SUPPLEMENTARY INFORMATION

An Ultra-High Affinity Small Organic Ligand of Fibroblast Activation Protein for Tu-
mor Targeting Applications

Jacopo Millula†, Gabriele Bassia†, Jacqueline Mockb†, Abdullah Elsayedb, Christian Pelle-
grinob, Aureliano Zanaa, Sheila Dakhel Plazaa, Lisa Nadala, Andreas Glogera, Eleonore
Schmidta, Ilaria Biancofiorea, Etienne J. Donckelea, Florent Samaina, Dario Nerib,c* & Sam-
uele Cazzamallia*

a) Philochem AG, Libernstrasse 3, CH-8112 Otelfingen (Switzerland)

b) Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology
(ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093 Zurich (Switzerland)
c) Philogen SpA, Piazza La Lizza 7, 53100 Siena (Italy)

†) These authors equally contributed to this work

*Corresponding authors:

Dario Neri
phone: +41 43 544 88 06
fax: +41 43 544 88 09
Email: [email protected]

Samuele Cazzamalli
phone: +41 43 544 88 19
fax: +41 43 544 88 09
Email: [email protected]

Author Contributions: J.Millul, D.N. and S.C. designed and planned the study. J.Millul per-
formed most of the experiments with the help of the other authors as follows. S.D.P., L.N.
and A.G. produced human and murine recombinant FAP. A.G., E.S., I.B., E.J.D. and F.S.
contributed to the in vitro characterization of novel FAP ligands. J.Millul and A.E. generated
S1
hFAP expressing tumor cell lines and performed FACS experiments. J.Millul and S.C. per-
formed confocal microscopy analysis. J.M. and A.Z. designed and performed the ex vivo
tumor and organ penetration experiment. J.Millul and S.C. performed the IVIS experiments.
G.B., J. Mock and S.C. performed the biodistribution studies with radiolabeled compounds.
J.Millul and C.P. performed the in vitro UniCAR-T killing experiments. J.Millul prepared the
figures. J.Millul, D.N. and S.C. wrote the manuscript.

Competing Interest Statement: D.N. is a co-founder and shareholder of Philogen

(www.philogen.com), a Swiss-Italian Biotech company that operates in the field of ligand-
based pharmacodelivery. J.M., G.B., A.Z., S.D.P., L.N., A.G., E.S., I.B., E.J.D., F.S. and S.C.
are employees of Philochem AG, daughter company of Philogen acting as discovery unit of
the group.

Classification: BIOLOGICAL SCIENCES/Medical Sciences

Keywords: Tumor Targeting, Small Molecule Therapeutics, Fibroblast Activation Protein,

Theranostics

This PDF file includes:

Supplementary information
Figures SI1 to SI6

S2
Table of Contents

Abbreviations ....................................................................................................................................................... 4

General Remarks and Procedures............................................................................................................... 6

Synthesis of Intermediate A ........................................................................................................................... 7

Synthesis of compound 1 ............................................................................................................................... 8

Synthesis of Intermediate B ......................................................................................................................... 10

Synthesis of compound 2 ............................................................................................................................. 11

Synthesis of compound 3 ............................................................................................................................. 12

Synthesis of compound 4 ............................................................................................................................. 13

Synthesis of compound 5 ............................................................................................................................. 14

Synthesis of compound 6 ............................................................................................................................. 15

Synthesis of compound 7 ............................................................................................................................. 16

LC/MS and HPLC profiles .............................................................................................................................. 18

IVIS Experiment ................................................................................................................................................. 21

Dose escalation study in SK-RC-52.hFAP .............................................................................................. 23

Therapy experiment with OncoFAP-Vedotin in combination with L19-IL2 in tumor-bearing

mice ........................................................................................................................................................................ 24

In vitro affinity measure of FAPI-04 ........................................................................................................... 25

Protein Sequence of Extracellular Domain Murine FAP and quality control ........................... 26

Protein Sequence of Extracellular Domain Human FAP and quality control ........................... 28

hFAP Lentiviral transfer vector and amino acid sequence ............................................................. 29

UniCAR-T in Vitro Killing Assay ................................................................................................................. 30

