jitc-2023-December-11-12--inline-supplementary-material-1-html.html

Supplementary methods

Flow cytometry and antibodies

The following antibodies and flow cytometry reagents were used: CD1d-PE (clone 51.1,

Thermo Fisher cat# 12-0016-42, RRID:AB_1724019), CD1d-APC (clone 51.1, Biolegend cat#

350308, RRID:AB_10642829), CD3-BV421 (clone SK7, BD Biosciences cat# 563797,

RRID:AB_2744383), CD14-

percpCy5.5 (clone MΦP9, BD Biosciences cat# 562692,

RRID:AB_2737726), CD19-APC (clone SJ25C1, BD Biosciences cat# 345791,

RRID:AB_2868817), CD45-AF700 (clone HI30, Biolegend cat# 304024, RRID:AB_493761),

CD56-BV510 (clone NCAM16.2, BD biosciences cat# 563041, RRID:AB_2732786), CD69-

PE (clone FN50, BD Biosciences cat# 555531, RRID:AB_395916), CD107a-PE (clone H4A3,

Thermo Fischer, cat# 12-1079-42, RRID:AB_10853326), CD163-BV421 (clone GHI/61, BD

Biosciences cat# 562643, RRID:AB_2737697), Streptavidin-APC (eBioscience cat# 17-4317-

82), anti-human Fc-PE (Thermo Fisher cat# 12-4998-82), Goat-anti-

mouse (GαM) Fab

APC

(Santa Cruz Biotech cat# SC-3818, RRID:AB_649051), rabbit anti-

Llama VHH (RαL)

iFluor647 (Genscript, cat# A02019), PSAP-unconjugated (clone 4D5F4, Thermo Fisher cat#

MA5-17159, RRID:AB_2538630), bavituximab (ADCC enhanced) (Creative Biolabs cat#

TAB-175, RRID:AB_2459753), rituximab (Amsterdam UMC pharmacy), recombinant

human(rh)TIM-1 Fc (R&D systems cat# 9319-TM), rhTIM-3 Fc (R&D systems cat# 2365-

TM), rhTIM-4 Fc (R&D systems cat# 9300-TM), 7-amino-actinomycin D (7-AAD) (Sigma

cat# A9400), annexin V-FITC (VPS diagnostics, the Netherlands, cat# A700), propidium iodide

(PI) (Thermo Fisher, cat# p16063), counting beads (Thermo Fisher cat# 01-1234-42),

carboxyfluorescein succinimidyl ester (CFSE) (Sigma cat# 21888). All antibodies were used at

empirically determined dilution factors. Staining was performed at 4 degrees Celsius in PBS

supplemented with 0.1% BSA and 0.02% sodium azide or annexin V-staining buffer unless

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, et al.

Lameris R

otherwise stated. Analyses were performed using a FACS LSRFortessa (BD Biosciences). Data

analysis was performed using Kaluza (Beckman Coulter) and FlowJo (BD Biosciences).

Cell lines

The lymphoblastic cell line C1R, WT or stably transduced with CD1d (generated previously

)

was grown in IMDM (Gibco, Life Technologies) supplemented with 10% (v/v) fetal calf serum

(HyClone, GE Healthcare), 0.05 mM β

-mercaptoethanol, 100 IU ml

-1

sodium penicillin, 100

μg ml

-1

streptomycin sulphate and 2.0 mM L-glutamine (Gibco, Life Technologies). C1R.GFP

cells expressing WT CD1d or mutant CD1d (Q62A, H68A, V72A, R79A, E83A, K86A, M87A,

V147A, Q150A, T157A, W160A) were previously generated.

The MM cell line MM.1s, WT

and stably transduced with CD1d were kind gifts from Dr. W. Song

and grown in RPMI-1640

(Gibco, Life Technologies) supplemented as above. The epithelial cell line HeLa, stably

transduced with CD1d, was a kind gift from Dr. M. Kronenberg

, and grown in DMEM (Gibco,

Life Technologies) supplemented as above. The αβTCR

-deficient T-ALL cell lines SKW-3

cells expressing the CD1d-restricted TCRs NKT12 or NKT15 and GFP were previously

generated and grown in RPMI-1640 supplemented as described above.

