
40
50
Time (days)
d
Trastuzumab-oxime-AF647 Trastuzumab-HIPS-AF647
eIn
tegr
ated
fluo
resc
ence
in
tens
ity (x
10 )
4
0
5
10
15
20
0 1 2 3 4Time (days)
Inte
grat
ed fl
uore
scen
ce
inte
nsity
(x10
)
4
0
4
8
12
0 1 2 3 4Time (days)
p=0.017 p=
0.0029
p=0.094
p<0.0001
p<0.0001
p>0.99
p=0.99
12
Supplementary Fig. 10. Synthesis of α-chloroacetamide-DBCO (2). DBCO-PEG3-amine was treated with chloroacetic acid in the presence of EDC in DCM to yield α-Chloroacetamide-DBCO.
NO
HNO
O NH2
Cl OHO
4
EDCDCM77%
NO
HNO
O NH4
ClO
2
13
Supplementary Fig. 11. Site-specific conjugation of the α-chloroacetamide-DBCO molecule onto ST sialidase to make ST-DBCO. a, α-chloroacetamide-DBCO was conjugated to ST sialidase under mildly reducing and basic conditions (20 mM TCEP in 50 mM ammonium bicarbonate buffer pH 8.3, rotating overnight). b, ESI-TOF mass spectrum of sialidase-DBCO. c, Summary of results from tryptic ST digestion followed by HCD on the Orbitrap Fusion: all cysteine-containing peptides were detected. Only the cysteine in the C-terminal (SLCTPSR) peptide had a mass shift correlating to α-chloroacetamide-DBCO addition. d, MS2 spectrum of the modified SLCTPSR peptide from (c).
LCTPSR
SH
a
LCTPSR
b
N
O
NH
O
OHN
4S
O
N
O
NH
O
OHN
OCl
4
Inte
nsity
(x10
) (a
u)4
m/z
Expected: 46559.8Observed: 46560.2,
not observed:di-modified: 47123.0tri-modified: 47686.3
ST-alkyl-DBCO
cYFRIPAMC[+57]TTSKIPAMC[+57]TTSK
VMDPTC[+57]IVANIQGR
C[+57]EGFGSENNIIEFNASLVDNIR
VGNASGAGYSC[+57]LSYRKVGNASGAGYSC[+57]LSYR
SYNSLC[+563]TPSRSYNSLC[+774]TPSRSYNSLC[+57]TPSRSYNSLC[+563]TPSRGSSYNSLC[+57]TPSRGS
Cys containing peptides MA
2.39E+085.64E+10
1.04E+11
1.45E+07
1.48E+096.23E+10
4.29E+103.73E+091.05E+109.24E+077.48E+07
fmol
163870
7162
1
1024274
294125672065
no DBCO modification detected
no DBCO modification detected
no DBCO modification detected
no DBCO modification detected
81.5% of peptides detected were modified with DBCO
dFL0018367 #22789-23161 RT: 69.15-69.31 AV: 2 NL: 4.98E7F: FTMS + c NSI d Full ms2 [email protected]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
y7
b2
y6
x2
y8
y5y4
y3
b6b3
S Y N S L C T P S R 10 9 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 9 10
25
75
100
Rela
tive
Abun
danc
e
0
50
200
45996.4
91992.4
0
5
10
15
20
40000 50000 60000 70000 80000 90000
25
46560.2
46810.2
46400 46800 47200
46560.2
0
1
2
3
4
5
6
40000 60000 80000 100000 120000 140000m/z
Expected: 45996.45Observed: 45996.4, 91992.4 (dimer)
ST-Sia
14
Supplementary Fig. 12. An enzyme/antibody ratio of ~1 is smaller demonstrates improved NK cell-mediated ADCC over an enzyme/antibody ratio of ~2. a, Gel of purified fractions after size exclusion chromatography of T-Sia 2 separating large aggregates from di-sialidase T-Sia 2, mono-sialidase T-Sia 2, and trastuzumab alone. (Right of gel): two isolated collected fractions with an EAR (Enzyme/Antibody Ratio) ~1 and ~2. b, NK cell-mediated ADCC with three different NK cell donors against BT-20 cells treated with PBS, ST sialidase (20 nM), trastuzumab (10 nM), T-Sia EAR ~2 (10 nM), and T-Sia EAR ~1 (10 nM), n=3 experimental replicates for each biological NK cell donor. NK cells were IL-2 treated, followed by and incubation with target BT-20 cells at E/T = 1. Percent cytotoxicity was determined from the LDH release after 8 h, statistical analysis by one way ANOVA with Tukey’s multiple comparisons reported. c, Paired t-test from the three biological donors shown in (b) reveals a 1.13-fold increase in NK cell-mediated ADCC by EAR~1 over EAR~2.
aggregates
EAR ~1
EAR =2
EAR =1
EAR =0
EAR~2
19898
62
49
b
a
0
5
10
15
20
0
5
10
15
20
0
5
10
15
20
% C
ytot
oxic
ity
c
0
5
10
15
20 p=0.035
EAR~2
EAR~1PBS ST sialidase Trastuzumab EAR ~2 EAR ~1
p=0.35
p=0.0008p=0.58
p=0.0001
p=0.92
p=0.62p=0.22
p=0.025
p=0.24
p<0.0001
p<0.0001p=0.029
15
Supplementary Fig. 13. T-Sia 2 maintained sialidase activity and did not significantly alter the binding thermodynamics of trastuzumab alone to HER2+ cells. a, ESI-TOF mass spectrum of conjugated sialidase heavy chain. b, HPLC trace comparing T-Sia 2 and trastuzumab (tras). c, Flow cytometry assay showing trastuzumab and T-Sia 2 binding curve to SK-BR-3 breast cancer cells, n=3 experimental replicates, least-squares one-site total binding curve generated on GraphPad Prism software. KD values (right) from the binding curves were not significantly different by two-tailed t test.
NN
NHN
OO
N
NN
N
O
NH
O
OHN
OS
NN
N
3
4
97027.8
15
10
5
0
Inte
nsity
x10
3
20
40000 60000 80000 100000 120000 m/z
Expected: 96881.5, 97025.5Observed: 96882.4, 97027.8
A
B
T-Sia 2Trastuzumab100
0
50
perc
ent b
ound
0.1 10.01 10antibody concentration
100time (min)
4 6 8 10 12 14 16
tras
T-Sia 2
LCHC-ST
HC
aggregates
18
C
0
1
2
3
4
5
expe
rimen
tal K
D (n
M)
tras T-Sia 2
nsp= 0.16
16
Supplementary Fig. 14. T-Sia 2 enhances NK cell-mediated and gamma-delta T cell-mediated ADCC in vitro. a, NK cell-mediated ADCC with BT-20 target cells treated with PBS, 20 nM free ST sialidase, 10 nM trastuzumab, or 10 nM T-Sia 2. Results are shown from BT-20 cells alone (left) and from incubation with NK cells isolated from three distinct biological donors, (n=3), E/T = 4, no IL-2 activation, detecting LDH release after 4 h; statistical analysis by two-tailed t tests. b, A paired t test (two-tailed) of the three biological NK donors mediating ADCC showed the expected increase in killing with T-Sia 2 over trastuzumab alone. c, A γδ T cell-mediated ADCC assay targeting SK-BR-3 cells incubated with PBS, trastuzumab (10 nM) or T-Sia 2 (10 nM), (n=3), E/T = 3, IL-2 activated, detecting LDH release after 6 h; statistical analysis by two-tailed t test.
PBS St Sialidase Trastuzumab T-Sia 20
6
12
18
0
6
12
18
0
6
12
18
0
6
12
18no NK cells NK Donor 1 NK Donor 2 NK Donor 3
% C
ytot
oxic
ity
p = 0.013
p = 0.034
p = 0.0004
p = 0.0002
p = 0.003
Tras T-Sia 20
6
12
18%
Cyt
otox
icity
p = 0.039
0
5
10
15
a
b c
17
Supplementary Fig. 15. ST sialidase can efficiently cleave Siglec ligands from the mouse EMT6 cancer line. (Above): representative (n=3) flow cytometry histograms of Siglec-Fc binding to EMT6 cancer cells reveals that treatment with high ST sialidase concentration (2 µM) can drastically reduce human Siglec-7 and -9, and mouse Siglec-F binding to cells, more so than even genetic knockout of the GNE protein in the EMT6 cells (GNE is essential for the biosynthesis of sialic acid, but GNE KO cells can still decorate their cell-surfaces with salvaged sialic acid from the media). Greater than 5,000 cells are reported on each histogram, normalized to mode. (Below): Averaged median fluorescence of the n=3 replicates of the experiments shown above demonstrating a clear decrease in Siglec binding to ST sialidase-treated cells.
wt EMT6
ST-treated EMT6
GNE KO EMT6
ST-treated GNE KO
WT
WT + SIA
GNE
GNE + SIA
0.0
5.0 105
1.0 106
1.5 106
Siglec-7-Fc Siglec-9-Fc Siglec-F-Fc
Me
dia
n A
F4
88
flu
ore
sce
nce
(a
u)
WT
WT + SIA
GNE
GNE + SIA
0
2 106
4 106
6 106
8 106
1 107
WT
WT + SIA
GNE
GNE + SIA
0
1 106
2 106
3 106
4 106
Alexa Fluor 488 - Siglec-Fc
103
105
106
104
102
107
103
105
106
104
102
107
103
105
106
104
102
107
18
Supplementary Fig. 16. T-Sia 2 treatment prolongs mouse survival with EMT6 tumors compared to trastuzumab or PBS treatment. a, Survival curve demonstrating that both T-Sia doses (10 mg/kg and 15 mg/kg) significantly prolong mouse survival compared to PBS or trastuzumab-treated mice. Statistics: log-rank (Mantel-Cox) test between T-Sia (10mg/kg) and trastuzumab control. N=5 (PBS-treated) or n=6 (trastuzumab/T-Sia 2-treated mice). b, Mouse weight measured 5x over the course of 30 days of treatment and tumor growth. Ordinary two-way ANOVA with Dunnet’s multiple comparisons to untreated mice, multiplicity-adjusted p-values for T-Sia (15 mg/kg) compared to PBS are shown at three time points, (mean + SD upper error bar shown).