S3
Abbreviations

AA Antibiotic Antimycotic
AAZ Acetazolamide
AMC 7-amido-4-methylcoumarin
Asp Aspartic Acid
Boc tert-Butyloxycarbonyl
Cit Citrulline
CT Chlorotrityl
Cys Cysteine
DIPEA N,N-Diisopropylethylamine
DCC N,N’-Dicyclohexylcarbodiimide
DCM Dichloromethane
DMAP 4-(Dimethylamino)pyridine
DMF N,N-Dimethylformamide
DMSO Dimethylsulfoxide
ESI Electrospray Ionization
eq Equivalents
FA Formic Acid
FBS Fetal Bovine Serum
FCS Fetal Calf Serum
Fmoc 9-Fluorenylmethoxycarbonyl
Glu Glutamic acid
Gly Glycine
HATU O-(7-azabenzotriazol-1-yl)-tetramethyl-uronium hexafluorophos-
phate
HBSS Hank’s Balanced Salt Solution
HPLC High Performance Liquid Chromatography
IC50 Half maximal inhibitory concentration
ID/g Injected dose / gram
KD Dissociation constant

S4
LC/MS Liquid Chromatography / Mass Spectrometry
Lys Lysine
m/z mass-to-charge ratio
MC Maleimido-caproyl
MeCN Acetonitrile
MMAE Monomethyl Auristatin E
MS Mass Spectroscopy
mQ Milli-Q
MW Molecular Weight
PAB para-Aminobenzoyl
PBS Phosphate-Buffered Saline
ppm Part per million
Pro Proline
RP Reverse Phase
RPMI Roswell Park Memorial Institute
r.t. Room Temperature
SD Standard Deviation
SEM Standard error of the mean
SPPS Solid Phase Peptide Synthesis
tBu tert-Butyl
TFA Trifluoroacetic Acid
THF Tetrahydrofuran
TIPS Triisopropylsilane
Trt Trityl
Val Valine
w.t. wild type
Z Benzyloxycarbonyl

S5
General Remarks and Procedures
Peptide grade N,N-dimethylformamide (DMF) for solid phase synthesis was bought from
VWR. All other solvents were used as supplied by Merck or Sigma Aldrich in HPLC or ana-
lytical grade. H-Cys(Trt)-2-CT-polystyrene resin was purchased from RAPP Polymere
(Ernst-Simon-Strasse 9, 72072 Tuebingen, Germany). Maleimidocaproyl-ValCit-p-amino-
benzylalcohol-MMAE was purchased from Levena Biopharma (No.9 Weidi Road, Qixia Dis-
trict, Nanjing, 210046, China).
All other reagents were purchased from Sigma Aldrich, Thermo Fisher, Fluorochem, Che-
matech or Astatech and used as supplied. Yields refer to chromatographically purified and
spectroscopically pure compounds, unless noted otherwise.
Liquid-Chromatography/Mass-Spectrometry (LC/MS) spectra presented were recorded on
an Agilent 6100 Series Single Quadrupole MS system combined with an Agilent 1200 Series
LC, using an InfinityLab Poroshell 120 EC-C18 Column, 2.7 µm, 4.6 × 50 mm at a flow rate
of 0.6 ml min-1, 10% MeCN in 0.1% aq. FA to 100% MeCN in 6 min.
Reversed-phase high-pressure liquid chromatography (RP-HPLC) were performed on an
Agilent 1200 Series RP-HPLC with PDA UV detector, using a Synergi 4μm, Polar-RP 80Å
10 × 150 mm C18 column at a flow rate of 5 ml min-1 with linear gradients of solvents A and
B (A = Millipore water with 0.1% TFA, B = MeCN with 0.1% TFA).
Size-exclusion chromatography was performed on a Superdex 200 Increase 10/300 GL
column on an ÄKTA FPLC (GE Healthcare).

S6
Synthesis of Intermediate A
NC O
HO O H
NC O Cl N O
HATU N
NH3 DIPEA
N
F
DMF/DCM F
N F r.t. 1h
F N
NH2
NH2