The lymphoblastic

natural killer (NK)-92 cell line stably transduced with CD16 and GFP

was grown in MEM

Alpha (Gibco, Life Technologies) supplemented with 12.5% FCS (v/v), 12.5% Horse Serum

(v/v) (Gibco, Life Technologies), 100 IU ml-1 s

odium penicillin, 100 μg ml

-1 streptomycin

sulphate and 2.0 mM L-glutamine, and 80 U/mL interleukin 2 (IL-2, Novartis, Basel,

Switzerland). CD1d expression of used transfectants is shown in

fig. S3g.

Generation of CD1d-specific VHHs

Human CD1d-specific VHHs were generated and screened as previously described.

Highly

purified and endotoxin free anti-CD1d VHH1D7 (control anti-CD1d VHH; VHH1D control),

VHH1D5, VHH1D17 (WT and single amino acid mutants) and VHH1D22 were produced by

UPE, the Netherlands. For detection of binding to (WT or mutant) CD1d, VHH1D17 was

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biotinylated using NHS-D-biotin (Sigma-

Aldrich) according to the manufacturer’s protocol.

1x10

C1R.GFP.CD1d (WT or mutant) cells were sequentially incubated with biotinylated

VHH1D17 (25 µg ml

-1

) and Streptavidin-APC, for subsequent flow cytometry analysis. Median

fluorescence was normalized for GFP expression and made relative to CD1d.WT. For binding

of (mutant) VHH1D17, 1×10

MM.1s.CD1d cells were sequentially incubated with 200 nM of

the VHHs and rabbit anti-

Llama VHH (RαL)

-iFluor647, for subsequent flow cytometry

analysis. Relative percentage of binding was calculated by dividing median fluorescence (MF)

[VHH1D17.mutant] by MF [VHH1D17.WT] multiplied by 100. For detachment of VHH1D17

at different pH, 1×10

C1R.CD1d cells were first incubated with 100 nM VHH1D17 (pH 7.4),

washed and incubated at indicated pH (citric acid buffer) for 1 h, washed (pH 7.4) and then

incubated with Streptavidin-APC, for subsequent flow cytometry analysis. Relative percentage

of binding was calculated by dividing median fluorescence (MF) [VHH1D17 indicated pH] by

MF [VHH1D17 pH 7.4] multiplied by 100.

TCR transfected cell stimulation

For stimulation assays of

CD1d(α

-GalCer)-restricted NKT12 and NKT15 TCR transfected

cells, 1×10

TCR-expressing SKW-3 cells were co-cultured overnight, with or without 1×10

C1R cells (either C1R WT or C1R.CD1d) and VHH1D17 (1 µM), vehicle control, α

-GalCer

(100 ng ml

-1

, Avanti Polar lipids cat #867000P) or a combination thereof. Activation of TCR-

expressing cells (GFP

CD3

CD19

) was then analyzed by assessing CD69 expression using

flow cytometry.

Generation of

PSAP KO C1R.CD1d

By using CRISPR-mediated genome editing, a PSAP KO C1R.CD1d cell line (C1R.CD1d.

KO/PSAP) was established essentially as described.