100
50
0
Days after tumor inoculation10 20 30 400 50
Perc
ent s
urvi
val*
a b
Mou
se w
eigh
t (g)
4 8 12 16 20 2420.0
22.5
25.0
27.5
30.0
Days after tumor inoculation
p = 0.021p = 0.069
p = 0.19
T-Sia 2 (10 mg/kg)T-Sia 2 (15 mg/kg)PBS
TrastuzumabTreatment
p = 0.0010
19
Supplementary Fig. 17. T-Sia 2 accumulates in the HER2+ EMT6 tumors in mice and can desialylate tumor cells even at low concentrations (~0.16 mg/kg, single injection). Live mice bearing HER2+ EMT6 subcutaneous tumors on their left flanks were photographed and their fluorescence was visualized and overlaid using the IVIS Lumina (ex: 700 nm, em: 790) at a, 48 h, and b, 4 days after 800-labeled T-Sia 2 IP
0
1
2
3
4
5500 pmol 100 pmol 20 pmolPBS
Tum
or
Sple
en
Heart
Liv
er
Kid
ney
Tum
or
Sple
en
Heart
Liv
er
Kid
ney
Tum
or
Sple
en
Heart
Liv
er
Kid
ney
Tum
or
Sple
en
Heart
Liv
er
Kid
ney
a
b
c d
e
500 pmol (~5 mg/kg) 100 pmol (~1 mg/kg) 20 pmol (~0.2 mg/kg) PBS control
Epi-fluorescence
2 d after injection
3.2
3.0
2.8
x107
Color scale
Min =2.72e7
Max =3.38e7
Amount of T-Sia injected
T-Sia injected (pmol)
500 100 20 0
Tumor
Spleen
Heart
Liver
Kidney
2.0 1.5 1.0 x107Color scale
Min =7.5e6
Max =2.3e7
Fold
change in m
axim
um
radia
nt
effic
iency o
ver
PB
S c
ontr
ol org
ans
****
**
******
SNA PNA MALII
T-S
ia inje
cte
d (
pm
ol)
0
20
100
500
Biotinylated lectins + AF647 streptavidin fluorescence (a.u.)
Epi-fluorescence
4 d after injection
4.0
3.5
3.0
x107
Color scale
Min =2.60e7
Max =4.48e7
****
20
injection. c, Representative photographs overlaid with fluorescent images of the tumor, spleen, heart, liver, and kidneys of these mice removed 4 days after conjugate injection. d, Maxium radient efficiency was quantified and normalized to the average of the PBS mouse organ control. A two-way ANOVA was performed with Dunnet’s multiple comparisons to the vehicle control, these results are shown as ****p<0.0001 and **p=0.0083, other p values not shown were all greater than p=0.15 and were deemed not significant (alpha = 0.05). e, Since some of the tumors were very small (particularly in the 500 pmol T-Sia-treated group), the three tumors taken from each mouse group were pooled, resuspended, and single tumor cells were analyzed for lectin staining by flow cytometry on an LSR II instrument. Although the SNA ligands in vivo were insensitive to T-Sia therapy at these doses, dramatic desialylation could be visualized at all doses by the more sensitive PNA (detecting exposed galactosyl (β-1,3) N-acetylgalactosamine) and MAL II (detecting α2,3 linked sialic acids) staining The three tumor samples from each mouse. Cell counts in flow cytometry were lowest for the 500 pmol-dosed mice, which had extremely small tumors at day 4. For MALII, SNA, and PNA staining staining n>2,600 for all groups except the 500 pmol dosed-mice had n= 827 and n=1,809, and n=234 cells represented, respectively. X-axis is displayed on a biexponential scale, y-axis is normalized to mode.
21
Supplementary Fig. 18. ST-LOF, a single alanine point mutation resulting in decreased ST sialidase activity, was expressed, purified, and conjugated to trastuzumab-HIPS-azide to make T-Sia-LOF. a, In vitro enzyme activity assay with the fluorogenic substrate 4-MU-NANA comparing the enzymatic activity of ST sialidase vs. ST-LOF sialidase, n=3 experimental replicates. b, Representative (n=3) RP-HPLC trace showing EAR = 1.1 for T-Sia-LOF. c, ESI-TOF mass spectrometry revealed unchanged light chain and an increased heavy chain mass indicative of LOF sialidase addition.
Time (min)M
ethy
lum
bellif
eron
ere
leas
ed (μ
M)
ST-wt
ST-LOF
a
100
50
00 2010 30
b
c
4 6 8 10 12 14 16 18Time (min)
EAR
1.1 0.1+
23440.4
0
1000
2000
3000
4000
5000
23000 23500 24000
96795.5
96944.0
100
200
300
400
96600 96800 97000 97200 97400
500
0
Inte
nsity
(au)
m/z
Expected: LC: 23443.1, HC: 96789.4, 96933.3Observed: LC: 23440.4, HC: 96795.5, 96944.0
m/z
T-Sia LOF
22
Supplementary Fig. 19. Development and characterization of Isotype-Sia, a nontargeting sialidase control. a, ESI-TOF mass spectrum of motavizumab expressed from Expi293 cells. b, ESI-TOF mass spectra of motavizumab light and heavy chains after partial heavy chain fGly conversion with tbFGE. c, ESI-TOF mass spectra of Isotype-HIPS-azide light chain (unchanged), and heavy chains (partially HIPS modified). d, ESI-TOF mass spectra of Isotype-Sia molecule. e, SDS-page gel of Isotype-Sia conjugate. f, Representative (n=3) RP-HPLC trace of Isotype-Sia conjugate, EAR = 1.2.
23123.850286.1
0.0
0.5
1.0
1.5
20000 40000 60000 80000m/z
Inte
nsity
(x10
) (a
u)5
Expected: LC: 23127.8 HC: 50308.0Observed: LC: 23123.8 HC: 50286.1
Isotype Antibody(Motavizumab)
Inte
nsity
(x10
) (a
u)4
23123.7
0
2
4
6
8
10
23000 23200 23400m/z
11
50268.150285.7
0
2
4
6
50200 50275 50350m/z
Expected: LC: 23123.8 HC: 50286.1 and 50268.1Observed: LC: 23123.8 HC: 50285.7 and 50268.1
a
bIsotype-fGly
196
96
26
38
49
62
Iso
Iso-
Sia
Iso
Iso-
Sia
full length reduced
e
f
0
5
10
15
20
25
30
50298.5
50687.3
0.0
0.2
0.4
0.6
0.8
50000 50400 50800 m/z
Expected: LC: 23123.8 HC: 50286.1 and 50685.4Observed: LC: 23124.1 HC: 50298.5 and 50687.3
Inte
nsity
(x10
) (a
u)3
1.0Isotype-HIPS
c
23124.1
22800 23000 23200 23400
97273.4
0
1
2
3
80000 100000 120000
4
Expected: LC: 23123.8 HC: 50286.1 and 97245.2Observed: LC: 23123.8 HC: 50298.1 and 97273.4
23123.8
23178.60
1
2
3
4
5
23050 23100 23150 23200 23250
6
50250.550298.1
50327.5
0
0.2
0.4
0.6
0.8
1
50200 50250 50300 50350
8 10 12 14 16
LCHC HC-Sia
no clear separation
Iso-Sia
Isotype antibody
Inte
nsity
(x10
) (a
u)3
d
EAR: 1.2 0.3+
m/z
23
Supplementary Fig. 20. Development and characterization of T-FcX-Sia, a control with reduced effector recruitment. a, ESI-TOF mass spectrum of T-FcX expressed from Expi293 cells. b, ESI-TOF mass spectra of T-FcX light and heavy chain after aldehyde conversion with tbFGE. c, ESI-TOF mass spectra of T-FcX-azide light and heavy chains. d, ESI-TOF mass spectra of T-FcX-Sia molecule. e, SDS-page gel of T-FcX-Sia conjugate. f, Representative (n=3) RP-HPLC spectrum of T-FcX-Sia, EAR = 1.1
23440.1
49863.6
0123456
20000 30000 40000 50000 60000 70000 80000 m/z
Inte
nsity
(x10
) (a
u)5
Inte
nsity
(x10
) (a
u)4
a
b
e
49846.1
49864.5
0.0
0.2
0.4
0.6
0.8
1.0
49800 49850 49900 49950 50000m/zm/z
23407.1
23438.9
0
10
20
30
40
50
23300 23400 23500
60
c
23440.0
23496.5
49873.2
50265.0
0
1
2
3
4
5
49500 50000 50500 51000m/z
Inte
nsity
(x10
) (
au)
3
6
0102030405060
23400 23450 23500 23550m/z
Expected: LC: 23443.1 HC: 49845.6 and 49863.6Observed: LC: 23438.9 HC: 49846.1 and 49864.5
Expected: LC: 23443.1 HC: 49863.6 and 50288.9Observed: LC: 23440.0 HC: 49873.2 and 50265.0
Expected: LC: 23443.1 HC: 49868.3Observed: LC: 23440.1 HC: 49863.6
196
96
26
38
49
62
T-Fc
X
T-Fc
X-Si
a
full length reduced
T-Fc
X
T-Fc
X-Si
a
f
23421.6
23439.5
23494.30
20
40
60
80
23350 23400 23450 23500 23550
49826.4
49873.1
49903.7
0
1
2
3
4
5
49750 49850 49950
96312.296453.6
96852.4
97265.10
0.2
0.4
0.6
0.8
1
96000 96500 97000 97500
4 6 8 10 12 14 16 18Time (min)
Expected: LC: 23443.1 HC: 49863.6 and 96853.3Observed: LC: 23439.5 HC: 49873.1 and 96852.4
Inte
nsity
(x10
) (
au)
3
d
EAR:
1.1 0.2+
m/z m/z m/z
24
Supplementary Fig. 21. NK cell-mediated ADCC with control constructs. NK cell-mediated ADCC performed as in Fig. 5b on two additional control cell lines. (Left): MDA-MB-468 HER2- control cell lines do not show any trends towards increased killing with increased antibody-sialidase construct concentration. (Right): SK-BR-3, the HER2-high expressing cell line demonstrates marked increase in ADCC with T-Sia 2, T-Sia LOF, and trastuzumab-treated cells, T-FcX is significantly reduced, and Isotype-Sia does not bind and desialylate target cells at these concentrations (Mean ± SD, n=3)
[Antibody conjugates] (pM)
Trastuzumab
T-Sia 2
T-Sia-LOF
Isotype-Sia
T-FcX-Sia
SK-BR-3
% C
ytot
oxic
ity
1 10 100 1000
0
10
20
30
40
50
[Antibody conjugates] (pM)
MDA-MB-468
1 10 100 10000
10
20
30
40
50
25
Supplementary Fig. 22. T-Sia 2, Isotype-Sia, T-FcX-Sia, T-Sia-LOF, and trastuzumab mouse experiment. a, Growth curves of the HER2+ EMT6 tumors of mice treated with T-Sia 2 (10 mg/kg) were consistent between the first experiment (shown in red, depicted in Fig. 4c, n=6 mice) and the second experiment (blue, depicted in Fig. 5e, n=7 mice); data are reported as mean ± SEM. b, Growth curves of mouse tumors (from Fig. 5d,e) extended out to the time of the first mouse death in each group (Mean + SEM, n=6-7). c, Lectin stain with PNA of exposed galactose from extracted tumor cells. Geometric mean ± SD of PNA/ConA, n=5-6, ordinary one-way ANOVA with Dunnet’s multiple comparisons to PBS control. d, Red and e, white blood cell counts for the mice (depicted in Fig. 5c) 2 days after first injection of conjugate therapy; ordinary one-way ANOVA (Mean counts ± SD, n=3-4). f, Platelet counts for the mice (depicted in Fig. 5c) 2 days after first injection of conjugate therapy; ordinary one-way ANOVA with Dunnet’s multiple comparison test ***p<0.0001 (multiplicity-adjusted) compared to trastuzumab-treated mice, ns indicates p>0.99 (Mean ± SD, n=3-4). g, Mouse weight measured 5x during treatment and tumor growth; ordinary two-way ANOVA revealed no significance in mouse weights over time (Mean ± SD, n=6-7).