In a 25mL round bottom flask, 8-aminoquinoline-4-carboxylic acid (100 mg, 0.531 mmol, 1
eq), (S)-1-(2-aminoacetyl)-4.4-difluoropyrrolidine-2-carbonitrile hydrochloride (132 mg,
0.585 mmol, 1.1 eq) and HATU (202 mg, 0.531 mmol, 1 eq) were suspended in 900 µL of
DMF and 4 mL of DCM.
DIPEA (371 µL, 2.127 mmol, 4 eq) was added dropwise and the reaction was stirred until
completion (checked via LC/MS with the method 90:10 Water/Acetonitrile 0.1% FA to 100%
Acetonitrile 0.1% FA in 3min, Positive).
The crude was diluted with DCM, washed with water, dried over Na2SO4, filtered and the
solvent evaporated under vacuum.
The dried crude was purified via CombiFlesh Nextgen 300+ (parameters: flow 50 ml/min, 40
gr silica column, 100% DCM to 85:15 DCM/MeOH in 5 min) to obtain an amber oil (172 mg,
0.478 mmol, 90% yield). MS (ES+) m/z 360.12 (M+H)+.

S7
Synthesis of compound 1

NC O
NC O H
H N O
N O N
N
O O DMAP
F
F O F
F THF N O
N 60°C, 6h
HN
NH2 OH
O

In a 25 mL round bottom flask, Intermediate A (48 mg, 0.134 mmol, 1 eq), succinic anhy-
dride (669 mg, 6.683 mmol, 50 eq) and DMAP (8 mg, 0.067 mmol, 0.5 eq) were dissolved
in 3 mL of THF. The reaction was heated at 60 °C for 6h and checked via LC/MS (method
90:10 Water/Acetonitrile 0.1% FA to 100% Acetonitrile 0.1% FA in 3min, Positive). The re-
action was dried under vacuum, diluted with water, extracted with DCM, dried over Na2SO4,
filtered and concentrated under. The dried crude was purified via CombiFlesh Nextgen 300+
(parameters: flow 30 ml/min, 24 gr silica column, DMC/MeOH 90:10 to 70:30 in 4 minutes)
to obtain an amber oil (58 mg, 0.127 mmol, 95% yield). MS (ES+) m/z 460.14 (M+H)+.

S8
1
H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H) NHSu, 9.16 (t, J = 6.0 Hz, 1H) NHGly, 9.03
(d, J = 4.3 Hz, 1H) CHQn2, 8.66 (dd, J = 7.8, 1.3 Hz, 1H) CHQn7, 8.00 (dd, J = 8.6, 1.3 Hz,
1H) CHQn5, 7.67 (d, J = 4.4 Hz, 1H), CHQn3, 7.65 (t, J = 8.2 Hz, 1H), CHQn6, 5.19 (dd, J
= 9.3, 2.7 Hz, 1H) -CHCN-, 4.41 – 4.29 (m, 1H) -NCH2CF2-, 4.23 – 4.09 (m, 1H) -NCH2CF2-,
4.26 (t, J = 5.9 Hz, 2H) CH2Gly, 3.04 – 2.79 (m, 2H) -CHCNCH2CF2-, 2.85 (t, J = 7.4 Hz,
3H) CH2Su, 2.60 (t, J = 7.4 Hz, 2H) CH2Su

13
C NMR (101 MHz, DMSO-d6) δ 174.31, 171.25, 168.27, 167.64, 148.91, 143.13, 138.71,
135.27, 129.90, 128.15, 127.44, 124.97, 124.71, 118.32, 117.34, 51.68, 44.72, 41.83, 36.88,
32.02, 29.36

S9
Synthesis of Intermediate B
NH2

N
SH
O O O O
F H H
N N OH
F N N N
N N
H H H H
N O OH O OH O
O
O O

Commercially available pre-loaded Fmoc-Cys(Trt) on Tentagel resin (500 mg, 0.415 mmol,
RAPP Polymere) was swollen in DMF (3 × 5 min × 5 mL), the Fmoc group removed with
20 % piperidine in DMF (1 × 1 min × 5 mL and 2 × 10 min × 5 mL) and the resin washed
with DMF (6 × 1 min × 5 mL). The peptide was extended with Fmoc-Asp(tBu)-OH, Fmoc-
Lys(NHBoc)-OH and Fmoc-Asp(tBu)-OH in the indicated order. For this purpose, the Fmoc
protected amino acid (2.0 eq), HATU (2.0 eq) and DIPEA (4.0 eq) were dissolved in DMF (5
mL) and reacted with the resin for 1 h under gentle agitation. After washing with DMF (6 ×
1 min × 5 mL) the Fmoc group was removed with 20 % piperidine in DMF (1 × 1 min × 5 min
and 2 × 10 min × 5 mL). Deprotection steps were followed by wash steps with DMF (6 × 1
min × 5 mL) prior to coupling with the next amino acid.