Five specific oligonucleotides were

designed using the online E-CRISP design tool

(5’

-GGACTGAAAGAATGCACCAG-

3’, 5’

GAAGACGGCGTCCGACTGCG-

3’,

5’

-GTGAAGACGGCGTCCGACTG-

3’

5’

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GCTCTAGCCGGCCCGGTCCT-

3’, 3’

-GCAGACCGTTTGGAACAAGC-

5’), an

nealed and

ligated into pSpCas9(BB)-2A-Puro (PX459) (Addgene plasmid #48139), transformed into XL-

10 gold E. coli cells (Agilent technologies cat# 200314), their sequences validated (Baseclear

BV, Leiden, The Netherlands) and electroporated into C1R.CD1d cells (Neon transfection

system, Thermo Fisher) according to the manufacturer’s instructions. Transfected cells were

positively selected using puromycin (1 µg ml-1). Indel mutations were detected using the

SURVEYOR nuclease assay (forward primer: 5’

-CACCTCCTCACGTTGGTTTT-

3’ and

reverse primer 5’

-CATCCTGTCTGCCACAAGAA-

3’) followed by clonal

-density dilution to

obtain clonal cell lines. C1R.CD1d clones containing homogeneous indel mutations were

identified by sequencing. Presence of PSAP was assessed by subjecting lysates of C1R.CD1d

cell (clones) containing equal amounts of total protein to SDS-PAGE followed by

immunoblotting with anti-PSAP antibody (polyclonal, Proteintech cat# 10801-1-AP) and anti-

rabbit IRDye 680RD (LI-COR cat #926-68071). Anti-GAPDH (clone 6C5, Abcam cat #8245)

and anti-mouse IRDye 800CW (LI-COR cat #926-32210) were used to confirm adequate cell

lysate loading.

Crystallization and structure determination

Purified CD1d-

m was generated as described.

2,9

Crystals of the VHH1D17-

CD1d(endogenous) complex were grown via the hanging drop vapor diffusion method, using a

protein-reservoir drop ratio of 1:1, at a protein concentration of 5 mg ml

-1

in 12% PEG 8k, 0.05

M Zn Acetate, 0.05 M MES (pH 6.2) at 20 °C. Crystals of the VHH1D17-CD1d(endo)

complexes were soaked in cryoprotectant solutions comprised of reservoir solution containing

10 % (v/v) ethylene glycol, before being flash frozen in liquid nitrogen. Data were collected at

the Australian Synchrotron at the MX2 beamline, using the EIGER X 16M detector (Dectris

Ltd).

Data were processed using XDS software

, and scaled using Aimless as part of the

CCP4i program suite.

A single complete data set was collected of the VHH1D17-CD1d(endo)

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complex. Crystal structures were solved via molecular replacement using Phaser as part of the

Phenix program suite

, with the structure of the CD1d(αGalCer)

-NKT15-TCR complex (PDB

code 2PO6)

used as model for solving CD1d, and structure of a camelid antibody fragment

(Chain B, PDB code 3P0G) used as a model for solving VHH1D17. Manual adjustments of the

models were conducted using the Coot graphics program

, followed by maximum-likelihood

refinement using Buster 2.10.

All molecular model representations were generated using

PyMOL. BSA values were calculated using Areaimol, and contact analysis conducted using the

CONTACT program, both found in the CCP4i program suite.

Resulting structural data was

deposited to the Protein Data Bank (PDB ID: 8SOS).

Definition of CDR and FR regions were

based on the IMGT numbering system.

Monocyte derived macrophages and phagocytosis assay

To generate moMacrophages

, monocytes were isolated from peripheral blood mononuclear

cells (PBMC) using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and

cultured in complete RPMI-1640 medium in the presence of 5 ng ml

-1

M-CSF (R&D systems

#216-MC) for 5-7 days. Cultures expressing >90% CD14 and CD163 where used for

experiments. To assess phagocytosis, MM.1s.CD1d cells were stained with CFSE (5 µM) and

incubated overnight in the presence or absence of anti-CD1d VHH (100 nM) or bortezomib (3

nM, positive control, Axxora cat# LKT-B5871). After confirmation of induction of annexin V

binding, MM.1s.CD1d cells were co-cultured with moMacrophages (1:1 effector to target ratio)

for 4 h on ice or 37 ºC after which the percentage of CD14

CFSE

of total CD14

cells was

determined by flow cytometry.