0 10 20 30
1.2
0.8
0.4
0
Tu
mo
r v
olu
me (
cm
)
3
Time (days after tumor injection)
b
c d
e
0 8 16 24 32
Mouse expt. 1 (Fig. 4)
Mouse expt. 2 (Fig. 5)
a
Tu
mo
r v
olu
me (
cm
)
3
1.0
0.5
0
Time (days after tumor injection)
0
5
10
15
Wh
ite
blo
od
ce
lls /n
L
p = 0.26
Tras T-Sia
LOF
T-Sia
2
T-FcX
Sia
Iso
Sia
pla
tele
ts/n
L b
loo
d
10
10
10
1
2
3ns
*** *** ***
Tras T-Sia
LOF
T-Sia
2
T-FcX
Sia
Iso
Sia
Tras T-Sia
LOF
T-Sia
2
T-FcX
Sia
Iso
Sia
9
10
11
12
13
Re
d b
loo
d c
ell
s /
pL
p = 0.5
f
Untreated
Trastuzumab
T-Sia
T-Sia LOF
Isotype-Sia
Fc-Dead Sia
Treatment
0 5 10 15 2015
20
25
30
Days since tumor inoculation
We
igh
t [g
]
p >0.99
Tras T-Sia
LOF
T-Sia
2
T-FcX
Sia
Isotype
Sia
PBS
0.25
0.5
0.75
1.0
PN
A/C
onA
ratio (
gM
FI)
0.0
p =
0.99p =
0.86
p =
0.0061 p =
0.10
p =
0.056
g
26
Supplementary Fig. 23. Analysis of TILs in the PBS-, trastuzumab-, and T-Sia 2-treated tumor microenvironment after sacrifice. Top: Analysis of tumor infiltrating leukocytes quantified from two independent mouse experiments (Figs. 4 and 5) revealed an increase in %CD69+ CD8+ T cells and NK cells, as well as increased %granzyme B+ NK cells and CD8+ T cells. Additionally, a decrease in %CD206+ tumor-associated macrophages (TAMs) and an increase in MHC II+ TAMs was demonstrated. The analysis of cell types was analyzed by an ordinary one-way ANOVA with p-values shown; when p < 0.05, a post hoc test (Dunnet’s) was applied and multiplicity-adjusted p-values are reported comparing PBS- and trastuzumab-treated tumors with T-Sia 2- treated mice (n=8-15 mice/group, mean ± SD). B = B cells, conv CD4 = conventional CD4+ T cells, CD8+ = CD8+ T cells, DC = dendritic cells. Below: pie charts of percentages of TILs from PBS-, trastuzumab-, and T-Sia 2-treated mice.
7.84% B cells1.98% NK cells2.23% Tregs7.11% conv CD42.54% CD832.81% TAMs5.21% DCs40.28% Uncharact.
7.00% B cells1.88% NK cells2.83% Tregs11.93% conv CD43.30% CD836.52% TAMs6.62% DCs29.92% Uncharact.
11.40% B cells2.21% NK cells1.76% Tregs12.89% conv CD44.21% CD829.06% TAMs7.89% DCs30.59% Uncharact.
Untreated Trastuzumab T-Sia 2
% T
AM
of
CD
45
% C
D8
+ o
f C
D4
5
% T
re
g o
f C
D4
0
1
2
3
4
% B
of C
D4
5
p = 0.57
% C
D2
06
+ T
AM
S
p = 0.043
p = 0.027 p = 0.013
p = 0.50
% M
HC
-II+
TA
MS
p = 0.23 p = 0.092p = 0.51
0
5
10
15
0
10
20
30
40
50
0
2
4
6
0
5
10
15
20
% D
C o
f C
D4
5
% N
K o
f C
D4
5
p = 0.083 p = 0.69
0
1
2
3
0
2
4
6
8
10
0
20
40
60
80
0
10
20
30
0
10
20
30
40
50
% C
D4
of
CD
45
% T
reg
of C
D45
p = 0.37 p = 0.15
PBS Tras T-Sia 2
% g
zmb
NK
cells
50
40
20
0
PBSTras
T-Sia 2
0
10
20
30
40
50
% g
zmb
CD
8 T
cel
ls p = 0.063p = 0.043
p = 0.011p = 0.017
10
30+
++
0
20
40
80
60
%C
D69
C
D8
T c
ells
p = 0.039p = 0.098
+
+
100
0
80
20% C
D69
N
K ce
lls
p = 0.0002p < 0.0001
40
60+
27
Supplementary Fig. 24. Siglec-Fc staining of HER2+ B16D5 cells. HER2+ B16D5 cells cultured in vitro bound to commercially available mouse Siglec-Fc proteins (Siglecs -E and -F-Fc). The Siglec ligands detected on the B16D5 cells sensitive to 2 µM ST sialidase treatment for 1 h. y-axis shows cell count normalized to mode, x-axis is on a bi-exponential scale.
Siglec EPBS
ST
Siglec FPBSST
AF647 anti mouse-Fc fluorescence (a.u.)
AF488 anti human-Fc fluorescence (a.u.)
28
Supplementary Materials and Methods Additional general synthetic chemistry instrumentation Molecular sieves (Sigma-Aldrich, 688363) were flame-dried under vacuum and used immediately after cooling. Thin layer chromatography was conducted on SiliCycle silica plates (TLG R10011B-624) or C18 silica gel TLC plates (Analtech, 55077), with detection of multiband UV-absorption (254 – 365 nm). Column chromatography was done with Biotage SNAP KP-Sil (FSK0-1107) or Ultra C18 (FSUL-0401) flash purification cartridges (10 - 120 g) and an Isolera Prime ACI automated fraction collector from Biotage. The preparative RP-HPLC instrument used in this manuscript consists of an Agilent Technologies ProStar 325 UV-vis detector, two PrepStar solvent delivery modules, and a 440-LC fraction collector. The column for prep RP-HPLC was a Varian Microsorb 100 Å C18, 8 µm, 21.4 × 250 mm Dynamax preparative column (R0080220G8). Proton nuclear magnetic resonance (1H NMR) and proton-decoupled carbon-13 nuclear magnetic resonance (13C {1H} NMR) spectra were obtained on Mercury-400 and Varian-400 NMR spectrometers at 25 °C, are reported in parts per million downfield from tetramethylsilane, and are referenced to the residual protium or carbon resonances of the NMR solvent (CDCl3: 7.26 (1H), and 77.16 (13C), [CHCl3]). MestReNova v12.0.3 was used for all chemical NMR analysis. Data are represented as follows: chemical shift, multiplicity, coupling constants in Hertz (Hz), and integration. Splitting patterns are designated as follows: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, sept = septet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet. NMR signals were assigned on the basis of 1H, 13C, COSY, and HSQC experiments. Low resolution mass spectra of small molecules were recorded using an Agilent 1260 Infinity Quaternary LC and 6120 Quadrupole LCMS System. High resolution mass spectra of small molecules and proteins were performed by the Stanford University Mass Spectrometry (SUMS) core facility and recorded on an Agilent 1260 HPLC, a Bruker MicroTOF-Q II ESI-Qq-TOF, and a Thermo Exactive benchtop Orbitrap mass spectrometer. Mass spectrometry of digested proteins was performed on Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Cloning of trastuzumab-FcX and motavizumab antibodies The humanized trastuzumab protein containing the aldehyde tag was generously provided by Catalent Pharma Solutions. To make the trastuzumab-Fc-X antibody, the plasmid DNA pCDNA3.1 containing the trastuzumab antibody with an HC C-terminal aldehyde tag sequence (DNA Sequences 1 and 2) was digested with NheI/BsrGI endonucleases and a gBlock gene fragment (Sequence 3) encoding the desired mutations was inserted by In-Fusion HD cloning (Takara), transformed into Stellar competent DH5α E. coli, and re-transformed for further purity into One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, C4040). Plasmid was isolated using the EndoFree Plasmid Maxi Kit (Qiagen) according to manufacturer’s protocol.