Compound 1 (2 eq) was coupled as described. The peptide was cleaved from the resin with
a cleavage cocktail composed by TFA (15%), m-Cresol (3.5%), Thioanisole (3.5%), Triiso-
propylsilane (14%), mQ water (4%) and DCM (60%). The crude product was purified by
reversed-phase HPLC (95:5 Water/Acetonitrile with 0.1%TFA to 20:80 Water/Acetonitrile
with 0.1% TFA in 20 min) and lyophilized, to obtain a white solid (15 mg; 16.3 µmol, 4%
yield). MS(ES+) m/z 921.29 (M+H)+.

S10
Synthesis of compound 2

NC O
H
N O
N

O O
F
F
N O OH O OH O
H H
HN N N
N N OH
H H
O O O
S
O
NH2 N
O O

O OH
HO

Intermediate B (2 mg, 2.17 µmol, 1.0 eq) was dissolved in PBS pH 7.4 (800 µL). Fluores-
cein-5-maleimide (1.8 mg, 4.34 µmol, 2.0 eq) was added as dry DMSO solution (200 µL).
The reaction was stirred for 3 h at room temperature, protected from the light. The crude
material was purified by reversed-phase HPLC (95:5 Water/Acetonitrile with 0.1%TFA to
20:80 Water/Acetonitrile with 0.1% TFA in 20 min) and lyophilized, to obtain a yellow solid
(420 nmol, 19.3% yield). MS(ES+) m/z 1348.36 (M+1H)1+

S11
Synthesis of compound 3

NC O
H
N O
N

O O
F
F
N O OH O OH O
H H
HN N N
N N OH
H H
O O O
S
O
NH2 N
O O

O NH2
H 2N
SO3H SO3H

Intermediate B (293 µg, 0.32 µmol, 1.0 eq) was dissolved in PBS pH 7.4 (300 µL). Alexa-
488-C5-Maleimide (200 µg, 0.29 µmol, 0.9 eq) was added as dry DMSO solution (1 mg /
mL). The reaction was stirred for 3 h at room temperature, protected from the light. The
crude material was purified by reversed-phase HPLC (95:5 Water/Acetonitrile with 0.1%TFA
to 20:80 Water/Acetonitrile with 0.1% TFA in 20 min) and lyophilized, to obtain an orange
solid (70 nmol, 21.9% yield). MS(ES+) m/z 1619.57 (M+1H)1+

S12
Synthesis of compound 4

NC O O COOH
H NC H
N O N O
N N
1.DCC, dry DMSO N COOH
OH
Overnight, r.t.
F N N N
F HO O F
2. DOTA-GA-NH2 F
N O HOOC
PBS, 1h, r.t. N O N
HN
H
OH HN N
N COOH
O H
O O

Compound 1 (15 mg, 32.67 µmol, 1 eq), N-hydroxysuccinimide (4.5 mg, 39.2 µmol, 1.2 eq)
and DCC (8.8 mg, 42.4 µmol, 1.3 eq) were dissolved in 400µL of dry DMSO and the reaction
was stirred overnight at room temperature, protected from the light. The reaction was filtered
and at the DMSO solution were added 100 µL of PBS solution containing DOTA-GA-NH2
(20 mg, 38.4 µmol, 1.2 eq) and the resulting solution was stirred at room temperature for 1h.
The crude material was purified by reversed-phase HPLC (95:5 Water/Acetonitrile with
0.1%TFA to 20:80 Water/Acetonitrile with 0.1% TFA in 20 min) and lyophilized, to obtain a
white powder (3 mg; 3.92 µmol; 10% yield). MS(ES+) m/z 960.39 (M+2H)2+

S13
Synthesis of compound 5
NC O
H
N O
N
F
O O
F
N O OH O OH O
H H
HN N N
N N OH
H H
O O O
S
O