NK-92.CD16 stimulation assay

To evaluate induction of degranulation of NK-92.CD16 cells, 5×10

C1R.CD1d cells, either

pre-incubated with VHH1D17 (24 h, 100 nM) or not, were incubated with medium,

bavituximab or rituximab (indicated concentration) for 15 min, and co-cultured with 5×10

NK-

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92.CD16 cells for 4 h in the presence of PE-labelled anti-CD107a, after which NK-92.CD16

(GFP

CD19

CD45

CD56

) cells were analyzed for CD107a expression.

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:e007631.

2023;

J Immunother Cancer

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Figure S1. CD1d-specific VHH1D17 enhances non-apoptotic annexin V binding by

inducing saposine dependent presentation of anionic phospholipids by CD1d.

Fold change in annexin V binding to CD1d transduced C1R, MM.1s or HeLa cells after 48

h culture plus negative control, anti-CD1d mAb (clone 51.1, ~33 nM) or VHH1D17 (100 nM)

(n=3 independent experiments).

Fold change in annexin V binding to CD1d transduced C1R

cells after 24 h culture plus VHH1D control or VHH1D17 (concentration range, n=3

independent experiments).

Relative viability of CD1d transduced C1R, MM.1s or HeLa cells

after 48 h culture plus negative control, anti-CD1d mAb (clone 51.1, ~33 nM) or VHH1D17

(100 nM) (n=3 independent experiments).

Relative number of living (7-AAD

) cells after 24

h culture plus negative control, anti-CD1d mAb (clone 51.1, ~33 nM) or VHH1D17 (100 nM)

(n=3 independent experiments), quantified by flow cytometry counting beads.

e,f,

Exemplifying gating strategy used to analyze annexin V binding (C1R.CD1d) (

) and relative

viability (MM.1s.CD1d) (

Fold change in annexin V binding to C1R.CD1d cells after 24

h culture plus negative control, anti-CD1d VHH1D22 (100 nM), VHH1D17 (100 nM) or a

combination thereof (n = 4 (negative control and VHH1D17) or n=3 independent experiments).

VHH1D17 > VHH1D22; VHH1D22 (500 nM) was added 1 h before readout at 4 degree

Celsius.

, Histograms depicting CD1d and PSAP expression on C1R.CD1d.shRNA/control

and C1R.CD1d.shRNA/PSAP.

, Histogram depicting CD1d expression on C1R.CD1d WT and

C1R.CD1d.KO/PSAP.

, Western blot analyses of PSAP in whole cell lysates of HeLa.CD1d,

C1R.CD1d WT (parental cell line) and two C1R.CD1d clonal cell lines whether or not with

PSAP KO (KO/PSAP and WT/PSAP respectively).

Fold change in annexin V binding to

C1R.CD1d WT or KO/PSAP cells after 24 h culture plus negative control or VHH1D17 (100

nM) (n=3 independent experiments).

The bars indicate the mean ± s.d; nonlinear regression ± 95% confidence bands (dotted lines)

and EC

(dashed line) (

a,c,d,

One-way ANOVA with Tukey

multiple comparisons test.

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Figure S2. Structural differences between VHH1D5 and VHH1D17.

Fold change in annexin V binding to C1R.CD1d cells after 24 h culture plus negative control

or anti-CD1d VHH1D5 (100 nM) (n=3 independent experiments).

Relative binding of

VHH1D17-biotinylated to CD1d WT and alanine substitution mutants expressed on C1R cells,

detected by streptavidin-APC (normalized for GFP expression and made relative to CD1d.WT;

n=5 independent experiments).

Fold change in CD69 expression on gated SKW-3 cell lines

transduced with the CD1d(α

-GalCer)-restricted NKT12 or NKT15 TCRs after overnight

coculture with either C1R.WT or C1R.GFP.CD1d plus negative control, α

-GalCer (100 ng

−

), VHH1D17 (1 μM) or a combination thereof (n=4 independen

t experiments).