29
For the motavizumab heavy chain plasmid, the CMV/R vector70 (a gift from the Peter Kim lab at Stanford) was PCR amplified in two segments (primer-01 and -02) and (primer-04 and -05) to incorporate an aldehyde tag at the end of the heavy chain. Following this an overlapping PCR with HiFi polymerase (using primer-01 and -04) on the two PCR amplicons was performed. An In-Fusion reaction of the backbone with a gBlock of the motavizumab heavy chain (Sequence 7) was performed and transformed into Stellar competent E. coli. To make the light chain plasmid, the D5-VRC01 plasmid was amplified with primers (Sequences 3 and 6), and an In-Fusion reaction was performed with the light chain motavizumab gBlock (Sequence 8), transformed into Stellar competent DH5α E. coli. Both plasmids were re-transformed for further purity into One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, C4040). Plasmids were isolated using the EndoFree Plasmid Maxi Kit (Qiagen) according to manufacturer’s protocol. Cloning of sialidases AU54pETd* was a gift from Dr. Søren Christensen and has been previously reported71. pET22b-T5-A99 was made using the NdeI and NotI cut-sites on the plasmid and inserting amplified CP sialidase DNA with primer-05 and primer-06. pGEX-T1-Neu2 is a girft from Dr. Jennifer Kohler, pGEX-T1-Neu3 was made using the BamHI and XhoI cut-sites and inserting the Neu3 sequence with primer-07 and primer-08. Expression of antibodies Antibodies were expressed using the Expi293 cell expression system (Thermo Fisher Scientific) following the manufacturer’s protocol. For the one plasmid system (trastuzumab-FcX antibody) 1 µg of DNA per mL culture volume was used, and for the two-plasmid system 0.5 µg of DNA per mL culture was used for each plasmid. Antibodies were harvested after 7 days, and supernatant was collected with brief centrifugation at 300 x g for 5 min. This was followed by prolonged centrifugation to clarify the supernatant at 3,200 x g for 30 min at 4 °C. Supernatant was then filtered through a 0.22 µm filter unit (Fisher Scientific 0974025A). Expi293 expression gave yields of ~100 mg/L (trastuzumab-FcX antibody) and ~45 mg/L (motavizumab) Protein A antibody purification Protein A - Sepharose® 4B (Thermo Fisher Scientific, 101041) beads (1-3 mL) were placed in chromatography columns (Bio-Rad 7321010), washed twice with 10 mL elution buffer (100 mM Glycine in MQ Water, pH: 2.8), and re-equilibrated with (3 x 10 mL) PBS washes. After filtering transfected Expi293 supernatant, the clarified supernatant was then loaded and run though the beads twice. Protein A beads were then washed 2-5x with 10 mL PBS, and antibodies were eluted (2 x 5 mL) into two 15 mL falcon tubes pre-loaded with 100 µL 1M Tris buffer pH 8 (Thermo Fisher Scientific, AM9856). Following this, antibodies were quickly buffer exchanged into TEAM buffer or PBS using PD-10 desalting columns (GE Life Sciences, 17085101). Expression of soluble tbFGE for aldehyde incorporation onto proteins. Our lab has previously reported expression of the formylglycine-generating enzyme from M. tuberculosis (tbFGE) for aldehyde conversion72, and in vitro aldehyde
30
conversions have been reported with other prokaryotic FGE enzymes73. Here we use a plasmid expressing an MBP-tbFGE fusion protein that maintains equal activity as tbFGE alone but has higher stability for in vitro conversion reactions, as well as a modified purification protocol. A 5 mL culture of His6-MBP-TEV-tbFGE in BL21 (DE3) E. coli (New England Biolabs, C2527I) in LB broth containing 100 µg/mL ampicillin was grown overnight. After 12-16 h, 2 culture flasks containing 1 L 2xYT media (BD Diagnostic Systems, 244020) were inoculated with 2 mL of the starter culture and grown at 37 °C with shaking at 200 rpm for 2-6 h until OD = 0.5. To assist in chaperone production, the flask was set in ice water to cool for 10 min to 4 °C, then IPTG was added to 100 µM final concentration, incubator temperature was reduced to 18 °C, and culture was shaken at 250 rpm overnight. After 20 h, the bacterial pellets were collected at 6,000 x g for 20 min, and frozen overnight at -80 °C. The next day, tbFGE was purified: bacterial pellets were resuspended in 100 mL lysis buffer (1xDPBS -Ca, -Mg, 150 mM NaCl, 10 mM imidazole, 1 mM freshly added TCEP, pH 7.5) + 2 protease inhibitor tablets (Roche, 0469315900) + 10 µL of Pierce nuclease (0.1 µL/mL) (Thermo Fisher Scientific, 88701). Pellet suspension was shaken at 150 rpm for 2 h at 4 °C in the presence of nuclease to reduce lysate viscosity. Cell lysis was accomplished by 3 passes through an Emulsiflex C3 homogenizer (Avestin) at 15 kPa, followed by centrifugation at 13,000 x g for 30 min. Meanwhile, nickel resin (Thermo Fisher Scientific, 88221) was equilibrated with water, 1x flow through, and lysis buffer 2x flow through. When lysed bacteria were finished centrifuging, supernatant was collected and equilibrated nickel resin was added, 3 mL per L of original culture. Resin was incubated at 4 °C for 30 min, then loaded onto a chromatography column, (Bio-Rad 7321010), washed with 900 mL washing buffer (1xDPBS -Ca, -Mg, 150 mM NaCl, 20 mM imidazole, 1 mM freshly added TCEP, pH 7.5), and eluted with 10 mL elution buffer (1xDPBS -Ca, -Mg, 150 mM NaCl, 250 mM imidazole, 1 mM freshly added TCEP, pH 7.5). Elution was concentrated with a 10,000 MWCO Amicon filter (Millipore Sigma, UFC901024) and buffer exchanged into TEAM buffer (25 mM TEA, 50 mM NaCl, pH 9) + 2 mM BME. Glycerol was added to a final concentration of 10% and tbFGE was flash-frozen in liquid nitrogen and stored as aliquots at -80 °C; final yield: 15 mg/L. Expression and purification of VC sialidase Escherichia coli C600 transformed with plasmid pCVD364 containing the V. cholerae sialidase gene was a generous gift from Eric R. Vimr (University of Illinois, Urbana–Champaign)74,75. Expression and purification was performed as previously reported 10. Representative expression and purification of sialidase with His-tag (ST, AU, CP) A 5 mL culture of N-His-ST Sialidase-C-Aldehyde tag in pET151 plasmid transformed into in BL21 (DE3) E. coli (New England Biolabs C2527I) was inoculated from a glycerol stock and grown overnight at 37 °C with shaking at 200 rpm in LB broth containing 100 µg/mL ampicillin (Sigma-Aldrich, A9518-25G). The following day, 3 L of Difco 2xYT media (BD Diagnostic Systems 244020) containing 100 µg/mL ampicillin were inoculated with the overnight culture transformed with the ST plasmid (1.5 mL/flask) cells were grown at 37 °C with shaking at 200 rpm for 4 h until the O.D. = 0.6, then IPTG was added (0.3 mM final concentration). The incubator temperature was then
31
reduced to 27 °C, with shaking at 250 rpm and the induced culture was allowed to grow for 16 h. Cultures were pelleted by centrifugation at 5,100 x g for 40 min, and pellet was frozen overnight at -80 °C. The next day, pellet was thawed and resuspended in 150 mL lysis buffer (PBS (Corning, 21-040-CMX12) + 150 mM NaCl, + 10 mM imidazole, pH 7.3) + 3 protease inhibitor tablets (Roche, 04693159001), and 15 µL Pierce nuclease (Thermo Fisher Scientific, 88701). The lysate was incubated with the nuclease at 4 °C with shaking at 150 rpm for 2 h to reduce viscosity. The bacterial lysate was then sent 3x through an EmulsiFlex-C3 homogenizer to lyse the bacteria and collected by centrifugation at 9,000 x g for 30 min. After this, supernatant was poured into a separate falcon tube and 5 mL of nickel resin (pre-equilibrated with 10 mL water followed by 10 mL lysis buffer) was added and incubated with the supernatant for 30 min at 4 °C. Nickel and supernatant were loaded onto a column (Bio-Rad) and washed with wash buffer (PBS (Corning) + 150 mM NaCl, + 20 mM imidazole, pH 7.3) until protein was no longer eluting as monitored by the Quick Start Bradford protein assay kit (Bio-Rad, 500-0202). Then protein was eluted with buffer containing 250 mM imidazole (11 mL), concentrated with a 10,000 MWCO Amicon filter (Millipore Sigma, UFC801024), and run 4x through endotoxin-removal resin (incubating 1 h each) (Thermo Fisher Scientific, 88275). Followed by buffer exchange using a PD-10 column ( Fisher Scientific, 17-0851-01). The final yield was 53 mg, which equates to 18 mg per L of bacterial culture. CP sialidase in pET22b-T5-A99 plasmid was purified following this same protocol, the AU sialidase in the AU54pET9d* plasmid was purified identically, except that kanamycin was used in the place of ampicillin as a selection marker. Procedure for GST purification of Neu2 and Neu3 proteins pGEX-T1-Neu2/Neu3 plasmids were transformed in to BL21 (DE3) E. coli (New England Biolabs C2527I). Cells were grown in Difco 2xYT media, supplemented with ampicillin (100 µg/mL), at 37 °C with shaking at 200 rpm overnight. The following day, a 1 L culture of Difco 2xYT broth containing ampicillin was inoculated from the overnight stock and grown for 3 h until OD = 0.6. Protein production was induced with 0.1 mM IPTG and cells were incubated at 30 °C with shaking at 200 rpm for 16 h. The next day, cell pellets were collected by centrifugation at 5,000 x g for 15 min. Cells were lysed in GST binding buffer (50 mM Tris-Cl (pH 8), 150 mM NaCl, 0.1 mM EDTA), with a dounce homogenizer followed by running 3x through an EmulsiFlex-C3 homogenizer. Clarified lysate was isolated by centrifugation at 13,000 x g for 40 min. Meanwhile the GST column was prepared by adding 0.5 mL glutathione sepharose beads (Sigma, GE17-0756-01) to a column (Bio-Rad, 7321010), and washing with 10 column volumes (CV) Elution buffer (50 mM Tris-Cl pH 8, 150 mM NaCl, 0.1 mM EDTA, and 10 mM freshly prepared reduced glutathione (Sigma)), followed by 30 CV of GST binding buffer. Clarified lysate was added to the prepared column, and beads were suspended by mixing. Beads were mixed with lysate for 30 min at RT on a rocker. The column was placed on a stand and beads were allowed to settle. The column was washed with GST binding buffer until no protein was detected in the flow through as monitored by the Quick Start bradford kit (Bio-Rad, 500-0202). Then, 0.5 CV GST Elution buffer was added to the beads and immediately eluted to make the first elution fraction. This was followed by a 10 min incubation with 0.5 CV GST Elution buffer on the beads before eluting to yield elution fraction 2. Beads were then washed with a further 1-5 CV of GST
32