N
NH2
O

O NH
O
O
HN
N NH2
H
HN O

O
HO
O N O
H
N N HN
N
O O O O
O

Intermediate B (2 mg, 1.83 µmol, 1.0 eq) was dissolved in PBS pH 7.4 (800 µL). MC-ValCit-
MMAE (2.5 mg, 1.8 µmol, 1.0 eq) was added as dry DMF solution (250 µL). The reaction
was stirred for 3 h at room temperature and monitored by LC/MS. The crude material was
purified by reversed-phase HPLC (95:5 Water/Acetonitrile with 0.1%TFA to 20:80 Water/Ac-
etonitrile with 0.1% TFA in 20 min) and lyophilized, to obtain a white powder (2.43 mg, 1.1
µmol, 60% yield). MS(ES+) m/z 2210.1 (M+H)+

S14
Synthesis of compound 6

NC O
H
N O
N

F O O
F
N O OH O OH O
H H
HN N N
N N OH
H H
O O O
S SO3H
O O3S
NH2 N
O O N
HN

SO3H
N

HO3S

Intermediate B (160 µg, 0.174 µmol, 1.0 eq) was dissolved in PBS pH 7.4 (800 µL).
IRDye750 (200µg, 0.174 µmol, 1.0 eq) was added as dry DMF solution (200 µL). The reac-
tion was stirred for 3 h. The crude material was purified by reversed-phase HPLC (95:5
Water/Acetonitrile with 0.1%TFA to 20:80 Water/Acetonitrile with 0.1% TFA in 20 min) and
lyophilized, to obtain a green solid (0.08 µmol, 50% yield). MS(ES+) m/z 1036.3 (M+2H)2+

S15
Synthesis of compound 7

HO O OH

O
F
F
O O
N N NH2 N

O O
O
HN O S
N O O
H H
O N N N OH
N N
H H
O OH O OH O
N
O O

(S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(3-(piperazin-1-yl)
propoxy)quinoline-4-carboxamide was synthetized as previously described in the literature.
Commercially available pre-loaded Fmoc-Cys(Trt) on Tentagel resin (500 mg, 0.415 mmol,
RAPP Polymere) was swollen in DMF (3 × 5 min × 5 mL), the Fmoc group removed with
20 % piperidine in DMF (1 × 1 min × 5 mL and 2 × 10 min × 5 mL) and the resin washed
with DMF (6 × 1 min × 5 mL). The peptide was extended with Fmoc-Asp(tBu)-OH, Fmoc-
Lys(NHBoc)-OH and Fmoc-Asp(tBu)-OH in the indicated order. For this purpose, the Fmoc
protected amino acid (2.0 eq), HATU (2.0 eq) and DIPEA (4.0 eq) were dissolved in DMF (5
mL) and reacted with the resin for 1 h under gentle agitation. After washing with DMF (6 ×
1 min × 5 mL) the Fmoc group was removed with 20 % piperidine in DMF (1 × 1 min × 5 min
and 2 × 10 min × 5 mL). Deprotection steps were followed by wash steps with DMF (6 × 1
min × 5 mL) prior to coupling with the next amino acid.
(S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(3-(piperazin-1-yl)
propoxy)quinoline-4-carboxamide (2 eq) was coupled as described. The peptide was
cleaved from the resin with a cleavage cocktail composed by TFA (15%), m-Cresol (3.5%),
Thioanisole (3.5%), Triisopropylsilane (14%), mQ water (4%) and DCM (60%). The crude
product was purified by reversed-phase HPLC (95:5 Water/Acetonitrile with 0.1%TFA to
20:80 Water/Acetonitrile with 0.1% TFA in 20 min) and lyophilized, to obtain a white solid.
The obtained peptide (2 mg, 1.9 µmol, 1.0 eq) was dissolved in PBS pH 7.4 (800 µL). Fluo-
rescein-5-maleimide (2 mg, 4.82 µmol, 2.5 eq) was added as dry DMSO solution (200 µL).
The reaction was stirred for 3 h at room temperature, protected from the light. The crude
S16
material was purified by reversed-phase HPLC (95:5 Water/Acetonitrile with 0.1%TFA to
20:80 Water/Acetonitrile with 0.1% TFA in 20 min) and lyophilized, to obtain a yellow solid
(420 nmol, 19.3% yield). MS(ES+) m/z 1474.46 (M+1H)1+

S17
LC/MS and HPLC profiles

Intermediate A

MS (ESI) m/z calculated for [C17H15F2N5O2] +: 360.12 [M+H] +, found: 360.20.

Compound 1

MS (ESI) m/z calculated for [C21H19F2N5O5] +: 460.14 [M+1H] 1+, found: 460.20.