VHH1D5

(green) and VHH1D17 (blue) and their CDR1 (red), CDR2 (orange), CDR3 (yellow) loops

overlaid, represented as ribbons.

, Electrostatic surface potential of VHH1D5 and VHH1D17

bound to CD1d(lipid) at indicated pH. The potential contours are shown on a scale from +5.0

(positive charge, blue) to −5.0 k

T e

−

(negative charge, red); white indicates a value close to 0

T e

−

(neutral charge).

Relative binding of VHH1D17-biotinylated to C1R.CD1d cells at

indicated pH, detected by streptavidin-APC (made relative to pH 7.4; n=3 independent

experiments).

Relative binding of VHH1D17 (WT or mutants) to MM.1s.CD1d cells,

detected by rabbit-anti-llama-iFluor647 (made relative to VHH1D17.WT; n=4 independent

experiments). The bars indicate the mean ± s.d.

One-

way ANOVA with Tukey’s multiple

comparisons test.

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Figure S3. VHH1D17 enhanced presentation of anionic phospholipids by CD1d results in

increased binding to bavituximab and TIM-molecules and does not trigger phagocytosis.

Percentage 7-AAD

MM.1s.CD1d cells after

24 h incubation with negative control, control

VHH1D (100 nM), VHH1D17 (100 nM) or bortezomib (3 nM) (n=3 independent experiments),

used for the phagocytosis experiment.

Exemplifying gating strategy used to analyze

phagocytosis.

Phagocytosis of CFSE labelled MM.1s.CD1d cells, pre-incubated for 24 h with

negative control, control VHH1D (100 nM), VHH1D17 (100 nM) or bortezomib (3 nM), by

monocyte derived (mo)Macrophages after 4 h coculture at 4 degree Celsius (n=3 independent

experiments).

Fold change in bavituximab binding to C1R.CD1d cells after 24 h culture plus

negative control (black) or VHH1D17 (50 nM, blue), detected by anti-Fc-PE.

, Expression (%)

of CD107a on NK-92.CD16 cells after 4 h co-culture with C1R.CD1d cells, pre-incubated for

24 h with negative control or VHH1D17 (100 nM), and ± bavituximab or rituximab (indicated

concentration) (n=4 independent experiments).

Fold change in binding of recombinant human

T-cell immunoglobulin and mucin domain containing protein (rhTIM)-1, -3 and -4 to

C1R.CD1d (WT or KO/PSAP) cells after 24 h culture plus negative control (black) or

VHH1D17 (100 nM, blue), detected by anti-Fc-PE (n=3 independent experiments).

Histograms depicting expression of CD1d on MM.1s WT and transfectant, C1R WT and

transfectant, and HeLA transfectant. The bars indicate the mean ± s.d. Box and whisker plots

indicate the median, 25th to 75th percentiles and minimum to maximum.

a,c,

One-way

ANOVA with Tukey multiple comparisons test.

Two-tailed pared t-test.

, two-way ANOVA

with Šídák multiple comparisons test.

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance

Supplemental material

placed on this supplemental material which has been supplied by the author(s)

J Immunother Cancer

doi: 10.1136/jitc-2023-007631

:e007631.

2023;

J Immunother Cancer

, et al.

Lameris R

Supplementary tables

Table S1 sequence alignment

FR1

CDR1

FR2

CDR2

FR3

1D5

EVQLVESGGGLVQAGGSLRLSCAAS

GSSFSSYT

MGWFRQAPGKEREIVAG

IRWSDESP

IY 60

1D17

VQLVESGGGLVQAGGSLRLSCAAS

GSSFSSYT

WFRQAPGKEREIVAG

IRWS

ESP

Y 60

FR3

CDR3

FR4

1D5

ADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC

AARLVPPGIPIPRTSESMRY

WGKG 120

1D17

ADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC

AARLVPPGIPI

ESMRY

WGKG 120

FR4

1D5

TLVTVSS 127

1D17

TLVTVSS 127

CDR according to IMGT numbering, CDR are bold and difference between VHH1D5 and

VHH1D17 marked in red

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance

Supplemental material

placed on this supplemental material which has been supplied by the author(s)

J Immunother Cancer

doi: 10.1136/jitc-2023-007631

:e007631.