Elution buffer and fractions were collected. Elution fractions were analyzed by SDS-PAGE and the purest fractions were collected to yield ~ 2mg protein / 1 L culture. Copper-click reaction for fluorophore labeling of antibodies SMARTag-labeled trastuzumab conjugated with the oxime-azide linker used to make T-Sia version 1, (Aminooxy-TEG-Azide, Berry & Associates, LK4270) or conjugated to the synthesized HIPS-azide (1) used to make T-Sia 2, were reacted with Alexa Fluor® 647 Alkyne (Thermo Fisher Scientific, A10278) using the following protocol: To azide-labeled antibody (265.5 µL, 14 µM) in PBS was added Alexa Fluor 647-alkyne (3 µL, 100 µM, from 10 mM DMSO stock). A premixed solution of 20 mM CuSO4 + 100 mM BTTAA in water was added to the reaction (1.5 µL, 0.1 mM CuSO4, 0.5 mM BTTAA). This was quickly followed by aminoguanidine (15 µL, 5 mM, from 100 mM PBS stock), and freshly dissolved sodium ascorbate (15 µL, 5 mM, from 100 mM PBS stock). The reactions were rotated slowly at RT, 1 h, under argon. After 1 h, EDTA was added (1 µL, 1.67 mM) to quench the copper, and proteins were buffer exchanged into PBS by repeated centrifugations and PBS washes in an Amicon Ultra-0.5 mL 50 kDa cutoff centrifugal filter unit (EMD Millipore, UFC505096). Equivalent conjugation to make the antibody-fluorophore conjugates trastuzumab-oxime-AF647 and trastuzumab-HIPS-AF647 was confirmed by absorbance at 280 nm and 650 nm wavelengths using the NanoDrop 2000 Spectrophotometer and calculating with the following equations: Protein concentration = (A280 – (A650 × 0.03)) / εprotein Dye ratio = A650 / 230,000 × protein concentration. Plasma stability assay Antibody-fluorophore conjugates were added to 80% human plasma at daily intervals and kept at 37 °C (final 0.56 mg/mL concentrations of antibodies in 80% plasma). On day 4, aliquots were diluted 1:193 in PBS and incubated on the surface of lifted HCC-1954 cells (Procedure #1) for 30 min on ice, followed by flow cytometry to detect conjugated fluorophore. Quantification was performed by comparing to a standard curve of cells incubated with antibody-fluorophores at 20, 10, 5, 1, and 0.1 nM. See supplemental methods for detailed information on the cell stability assay. Cell stability assay Trastuzumab-oxime-647 and trastuzumab-HIPS-647 were assessed for their stability on living HER2+ HCC-1954 cells. Briefly, HCC-1954 cells were plated at high confluence (4×104 cells/well) on a black-walled, clear-bottomed 96 well plate (Corning), and allowed to adhere for 24 h in normal growth conditions. In some experiments, cells were then fixed by washing 2x with PBS containing Ca2+ and Mg2+, followed by 15 min fixation in 4% PFA at RT, and 3x PBS washes; in other experiments, cells were kept alive without fixation. Antibody-fluorophores (50 nM) were then added in phenol-red free RPMI buffer (100 µL /well) containing 5 µM CellTracker Green CMFDA (Thermo Fisher Scientific, C7025) and incubated for 30 min 37 °C, 5% CO2. Then free antibodies were removed and replaced with normal growth media (phenol-red free). In some experiments, 100 µM leupeptin (EMD Millipore, EI8) was included in the media as a protease inhibitor. Fluorescence was monitored in an IncuCyte S3 Live Cell Analysis System (Sartorius) residing within a Thermo Fisher Scientific tissue culture incubator maintained at 37 °C
33
with 5% CO2. Data were acquired using a 10x objective lens in phase contrast, and red fluorescence (ex. 585 ± 20, em: 665 ± 40, acquisition time: 800 ms) channels. Images (1392 x 1040 pixels at 1.22 µm/pixel) were acquired from each well at 2 h intervals. The quantification of red fluorescence on live cells was analyzed with the following settings: Top-Hat segmentation with 100 µm radius and threshold adjustment: 0.2; Edge split on; Edge sensitivity -25; Hole fill 200 µm2; Filter area maximum 2600 µm2, mean intensity maximum 2.4 µm2. For fixed cells the analysis was: Top-Hat segmentation with 100 µm radius and threshold adjustment: 0.3; Edge split on; Edge sensitivity -35; Hole fill 0 µm2. The total red object integrated intensity (RCU × µm²/Image) from PBS-treated samples was subtracted from the integrated intensity of the antibody-fluorophore wells and is reported as “Integrated Fluorescence Intensity”. Trypsin digest and mass spectrometry of digested proteins Reduction and alkylation were performed according to ProteaseMax (Promega) protocols. Briefly, samples (5 µg) were diluted to 93.5 µL with 50 mM ammonium bicarbonate. Then, 1 µL of 0.5 M DTT was added and the samples were incubated at 56 °C for 20 min, followed by the addition of 2.7 µL of 0.55 M iodoacetamide at room temperature for 15 min in the dark. Digestion was completed by adding sequencing-grade trypsin (Promega) in a 1:20 enzyme:protein ratio overnight at 37 °C and quenched by adding 0.3 µL of glacial acetic acid. C18 clean-up was performed using SPEC tips (Agilent). Each tip was wet with 200 µL of methanol three times, followed by three 200 µL rinses of buffer A (5% formic acid in water). The samples were diluted to 200 µL in buffer A and loaded through the column 5-6 times, then rinsed three times with buffer A. Finally, the samples were eluted with three rinses of 100 µL buffer B (5% formic acid, 80% acetonitrile) and dried by speedvac. Samples were reconstituted in 10 µL of 0.1% formic acid (Thermo Fisher Scientific) and were analyzed by online nanoflow liquid chromatography-tandem MS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific). A portion of the sample (4 µL) was loaded via autosampler onto a 20-µL sample loop and injected at 0.3 µLŊmin−1 onto a 75-µm x 250-mm EASY-Spray column (Thermo Fisher Scientific) containing 2 µm C18 beads. The column was held at 40 °C using a column heater in the EASY-Spray ionization source (Thermo Fisher Scientific). The samples were eluted at 0.3 µLŊmin−1 using a 90-min gradient and a 185-min instrument method. Solvent A was composed of 0.1% formic acid in water, whereas solvent B was 0.1% formic acid in acetonitrile. The gradient profile was as follows (min/%B): 0:3, 3:3, 93:35, 103:42, 104:98, 109:98, 110:3, and 185:3. The instrument method used an MS1 resolution of 60,000 at FWHM of 400 m/z, an automatic gain control (AGC) target of 3e5, and a mass range from 300 to 1,500 m/z. Dynamic exclusion was enabled with a repeat count of 3, repeat duration of 10 s, and exclusion duration of 10 s. Only charge states 2–6 were selected for fragmentation. MS2 resolutions were generated at top speed for 3 s. HCD was performed on all selected precursor masses with the following parameters: isolation window of 2 m/z, 28–30% collision energy, orbitrap (resolution of 30,000) detection, and AGC target of 1e4 ions. All cysteine-containing peptides were manually sequenced using Xcalibur software and confirmed the presence of carbidomethyl (+57), DBCO
34
(+774), or reduced DBCO (+563). Extracted ion chromatograms were used to calculate relative abundance of the peptides for estimation of reaction efficiency. Antibodies and Usage Antibody or Lectin Source (#) Usage, dilution Alexa Fluor® 647 anti-human Her2 antibody Biolegend (324412) FC, 1:100 FITC Conjugated Sambucus nigra (Elderberry Bark) -SNA-I-, 1mg
EY Laboratories (F-6802-1) FC, 1:100
Siglec-7-Fc Chimera protein R&D Systems (1138-SL-050) FC, 1:50 Siglec-9-Fc Chimera protein R&D Systems (1139-SL-050) FC, 1:50 Siglec-F-Fc Chimera protein R&D Systems (1706-SF-050) FC, 1:50 Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific
Jackson ImmunoResearch (109-545-008)
FC, 1:375
PE/Dazzle™ 594 anti-mouse CD3 Antibody Biolegend (100246) FC, 1:100 Brilliant Violet 605™ anti-mouse CD4 antibody Biolegend (100548) FC, 1:100 Brilliant Violet 785™ anti-mouse CD8a Antibody
Biolegend (100750) FC, 1:100
Alexa Fluor® 488 Rat Anti-CD11b BD Pharmingen (557672) FC, 1:100 PE-Cy™7 Hamster Anti-Mouse CD11c BD Biosciences (561022) FC, 1:100 BV421 Rat Anti-Mouse CD19 BD Pharmingen (562701) FC, 1:100 FITC anti-mouse CD25 Antibody Biolegend (102005) FC, 1:100 CD45 Monoclonal Antibody (30-F11), PerCP-Cyanine5.5
eBioscience (45-0451-82) FC, 1:100
APC anti-mouse CD69 Antibody Biolegend (104514) FC, 1:100 APC anti-mouse CD206 (MMR) Antibody Biolegend (141708) FC, 1:50 PE Rat Anti-Mouse CD335 (NKp46) Clone 29A1.4
BD (560757) FC, 1:50
Alexa Fluor® 647 anti-mouse/rat/human FOXP3 Antibody
Biolegend (320014) FC, 1:100
APC/Cyanine7 anti-mouse I-A/I-E Antibody Biolegend (107628) FC, 1:200 Brilliant Violet 421™ anti-mouse F4/80 Antibody
Biolegend (123132) FC, 1:100
FITC Mouse anti-Human Granzyme B Clone GB11
BD (560211) FC, 1:100
PE/Cy7 Streptavidin Biolegend (405206) FC, 1:400 Biotinylated Concanavalin A (Con A) Vector Labs (B-1005) FC, 10 µg/mL Biotinylated Peanut Agglutinin (PNA) Vector Labs (B-1075) FC, 10 µg/mL Synthesis of Azido-PEG3-HIPS Compounds 3 – 8 were synthesized as previously described 13.
3-(2-((2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-1,2- dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoic acid (9): Prepared using a published procedure 13 with modified
N
COOH
N NFmoc
N
COOH
H
O
8 9
NH
FmocN
1,2-dichloroethane, 5 h
Na(OAc)3BH4Å MS
3
35
purification. To a solution of 8 (423 mg, 1.95 mmol, 1 equiv.) and (9H-fluoren-9yl)methyl 1,2-dimethylhydrazinecarboxylate, 3, (1.08 g, 3.83 mmol, 2 equiv.) in 1,2-dichloroethane (53 mL) with 4Å MS was added sodium triacetoxyborohydride (519 mg, 2.45 mmol, 1.3 equiv.). The resulting yellow suspension was stirred for 5 h and then quenched with NaHCO3 (saturated aqueous solution, 10 mL), followed by addition of HCl (1 M aqueous solution) to pH 4. The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (5 x 10 mL). The pooled organic extracts were dried over Mg2SO4, filtered, and concentrated to an orange oil. Purification by HPLC (20-90% ACN in water) gave 9 as an orange solid (821 mg, 1.70 mmol, 87%). The spectral data were in agreement with literature values36.
(9H-Fluoren-9-yl)methyl 2-((1-(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazinecarboxylate (10): A solution of 9 (1.2 g, 2.48 mmol, 1 equiv.) and 11-azido-3,6,9-trioxaundecan-1-amine (Azido-PEG3-amine) (819 µL, 3.72 mmol, 1.5 equiv.) in anhydrous dimethylacetamide (DMA) (9.92 mL) was cooled to 0 ˚C. Then, 2,4,6-trimethylpyridine (TMP) (655 µL, 4.96 mmol, 2 equiv.) and COMU (1.09 g, 2.48 mmol, 1 equiv.) were added and the reaction was stirred at 0 °C for 30 min, then warmed to RT and stirred for 4 h. The reaction mixture was diluted with ethyl acetate (150 mL), washed with 1 M HCl (3x 25 mL), 1 M NaHCO3 (3x 25 mL), and brine (3x 25 mL), dried over MgSO4, filtered, and concentrated to a viscous orange oil. Purification via C18 reversed-phase silica gel flash column chromatography (0-90% ACN in water) gave 10 as a light brown oil (1.26 g; 1.84 mmol; 75%). TLC: (water:ACN, 10:90 v/v) Rf = 0.72; (MeOH:DCM, 10:90 v/v) Rf = 0.52. 1H NMR: (400 MHz, CDCl3) δ 7.69 (d, J = 7.6 Hz, 2H, 2x Ar-CH Fmoc), 7.54 – 7.39 (m, 3H, 2x Ar-CH Fmoc & Ar-CH indole), 7.38 – 7.28 (m, 3H, Ar-CH indole & 2x Ar-CH Fmoc), 7.27 – 7.15 (m, 2H, 2x Ar-CH Fmoc), 7.15 – 7.02 (m, 2H, Ar-CH indole & NHCO ), 6.99 (t, J = 7.4 Hz, 1H, Ar-CH indole), 6.28 (s, 0.68H, Ar-CH indole), 5.95 (s, 0.44H), 5.82 (s, 0.38H), 4.65 – 4.21 (m, 4H, CH2 Fmoc & CH2-N indole), 4.15 (t, J = 6.1 Hz, 1H, CH Fmoc), 4.06 – 3.93 (m, 1H, CH2-NMe), 3.55 – 3.41 (m, 6H, 3x CH2 PEG), 3.39 (dd, J = 5.8, 3.5 Hz, 2H, CH2 PEG), 3.34 – 3.14 (m, 8H, 2x CH2 PEG & CH2-NHCO & CH2-N3), 2.82 – 2.72 (m, 3H, N-Me), 2.68 – 2.35 (m, 5H, CH2-CO & N-Me). 13C NMR: (101 MHz, CDCl3) δ 171.68 (NHCO), 155.50 (NCOO), 143.72 (2x Ar-C Fmoc), 141.17 (2x Ar-C Fmoc), 137.03 (Ar-C indole), 134.63 (Ar-C indole), 127.62 (2x Ar-CH Fmoc), 127.21 (Ar-C indole), 127.00 (2x Ar-CH Fmoc), 124.83 (2x Ar-CH Fmoc), 121.70 (Ar-CH indole), 120.39 (Ar-CH indole), 119.87 (2x Ar-CH Fmoc), 119.43 (Ar-CH indole), 109.52 (Ar-CH indole), 103.32 (Ar-CH indole), 70.34 (CH2 PEG), 70.26 (CH2 PEG), 70.17 (CH2 PEG), 69.83 (CH2 PEG), 69.71 (CH2 PEG), 69.28 (CH2 PEG), 67.15 (CH2-N indole), 50.63 (CH2-NMe), 50.41 (CH2-N3), 47.07 (CH Fmoc), 40.34 (CH2-OCO), 39.82 (CH3-N), 39.21 (CH2-NHCO), 36.86 (CH2CONH), 30.75 (CH3-N). ESI-HRMS: calc’d for C37H45N7NaO6 [M+Na]+:706.3324; found: 706.3339.