Intermediate B

MS (ESI) m/z calculated for [C38H46F2N10O13S] +: 921.29 [M+H] +, found: 921.30.

Compound 2

S18
MS (ESI) m/z calculated for [C62H59F2N11O20S] +: 1348.36 [M+H] +, found: 1348.40.

Compound 3

MS (ESI) m/z calculated for [C68H72F2N14O25S3] +: 1619.39 [M+H] +, found: 1618.30.

Compound 4

MS (ESI) m/z calculated for [C42H55F2N11O13] +: 960.39 [M+H] +, found: 960.40

Compound 5

MS (ESI) m/z calculated for [C106H151F2N21O28S] +: 1118.9 [M+2H] 2+, found: 1119.1

S19
Compound 6

MS (ESI) m/z calculated for [C93H112F2N14O28S5] +: 2071.64 [M+H] +, found: 1034.20 [M+H]2+

lutetium-177-OncoFAP

Radio-HPLC profile of lutetium-177-OncoFAP

S20
IVIS Experiment

SK-MEL-187 or SK-RC-52.wt xenografted tumors were implanted into the right flank of fe-
male athymic Balb/c AnNRj-Foxn1 mice (6-8 weeks of age) as described above, and allowed
to grow to an average volume of 200 mm3.
SK-RK-52.hFAP or HT-1080.hFAP xenografted tumors were implanted into the left flank of
female athymic Balb/c AnNRj-Foxn1 mice (6-8 weeks of age) as described above, and al-
lowed to grow to an average volume of 200 mm3.
Mice were injected intravenously with compound 4 (150 nmol/Kg, as 30 μM solutions pre-
pared in sterile PBS, pH 7.4). One hour after the intravenous injection, mice were sacrificed
by CO2 asphyxiation. Blood, heart, muscle, lung, kidneys, liver, spleen, stomach, a section
of the intestine, SK-MEL-187 tumor, SK-RC-52.hFAP tumor, SK-RC-52.wt tumor and HT-
1080.hFAP tumor were collected and fluorescence images acquired on an IVIS Spectrum
imaging system (Xenogen, exposure 1s, binning factor 8, excitation at 745 nm, emission
filter at 800 nm, f number 2, field of view 13.1).

A B

SI 1 (A) Near-infrared fluorescence imaging evaluation of the targeting performance of tar-

geted dye compound 6 (250 nmol/kg) in mice bearing SK-RC-52.hFAP tumors (right flank
of the animals). SK-MEL-187 tumors were implanted in the left flank of the animals and used
as negative control (no expression of FAP). Images were collected 1 h after the intravenous
injection. (B) Near-infrared fluorescence imaging evaluation of the targeting performance of
targeted dye compound 6 (250 nmol/kg) in mice bearing HT-1080.hFAP tumors (right flank
of the animals). SK-RC-52.wt tumors were implanted in the left flank of the animals and used
as negative control (no expression of FAP). Images were collected 1 h after the intravenous

S21
injection. OncoFAP-IRDye750 (compound 6) selectively accumulated in FAP-positive tu-
mors (HT-1080.hFAP and SK-RC-52.hFAP).

S22
Dose escalation study in SK-RC-52.hFAP

A dose escalation study in order to assess the tolerability of OncoFAP-Vedotin has been
performed. SK-RC-52.hFAP tumors were implanted into the right flank of female athymic
Balb/c AnNRj-Foxn1 mice (6-8 weeks of age) as described in the manuscript, and allowed
to grow to an average volume of 100 mm3. Mice received daily intravenous injections of 125
nmol/kg, 250 nmol/kg, 500 nmol/kg and 1000 nmol/kg of OncoFAP-vedotin (compound 5).
as sterile PBS solution with 2% of DMSO. Animals were weighted daily to assess tolerability
of the treatment. Animals were sacrificed when one or more termination criteria indicated by
the experimental license were reached (e.g. weight loss > 15%).

Dose Escalation of OncoFAP-Vedotin

20
Body Weight change (%)

OncoFAP-Vedotin (125 nmol/Kg)

10 OncoFAP-Vedotin (250 nmol/Kg)
OncoFAP-Vedotin (500 nmol/Kg)
0 OncoFAP-Vedotin (1000 nmol/Kg)
0 2 4 6 8

-10

-20
Days after tumor implantation

SI 2 Body weight change of mice treated with different doses of OncoFAP-Vedotin. At 1000
nmol/kg, OncoFAP-Vedotin was highly toxic. All the other treatments were well tolerated.