2023;

J Immunother Cancer

, et al.

Lameris R

Table S2. Data collection and refinement statistics

VHH1D17

–

CD1d(sphingomyelin)

Space Group

P 32 2 1

Resolution Range (Å)

44.66

–

2.33 (2.41

–

2.33)

Cell Dimensions (Å, °)

a=b=136.45, c=166.77

α=β=90, γ=120

Total no. of reflections

154080 (15262)

No. of unique reflections

77042 (7630)

Multiplicity

2.0 (2.0)

Completeness (%)

99.90 (99.95)

CC (1/2)

0.998 (0.795)

pim

(%)

2.86 (35.90)

Mean

(

)

20.0 (2.2)

factor

free

(%)

20.97 / 22.99

Non-hydrogen atoms

8568

Macromolecules

7828

Ligand

168

Solvent

572

Protein Residues

989

Bond Length (Å)

0.002

Bond Angles (°)

0.48

Ramachandran Plot

Favored Region (%)

97.94

Allowed Region (%)

1.96

Outliers (%)

0.10

B-Factors (Å

)

Average B-factors

55.67

Macromolecules

56.12

Ligands

49.41

Solvent

51.41

PDB ID

8SOS

pim

= ∑

hkl [1/(N - 1)]

1/2

∑

i |

hkl, i - <Ihkl>

|/∑

hkl <Ihkl>.

σ(

) is the estimated standard

deviation of the integrated intensity (

). c

factor =

∑

hkl||

o| - |

||/∑

hkl|

o| for all data

except 5%, which were used for

free calculation. Highest resolution shell is shown in

parenthesis.

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance

Supplemental material

placed on this supplemental material which has been supplied by the author(s)

J Immunother Cancer

doi: 10.1136/jitc-2023-007631

:e007631.

2023;

J Immunother Cancer

, et al.

Lameris R

Table S3. VHH1D17 contacts with CD1d and sphingomyelin

VHH region

VHH residues

CD1d residues

Bond type

CDR1

S28

H68

VDW

CDR1

S31

T65

VDW

CDR2

W53-N

T65-O



CDR2

W53

Q61, Q62, T65

VDW

CDR2

S54-O, -O



Q61-O



CDR2

S54

Q61

VDW

N74-N

Q61-N

VDW

CDR3

R99-N

-NH2

E156-O

CDR3

R99-N



-NH2

E156-O

2, W153-O

CDR3

R99

W153, E156, T157,

W160

VDW

CDR3

L100

H68, W160

VDW

CDR3

V101

T65

VDW

CDR3

P102

I69, W160, L161,

T165

VDW

CDR3

P103-O

T165-O



CDR3

P103

F58, Q62, L66, T165 VDW

CDR3

G104

Q168

VDW

CDR3

I105

W160

VDW

CDR3

S113-O



E156-O

CDR3

S113

W153, E156, W160

VDW

CDR3

M114

W153

VDW

CDR3

R115

W153

VDW

VHH region

VHH residues

Sphingomyelin

atom

Bond type

CDR3

R115-CZ

VDW

CDR3

R115-NH1

VDW

CDR3

R115-NH2

O3, P1

HB, VDW

VDW: van der Waals interaction (cut-off of 4.0 Å), HB: Hydrogen Bond (Cut-off of 3.5 Å),

SB: Salt Bridge (Cut-off of 4.5 Å)

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance

Supplemental material

placed on this supplemental material which has been supplied by the author(s)

J Immunother Cancer

doi: 10.1136/jitc-2023-007631

:e007631.

2023;

J Immunother Cancer

, et al.

Lameris R