N
COOH
N NFmoc
9
NN
FmocN
HN
OON3
3
Azido-PEG3-amine, TMP, COMU
10
DMA, 0 ºC to rt, 4 h
36
N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanamide (1): 10 (95 mg, 138 µmol, 1 equiv.) was added to a stirred solution of piperidine (274 µL, 2.76 mmol, 20 equiv.) in DMA (1.38 mL) at RT. The reaction mixture was stirred for 1 h and directly purified via C18 reversed-phase silica gel flash column chromatography (0-100% ACN in water) affording azido-PEG3-HIPS (1) as light yellow solid (54.6 mg, 118 µmol, 86%) containing some impurities. This compound appeared to degrade in light and air and was therefore used at this purity and stored at -80 °C under argon. TLC: (water:ACN, 10:90 v/v) Rf = 0.72; (MeOH:DCM, 10:90 v/v) Rf = 0.52. 1H NMR: (400 MHz, CDCl3) δ 7.47 (d, J = 7.5 Hz, 1H, Ar-CH), 7.30 (d, J = 8.4 Hz, 1H, Ar-CH), 7.11 (t, J = 7.6 Hz, 1H, Ar-CH), 7.00 (t, J = 7.4 Hz, 1H, Ar-CH), 6.31 (s, 1H, Ar-CH), 6.05 (s, 1H, NH), 4.54 (t, J = 7.5 Hz, 2H, CH2-N indole), 3.80 (s, 2H, CH2-NMe), 3.53 (m, 6H, 3x CH2 PEG), 3.49 – 3.44 (m, 2H, CH2 PEG), 3.39 – 3.34 (m, 2H, CH2 PEG), 3.27 (d, J = 9.7 Hz, 6H, CH2 PEG & CH2-N3 & CH2-NHCO), 2.63 (t, J = 7.2 Hz, 2H, CH2-CO), 2.53 (s, 3H, N-Me), 2.35 (s, 3H, N-Me). 13C NMR: (101 MHz, CDCl3) δ 170.82 (CONH), 137.19 (Ar-C), 135.92 (Ar-C), 127.68 (Ar-C), 121.70 (Ar-CH), 120.49 (Ar-CH), 119.66 (Ar-CH), 109.62 (Ar-CH), 103.17 (Ar-CH), 70.78 (CH2 PEG), 70.66 (CH2 PEG), 70.60 (CH2 PEG), 70.25 (CH2 PEG), 70.13 (CH2 PEG), 69.74 (CH2 PEG), 55.93 (CH2-NMe), 50.75 (CH2-N3), 43.47 (CH3-N), 40.21 (CH2-N indole), 39.36 (CH2-NHCO), 37.20 (CH2-CO), 35.24 (CH3-N). ESI-HRMS: calc’d for C22H36N7O4 [M+H]+: 462.2823; found: 462.2815.
Synthesis of chloroacetamide-DBCO-PEG4 To a solution of chloroacetic acid (18.9 mg, 200 µmol, 1.2 equiv.) in DCM (189 µL) was added DBCO-PEG4-amine (87 mg, 167 µmol, 1 equiv.) in DCM (350 µL). The mixture was cooled in an ice bath to 0 °C and EDC (51.2 mg, 267 µM, 1.6 equiv.) in DCM (1 mL) was added. The mixture gradually came to RT and was stirred overnight. The mixture was concentrated in vacuo and then purified by flash chromatography on silica gel (0-10% MeOH in DCM) to give α-chloroacetamide-DBCO (2) as a yellow oil (77 mg, 77%), which was dissolved in DMSO and stored in aliquots at -80 °C. TLC:
NN
FmocN
HN
OON3
3
10
NN
HN
HN
OON3
3
1
piperidineDMA, 1 h
NHN O NH2
O O 4
OOHCl
EDC
2
DCMN
HN O N
HO O 4
OCl
37
(DCM:MeOH, 9:1 v/v) Rf = 0.4. 1H NMR: (400 MHz, CDCl3) δ 7.66 (d, J = 7.4, 1H), 7.44-7.23 (m, 7H), 7.10 (br s, 1H), 6.54 (t, J = 5.6 Hz, 1H), 5.12 (d, J = 13.9 Hz, 1H), 4.03 (s, 2H), 3.71 – 3.54 (m, 15H), 3.52 – 3.45 (m, 4H), 3.36-3.21 (m, 2H), 2.55-2.44 (m, 1H), 2.35 – 2.26 (m, 2H), 2.03 – 1.86 (m, 1H). 13C NMR: (101 MHz, CDCl3) δ 172.1, 171.1, 166.2, 151.2, 148.2, 132.2, 129.2, 128.7, 128.5, 128.4, 127.9, 127.3, 125.7, 123.2, 122.6, 114.9, 107.9, 70.71, 70.68, 70.63, 70.48, 70.45, 70.3, 69.5, 67.2, 55.6, 42.8, 39.7, 37.0, 35.3, 34.9. ESI-HRMS: calc’d for C31H38ClN3O7 [M+H+]: 600.2476, found: 600.2466. Antibodies and Usage Antibody or Lectin Source (#) Usage,
dilution Alexa Fluor® 647 anti-human Her2 antibody
Biolegend (324412) FC, 1:100
Alexa Fluor® 488 anti-human Her2 antibody
Biolegend (324410) FC, 1:100
FITC Conjugated Sambucus nigra (Elderberry Bark) -SNA-I-, 1mg
EY Laboratories (F-6802-1) FC, 1:100
Siglec-E-Fc Chimera protein R&D Systems (5806-SL-050) FC, 1:50 Siglec-7-Fc Chimera protein R&D Systems (1138-SL-050) FC, 1:50 Siglec-9-Fc Chimera protein R&D Systems (1139-SL-050) FC, 1:50 Siglec-F-Fc Chimera protein R&D Systems (1706-SF-050) FC, 1:50 Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific
Jackson ImmunoResearch (109-545-008)
FC, 1:375
Alexa Fluor® 647 AffiniPure Goat Anti-mouse IgG, Fcγ fragment
Jackson ImmunoResearch (115-605-071)
FC, 1:375
PE/Dazzle™ 594 anti-mouse CD3 Antibody
Biolegend (100246) FC, 1:100
Brilliant Violet 605™ anti-mouse CD4 antibody
Biolegend (100548) FC, 1:100
Brilliant Violet 785™ anti-mouse CD8a Antibody
Biolegend (100750) FC, 1:100
Alexa Fluor® 488 Rat Anti-CD11b BD Pharmingen (557672) FC, 1:100 PE-Cy™7 Hamster Anti-Mouse CD11c BD Biosciences (561022) FC, 1:100 BV421 Rat Anti-Mouse CD19 BD Pharmingen (562701) FC, 1:100 FITC anti-mouse CD25 Antibody Biolegend (102005) FC, 1:100 CD45 Monoclonal Antibody (30-F11), PerCP-Cyanine5.5
eBioscience (45-0451-82) FC, 1:100
APC anti-mouse CD69 Antibody Biolegend (104514) FC, 1:100 APC anti-mouse CD206 (MMR) Antibody
Biolegend (141708) FC, 1:50
PE Rat Anti-Mouse CD335 (NKp46) Clone 29A1.4
BD (560757) FC, 1:50
Alexa Fluor® 647 anti-mouse/rat/human FOXP3 Antibody
Biolegend (320014) FC, 1:100
38
APC/Cyanine7 anti-mouse I-A/I-E Antibody
Biolegend (107628) FC, 1:200
Brilliant Violet 421™ anti-mouse F4/80 Antibody
Biolegend (123132) FC, 1:100
FITC Mouse anti-Human Granzyme B Clone GB11
BD (560211) FC, 1:100
PE/Cy7 Streptavidin Biolegend (405206) FC, 1:400 Biotinylated Concanavalin A (Con A) Vector Labs (B-1005) FC, 10
µg/mL Biotinylated Peanut Agglutinin (PNA) Vector Labs (B-1075) FC, 10
µg/mL Biotinylated Maackia Amurensis Lectin II (MAL II)
Vector Labs (B-1265) FC, 10 µg/mL
Biotinylated Sambucus Nigra Lectin (SNA, EBL)
Vector Labs (B-1305) FC, 10 µg/mL
Streptavidin Streptavidin, Alexa Fluor™ 647 conjugate
Thermo (S21374) FC: 2 µg/mL
Protein Sequences Protein Amino Acid sequence Trastuzumab light chain (and T-FcX light chain)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Trastuzumab heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS
T-Fc-X heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS
Motavizumab light chain
DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Motavizumab heavy chain
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS
ST Sialidase MHHHHHHGKPIPNPLLGLDSTENLYFQGTVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYF
39
(N-HIS C-Ald) RIPAMCTTSKGTIVVFADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDDGVTFSKVETNIHDIVTKNGTISAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAHNLYSGEVKLIDAFYPKVGNASGAGYSCLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYNSLCTPSRGS
ST Sialidase Y369A (N-HIS C-Ald)
MHHHHHHGKPIPNPLLGLDSTENLYFQGTVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTSKGTIVVFADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDDGVTFSKVETNIHDIVTKNGTISAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAHNLYSGEVKLIDAFYPKVGNASGAGASCLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYNSLCTPSRGS
ST Sialidase (original construct, no aldehyde Fig. 2a and Supplementary Fig. 3,14a)