S23
Therapy experiment with OncoFAP-Vedotin in combination with L19-IL2 in tumor-
bearing mice

Statistical analysis of tumor size (mm3) differences is performed using Prism 6 software
(GraphPad Software, regular two-way ANOVA followed by Bonferroni test).

OncoFAP-Vedotin + L19-IL2 vs L19--IL2

Day 19 p < 0.05
From day 20 p < 0.0001

OncoFAP-Vedotin + L19-IL2 vs OncoFAP-Vedotin

Day 29 p < 0.001
From day 30 p < 0.0001

OncoFAP-Vedotin + L19-IL2 vs vehicle

Day 12 p < 0.05
From day 13 p < 0.0001

OncoFAP-Vedotin vs L19-IL2
Day 15 p < 0.01

OncoFAP-Vedotin vs vehicle
Day 12 p < 0.05
From day 13 p < 0.0001

L19-IL2 vs vehicle
From day 15 p < 0.0001

S24
In vitro affinity measure of FAPI-04

The in vitro KD measurement of FAPI-04 fluorescent derivative is shown below.

Fluorescence Polarization - Compound 7 Fluorescence Polarization - Compound 7

250 300
200
200
Anisotropy

150

Anisotropy
100
100
50

0 0

-50 10-9 10-8 10-7 10-6 10-5 10-9 10-8 10-7 10-6 10-5
-100
[human FAP], M [murine FAP], M

SI 3 Affinity of Compound 7 for human and murine FAP was measured by fluorescence

polarization. Compound 7 shows a lower affinity for human and murine FAP (KD,humanFAP =

1.02 nM, KD,murineFAP = 30.9 nM) compared with OncoFAP-Fluorescein, [Figure 1C] (KD,hu-

manFAP = 0.68 nM, KD,murineFAP = 11.6 nM).

S25
Protein Sequence of Extracellular Domain Murine FAP and quality control

LRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISE-
QEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRY
SYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNI-
YLKQRPGDPPFQITY-
TGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQY
PRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLT-
WVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVS
TPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSS-
NEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGL
PISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLT-
FWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGT
AFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALAS-
GTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDY
LLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTH-
FLKQCFSLSD

mFAP Quality control

SDS-page and size-exclusion chromatography were performed in order to confirm the qual-
ity of the protein.

MW
(KDa) NR R

250
150 6
5
100
75 4
mAU

3
2
1
0
6 8 10 12 14 16 18 20
Elution volume (mL)

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SI 3: The biochemical properties of murine FAP protein were assessed by SDS-PAGE
(A), size exclusion chromatography eluting after around 13 mL (elution volume) (B) and by
ESI-MS (C). NR, non-reducing conditions; R, reducing conditions.

S27
Protein Sequence of Extracellular Domain Human FAP and quality control

LRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNI-
VLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSN
GEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGI-
PDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAG
AKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRV-
QNVSVL-
SICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIH
YIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNI-
YRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEI
KILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGG-
PCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITA
VRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTER-
FMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQV
DFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

hFAP Quality control

SDS-page and size-exclusion chromatography were performed in order to confirm the qual-
ity of the protein.
MW
(kDa) NR R
250 6
150
100
4
75
mAU

0
0 5 10 15 20 25
Volume (mL)

SI 4: The biochemical properties of human FAP protein were assessed by SDS-PAGE

(A), size exclusion chromatography eluting after around 13 mL (elution volume) (B) and by
ESI-MS (C). NR, non-reducing conditions; R, reducing conditions.

S28
hFAP Lentiviral transfer vector and amino acid sequence

The strategy to generate the lentiviral transfer vector with hFAP in shown below, together
with the amino acid sequence.

SI 5: The graphic representation of the transfer viral vector with hFAP is presented, to-
gether with the aminoacidic sequence of the cytoplasmic domain (A), the transmembrane
– signal anchor (B) and the extra-cellular domain (C).

S29
UniCAR-T in Vitro Killing Assay

The gating strategy to determine the killing rate is shown below.

All cells Single cells Stained Tumour cell line Live cells Dead cells

CAR T-cells

SI 6: The gating strategies used to determine the UniCAR T-cell induced tumor cell lysis.

S30