MHHHHHHGKPIPNPLLGLDSTENLYFQGTVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTSKGTIVVFADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDDGVTFSKVETNIHDIVTKNGTISAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAHNLYSGEVKLIDAFYPKVGNASGAGASCLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYN
VC sialidase MRFKNVKKTALMLAMFGMATSSNAALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGDVIFRGPDRIPSIVASSVTPGVVTAFAEKRVGGGDPGALSNTNDIITRTSRDGGITWDTELNLTEQINVSDEFDFSDPRPIYDPSSNTVLVSYARWPTDAAQNGDRIKPWMPNGIFYSVYDVASGNWQAPIDVTDQVKERSFQIAGWGGSELYRRNTSLNSQQDWQSNAKIRIVDGAANQIQVADGSRKYVVTLSIDESGGLVANLNGVSAPIILQSEHAKVHSFHDYELQYSALNHTTTLFVDGQQITTWAGEVSQENNIQFGNADAQIDGRLHVQKIVLTQQGHNLVEFDAFYLAQQTPEVEKDLEKLGWTKIKTGNTMSLYGNASVNPGPGHGITLTRQQNISGSQNGRLIYPAIVLDRFFLNVMSIYSDDGGSNWQTGSTLPIPFRWKSSSILETLEPSEADMVELQNGDLLLTARLDFNQIVNGVNYSPRQQFLSKDGGITWSLLEANNANVFSNISTGTVDASITRFEQSDGSHFLLFTNPQGNPAGTNGRQNLGLWFSFDEGVTWKGPIQLVNGASAYSDIYQLDSENAIVIVETDNSNMRILRMPITLLKQKLTLSQN
AU sialidase MGHHHHHHHHHHSSGHIEGRHMLEAPTPPNSPTLPPGSFSETNLAADRTAANFFYRIPALTYLGNDVVLAAWDGRPGSAADAPNPNSIVQRRSTDGGKTWGPVQVIAAGHVADASGPRYGYSDPSYIYDAEANKVFAFFVYSKDQGFGGSQFGNDDADRNVISSAVIESSDAGVTWSQPRLITSVTKPGTSKTNPAAGDVRSNFASSGEGIQLKYGPHKGRLIQQYAGDVRQADGSNKIQAYSVYSDDHGVTWHKGANVGDRMDENKTVELSDGRVLLNSRDNANRGYRKVAVSTDGGATYGPVSQDTELPDPANNGAIARMFPNAAQGSADAKKLIFTNANSKTGRENVSARVSCDDGETWPGVRTIRSGFSAYSTVTRLADGKFGVLYEGNYTDNMPFATFDDAWLNYVCAPLAVPAVNIAPSATQEVPVTVTNQEATTLSGATATVYTPSGWSATTVPVPDVAPGASVTVTVALTAPADASGPRSLNAAFTTADGRVSQFTFTATTPVAPQVGLTI
CP sialidase MRGSHHHHHHTDPCNKNNTFEKNLDISHKPEPLILFNKDNNIWNSKYFRIPNIQLLNDGTILTFSDIRYNGPDDHAYIDIASARSTDFGKTWSYNIAMKNNRIDSTYSRVMDSTTVITNTGRIILIAGSWNTNGNWAMTTSTRRSDWSVQMIYSDDNGLTWSNKIDLTKDSSKVKNQPSNTIGWLGGVGSGIVMDDGTIVMPAQISLRENNENNYYSLIIYSKDNGETWTMGNKVPNSNTSENMVIELDGALIMSTRYDYSGYRAAYISHDLGTTWEIYEPLNGKILTGKGSGCQGSFIKATTSNGHRIGLISAPKNTKGEYIRDNIAVYMIDFDDLSKGVQEICIPYPEDGNKLGGGYSCLSFKNNHLGIVYEANGNIEYQDLTPYYI
MBP-tbFGE MKSSHHHHHHGSSMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEENLYFQSNAMVLTELVDLPGGSFRMGSTRFYPEEAPIHTVTVRAFAVERHPVTNAQFAEFVSATGYVTVAEQPLDPGLYPGVDAADLCPGAMVFCPTAGPVDLRDWRQWWDWVPGACWRHPFGRDSDIADRAGHPVVQVAYPDAVAYARWAGRRLPTEAEWEYAARGGTTATYAWGDQEKPGGMLMANTWQGRFPYRNDGALGWVGTSPVGRFPANGFGLLDMIGNVWEWTTTEFYPHHRIDPPSTACCAPVKLATAADPTISQTLKGGSHLCAPEYCHRYRPAARSPQS
40
DNA oligomers Number DNA sequence primer-01 GGAATGTACACCGGTTGCAGTTGCTACTAGAAAAAG primer-02 TCTCTCTGCACCCCCTCCCGAGGTTCATGATGAGGATCCAGATCTGCTGTG primer-03 GGGGGTGCAGAGAGAACCCGGAGACAGGGAGAGG primer-04 GCGTCGACCAAGGGCCCATCGGTCTTC primer-05 GAATTCATTAAAGAGGAGAAATTACATATGCGTGGGTCGCATCACCACCACCACCA primer-06 GGTGGTGCTCGAGTGCGGCCGCCGCCAAGCTAGCTTGGATTTTATT primer-07 TCGGATCTGGTTCCGCGTGGATCCGAGGAGGTTACCACCTGCAGC primer-08 GTCAGTCACGATGCGGCCGCTCGAGGTTAGACTTGAATTGGGAGGG DNA sequences Name Sequence Sequence 1 Trastuzumab light chain DNA sequence including signal peptide (also the T-FcX light chain sequence)
ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCACGATGTGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCCTTCCTGTACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA
Sequence 2 Trastuzumab heavy chain DNA sequence including signal
ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCCACTCCGAAGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCAGGGGGCAGCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCACTGGGTCCGCCAGGCACCAGGCAAGGGACTGGAATGGGTGGCCCGGATCTACCCTACCAACGGCTACACCAGATACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTACCTGCAGATGAATTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTG
QDTATTHIGFRCVADPVSG
GST-thrombin-Neu2
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSMASLPVLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLNDRARSLVVPAYAYRKLHPIQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQLERPHRD
GST-thrombin-Neu3
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSEEVTTCSFNSPLFRQEDDRGITYRIPALLYIPPTHTFLAFAEKRSTRRDEDALHLVLRRGLRIGQLVQWGPLKPLMEATLPGHRTMNPCPVWEQKSGCVFLFFICVRGHVTERQQIVSGRNAARLCFIYSQDAGCSWSEVRDLTEEVIGSELKHWATFAVGPGHGIQLQSGRLVIPAYTYYIPSWFFCFQLPCKTRPHSLMIYSDDLGVTWHHGRLIRPMVTVECEVAEVTGRAGHPVLYCSARTPNRCRAEALSTDHGEGFQRLALSRQLCEPPHGCQGSVVSFRPLEIPHRCQDSSSKDAPTIQQSSPGSSLRLEEEAGTPSESWLLYSHPTSRKQRVDLGIYLNQTPLEAACWSRPWILHCGPCGYSDLAALEEEGLFGCLFECGTKQECEQIAFRLFTHREILSHLQGDCTSPGRNPSQFKSNLERPHRD
41
peptide and C-terminal aldehyde tag NheI cut site in red BsrGI cut site in blue Used for generating T-FcX antibody
GGGCCAGGGCACCCTGGTCACAGTGTCCTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTCTCTCTGCACCCCCTCCCGAGGTTCATGA
Sequence 3 gBlock used to replace the amino acids “ELLG” with “PVA-“ in the trastuzumab heavy chain to make Trastuzumab-FcX antibody.
AGTGTCCTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
Sequence 4 gBlock for Motavizumab (Isotype) antibody heavy chain variable sequence
ACCGGTGTACATTCCCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCGTCGACCAAGGGC
Sequence 5 gBlock for Motavizumab light chain variable sequence
ACCGGTGTACATTCAGACATACAGATGACCCAATCACCTTCCACGCTCAGCGCGTCTGTGGGCGACCGCGTCACGATTACTTGTTCAGCGTCCTCTCGGGTCGGGTACATGCACTGGTACCAGCAAAAGCCCGGTAAAGCACCGAAGTTGCTGATTTACGATACCTCCAAACTCGCTTCTGGTGTCCCATCCCGCTTCAGCGGTTCAGGGAGTGGTACCGAGTTTACACTGACTATTAGCAGTTTGCAGCCCGATGATTTCGCAACATACTACTGTTTTCAGGGGAGTGGATACCCATTTACGTTTGGCGGGGGTACAAAGGTGGAGATAAAGCGTACGGTGGCTGCA
MBP-TEV-tbFGE ATGAAATCTTCTCACCATCACCATCACCATGGTTCTTCTATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAA
42
CGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTCGGGATCGAGGAAAACCTGTACTTCCAATCCAATgcaATGGTGCTGACCGAGTTGGTTGACCTGCCCGGCGGATCGTTCCGCATGGGCTCGACGCGCTTCTACCCCGAAGAAGCGCCGATTCATACCGTGACCGTGCGCGCCTTTGCGGTAGAGCGACACCCGGTGACCAACGCGCAATTTGCCGAATTCGTCTCCGCGACAGGCTATGTGACGGTTGCAGAACAACCCCTTGACCCCGGGCTCTACCCAGGAGTGGACGCAGCAGACCTGTGTCCCGGTGCGATGGTGTTTTGTCCGACGGCCGGGCCGGTCGACCTGCGTGACTGGCGGCAATGGTGGGACTGGGTACCTGGCGCCTGCTGGCGCCATCCGTTTGGCCGGGACAGCGATATCGCCGACCGAGCCGGCCACCCGGTCGTACAGGTGGCCTATCCGGACGCCGTGGCCTACGCACGATGGGCTGGTCGACGCCTACCGACCGAGGCCGAGTGGGAGTACGCGGCCCGTGGCGGAACCACGGCAACCTATGCGTGGGGCGACCAGGAGAAGCCGGGGGGCATGCTCATGGCGAACACCTGGCAGGGCCGGTTTCCTTACCGCAACGACGGTGCATTGGGCTGGGTGGGAACCTCCCCGGTGGGCAGGTTTCCGGCCAACGGGTTTGGCTTGCTCGACATGATCGGAAACGTTTGGGAGTGGACCACCACCGAGTTCTATCCACACCATCGCATCGATCCACCCTCGACGGCCTGCTGCGCACCGGTCAAGCTCGCTACAGCCGCCGACCCGACGATCAGCCAGACCCTCAAGGGCGGCTCGCACCTGTGCGCGCCGGAGTACTGCCACCGCTACCGCCCGGCGGCGCGCTCGCCGCAGTCGCAGGACACCGCGACCACCCATATCGGGTTCCGGTGCGTGGCCGACCCGGTGTCCGGGTAG
AU Sialidase from AU54pETd*
ATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGTCATATGCTCGAGGCCCCCACTCCGCCCAATTCGCCCACGCTTCCACCGGGCAGCTTCTCTGAAACCAATCTGGCGGCCGACCGCACGGCGGCGAATTTCTTCTACCGGATTCCCGCGCTTACCTACCTTGGCAACGACGTGGTCCTTGCAGCGTGGGACGGTCGCCCGGGTTCGGCGGCGGACGCCCCGAACCCGAACTCGATCGTCCAGCGCCGAAGCACGGACGGTGGCAAGACCTGGGGGCCGGTCCAAGTGATCGCCGCAGGCCACGTCGCCGATGCCAGCGGCCCTCGATACGGCTACAGCGATCCCTCGTACATCTACGACGCGGAAGCCAACAAGGTCTTCGCTTTCTTCGTGTACTCGAAGGACCAAGGCTTTGGCGGCAGTCAGTTCGGCAACGACGACGCGGACCGGAACGTCATTTCCTCCGCCGTCATCGAGTCTTCCGACGCCGGCGTGACATGGAGCCAGCCCCGCCTCATCACCTCCGTCACCAAGCCGGGTACCAGCAAGACCAACCCGGCAGCCGGCGACGTCCGCTCCAACTTTGCCTCCTCCGGTGAGGGCATCCAGCTCAAATACGGCCCGCACAAGGGCCGTCTCATCCAGCAGTACGCCGGCGACGTGCGGCAAGCTGACGGAAGCAACAAGATCCAGGCCTACAGCGTCTATTCAGACGATCACGGCGTCACGTGGCACAAGGGTGCCAACGTGGGCGACCGGATGGACGAGAACAAGACTGTGGAACTGTCCGACGGTCGGGTCCTGCTCAACTCCCGGGACAACGCCAACCGGGGCTACCGCAAGGTGGCCGTCTCCACGGACGGCGGAGCCACGTACGGCCCCGTCAGCCAGGACACGGAATTGCCGGACCCTGCCAACAACGGTGCAATCGCCCGCATGTTCCCCAACGCGGCGCAGGGCTCCGCAGACGCGAAGAAACTGATCTTCACCAACGCAAACTCCAAGACCGGCCGCGAAAACGTCTCGGCCCGGGTCTCCTGTGACGACGGCGAAACCTGGCCGGGCGTCCGCACCATCCGTTCCGGCTTCTCGGCCTACTCAACAGTGACCCGCCTGGCGGACGGAAAGTTCGGCGTCCTCTACGAGGGCAACTACACGGACAACATGCCCTTCGCCACCTTCGACGACGCGTGGTTGAACTACGTCTGCGCTCCCTTGGCAGTACCGGCAGTCAACATCGCCCCGAGCGCAACGCAGGAGGTTCCGGTGACCGTCACTAACCAGGAAGCAACCACGCTTTCCGGCGCGACCGCAACTGTCTATACGCCGTCGGGGTGGTCTGCCACCACGGTGCCCGTGCCCGACGTCGCCCCCGGCGCGTCCGTCACCGTGACCGTTGCACTGACCGCACCGGCGGACGCCAGTGGCCCGCGCAGCCTCAACGCGGCATTCACGACGGCGGATGGCCGGGTTTCGCAGTTCACCTTCACCGCCACCACGCCCGTGGCTCCGCAAGTGGGCCTTACCATCTAA
CP sialidase pET22b-T5-A99
ATGCGTGGGTCGCATCACCACCACCACCACACGGATCCTTGTAATAAGAATAACACATTCGAGAAAAACCTGGATATTTCTCACAAACCGGAACCTCTGATTCTGTTTAATAAGGATAACAACATTTGGAATTCCAAATACTTTCGTATTCCAAACATTCAACTCCTGAATGACGGTACGATTCTTACCTTTTCCGACATCCGGTATAATGGGCCGGATGATCACGCATATATTGATATCGCGAGCGCTCGCTCTACCGACTTTGGTAAAACGTGGAGCTATAACATTGCGATGAAAAACAACCGGATTGACAGTACATATTCACGTGTCATGGATTCAACGACCGTAATTACTAACACCGGCCGTATTATTCTGATCGCAGGCTCGTGGAATACAAACGGTAATTGGGCAATGACGACTTCTACCCGTCGTTCTGACTGGAGCGTTCAGATGATCTACAGTGACGATAACGGACTGACGTGGTCCAATAAAATCGATCTGACGAAAGACTC
43
AAGCAAAGTGAAGAACCAGCCTTCAAATACCATTGGCTGGCTTGGTGGGGTTGGTTCGGGTATTGTTATGGATGATGGCACCATTGTTATGCCTGCGCAGATCAGTTTACGCGAAAATAATGAGAACAATTATTATAGCCTGATTATTTATTCAAAGGATAACGGCGAAACGTGGACTATGGGCAATAAAGTGCCGAACAGTAACACCTCAGAGAACATGGTGATCGAGCTGGATGGTGCTCTGATTATGTCCACCCGTTACGATTACAGCGGGTATCGCGCCGCCTACATCTCGCATGATCTGGGCACCACGTGGGAGATTTATGAACCTCTGAACGGAAAAATTTTGACTGGTAAAGGTTCAGGCTGCCAAGGCTCTTTCATTAAAGCGACCACCAGCAACGGGCATCGTATCGGACTGATTTCTGCCCCTAAAAACACCAAAGGCGAGTACATTCGCGATAACATCGCCGTGTACATGATTGATTTTGATGACCTCTCAAAAGGCGTGCAGGAGATTTGCATTCCGTATCCAGAAGATGGCAACAAACTGGGTGGGGGGTACTCGTGTCTGTCATTTAAAAATAACCATCTGGGAATTGTGTACGAAGCGAACGGGAATATTGAATATCAGGACCTGACCCCGTACTATATC
GST-thrombin-Neu2
ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGTTCCGCGTGGATCCATGGCGTCCCTTCCTGTCCTGCAGAAGGAGAGCGTGTTCCAGTCGGGAGCCCATGCCTACAGAATCCCTGCCCTGCTCTACCTGCCTGGGCAGCAGTCCCTGCTGGCCTTCGCGGAACAGCGGGCAAGCAAGAAGGATGAGCACGCAGAGCTGATTGTCCTGCGCAGAGGAGACTACGACGCACCCACCCACCAGGTTCAGTGGCAAGCTCAGGAGGTGGTGGCCCAGGCCCGGCTGGATGGCCACCGGTCCATGAACCCATGCCCCTTGTATGACGCGCAGACGGGGACCCTCTTCCTCTTCTTCATTGCCATCCCTGGGCAAGTCACGGAGCAACAGCAGCTGCAGACCAGGGCCAATGTGACGCGGCTGTGCCAAGTCACCAGCACTGACCACGGGAGGACCTGGAGCTCCCCCAGAGACCTCACTGATGCGGCCATCGGCCCAGCCTACCGGGAGTGGTCCACCTTTGCAGTGGGCCCGGGGCATTGTTTGCAGCTTAACGACAGGGCCCGGAGCCTGGTGGTGCCCGCCTACGCCTACCGGAAACTTCACCCCATCCAAAGGCCGATCCCCTCTGCCTTCTGCTTCCTCAGCCATGACCATGGGCGCACGTGGGCGCGAGGGCACTTTGTGGCCCAGGACACCCTGGAGTGCCAGGTGGCCGAAGTCGAGACTGGGGAGCAGAGGGTGGTGACCCTCAACGCGAGAAGCCACCTCCGAGCCAGGGTCCAGGCCCAGAGCACCAATGACGGGCTTGATTTCCAGGAGTCTCAGCTGGTGAAGAAGCTGGTGGAGCCGCCGCCCCAGGGCTGCCAGGGGAGCGTCATCAGCTTCCCCAGCCCCCGCTCGGGGCCTGGCTCCCCAGCCCAGTGGCTGCTCTACACTCACCCCACACACTCCTGGCAGAGGGCCGACCTGGGTGCCTACCTCAACCCGCGACCTCCAGCCCCTGAGGCCTGGTCAGAGCCGGTACTGCTGGCCAAGGGCAGCTGTGCCTACTCAGACCTCCAGAGCATGGGCACCGGCCCTGATGGGTCCCCCTTGTTTGGGTGTCTGTACGAAGCCAATGATTACGAGGAGATTGTCTTTCTCATGTTCACCCTGAAGCAAGCCTTCCCAGCTGAGTACCTGCCTCAGCTCGAGCGGCCGCATCGTGAC
GST-thrombin-Neu3
ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGTTCCGCGTGGATCCGAGGAGGTTACCACCTGCAGCTTTAACTCCCCTTTGTTTCGCCAGGAAGATGATCGCGGGATTACATATCGCATTCCCGCCCTTTTGTACATTCCCCCAACCCATACTTTTTTGGCATTTGCTGAGAAGCGTTCCACCCGCCGTGATGAGGATGCGTTACACTTAGTGTTGCGCCGTGGTCTTCGCATCGGACAATTAGTACAGTGGGGTCCTTTAAAACCGCTTATGGAAGCGACCTTACCAGGACATCGTACTATGAACCCCTGTCCTGTGTGGGAACAGAAATCTGG
44
CTGCGTGTTCTTATTCTTTATCTGCGTACGTGGTCACGTAACAGAACGCCAGCAAATTGTAAGTGGCCGTAACGCGGCCCGCTTATGTTTTATCTACTCGCAGGATGCGGGATGTTCTTGGTCAGAGGTCCGTGACCTGACAGAGGAGGTTATCGGGTCTGAGTTGAAACACTGGGCCACTTTCGCCGTAGGTCCAGGTCATGGGATCCAGTTGCAGTCTGGACGCCTGGTTATTCCGGCTTATACTTACTATATCCCGTCGTGGTTCTTTTGTTTTCAACTGCCCTGTAAGACGCGTCCACACTCGCTGATGATCTATAGCGATGATTTGGGAGTTACGTGGCATCATGGACGTTTGATCCGCCCGATGGTCACAGTCGAGTGTGAGGTTGCCGAAGTGACTGGCCGCGCAGGACATCCAGTGCTGTACTGTTCAGCGCGTACGCCAAATCGCTGTCGTGCCGAAGCACTTTCAACGGATCATGGTGAAGGCTTTCAACGCCTTGCGTTATCACGCCAACTGTGCGAACCGCCTCATGGGTGTCAGGGCAGCGTGGTTTCATTCCGCCCACTGGAAATTCCCCATCGTTGTCAAGACTCCTCTAGCAAAGATGCCCCTACGATCCAACAGTCGAGTCCTGGTAGTAGCCTGCGCCTTGAGGAAGAAGCAGGAACGCCGTCTGAGTCTTGGTTATTATACAGCCATCCAACGTCTCGTAAGCAGCGTGTGGACTTAGGTATCTACTTAAACCAAACTCCCCTTGAGGCCGCCTGTTGGAGCCGTCCCTGGATTCTGCACTGTGGCCCTTGCGGATATTCAGATTTGGCGGCCCTTGAAGAGGAAGGGCTGTTCGGCTGTTTATTTGAATGTGGCACAAAGCAAGAATGCGAACAAATCGCCTTCCGTTTATTTACTCATCGCGAGATTTTGAGCCACTTACAGGGAGACTGTACGTCGCCTGGACGTAATCCCTCCCAATTCAAGTCTAACCTCGAGCGGCCGCATCGTGACTGA
45
NMR Spectra
NN
FmocN
HN
OON3
3
10
NN
FmocN
HN
OON3
3
10
C NMR101 MHzCDCl
13
3
H NMR400 MHzCDCl
1
3
46
NN
HN
HN
OON3
3
1
H NMR400 MHzCDCl
1
3
C NMR101 MHzCDCl
13
3
NN
HN
HN
OON3
3
1
47
2
NHN O N
HO O 4
OCl
2
NHN O N
HO O 4
OCl
H NMR400 MHzCDCl
1
3
C NMR101 MHzCDCl
13
3
48
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70. Barouch, D. H. et al. A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates. J. Virol. 79, 8828–8834 (2005).
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72. Carrico, I. S., Carlson, B. L. & Bertozzi, C. R. Introducing genetically encoded aldehydes into proteins. Nat. Chem. Biol. 3, 321–322 (2007).
73. Holder, P. G. et al. Reconstitution of formylglycine-generating enzyme with copper(II) for aldehyde tag conversion. J. Biol. Chem. 290, 15730–15745 (2015).
74. Taylor, G., Vimr, E., Garman, E. & Laver, G. Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2. J. Mol. Biol. 226, 1287–1290 (1992).
75. Vimr, E. R., Lawrisuk, L., Galen, J. & Kaper, J. B. Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli. J. Bacteriol. 170, 1495–1504 (1988).
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