Cells

Fibrosarcoma cells (FSA) were a gift from the Department of Radiation Oncology at UCLA originally isolated from a methylcholanthrene-induced fibrosarcoma in C3Hf/He (C3H/Sed/Kam) mice [16]. Human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum (Omega Scientific, Tarzana, CA, USA) at 37ºC and 5% CO2. To generate cells overexpressing murine FAP (mFAP), FSA cells were transduced with the lentiviral vector pLV-mFAP and dilution cloning was performed to select clones with low (FSA-Flow), medium (FSA-Fmed) and high (FSA-Fhi) levels of mFAP (Supplemental Fig. 1). Cell media was tested every 2–4 weeks for mycoplasma contamination using MycoAlert (LT07-710, Lonza, Basel, Switzerland).

In vitro expression levels of FAP, PD-L1 and H-2 K (MHC class I) in FSA cells

FSA cells were seeded in 6-well plates (3 × 105 / well) and treated after 24 h with either 10 ng/mL murine interferon-g (IFNγ) or with 8 Gy X-rays using a Gulmay RS320 X-ray unit at 300 kV and 10 mA (Gulmay Medical Ltd., Surrey, UK). Dosimetry was based on a Capintec ionization chamber calibrated to NIST standards and film (GAFCHROMIC EBT2, International Specialty Products, Wayne, NJ, USA). Treated cells and controls were collected 24 h after incubation and analyzed by either flow cytometry or immunoblot.

To quantify cell surface protein expression, 0.5 × 106 cells were stained for 30 min on ice with anti-FAP (clone 73.3, 1:1000, Sigma-Aldrich, St. Louis, MO, USA), anti-PD-L1-PE (clone 10 F.9G2, 1:200, BioLegend, San Diego, CA, USA) or anti-H-2 K (MHC class I; clone Y-3, 1:40, Sigma-Aldrich) antibodies. Anti-IgG1-APC (15 min on ice; clone RMG1-1, 1:200, BioLegend) or anti-mIgG-FITC (15 min on ice; clone H + L, 1:1000, Invitrogen, Waltham, MA, USA) antibodies were used as secondary antibody for the detection of FAP and H2-K, respectively. All samples were measured on a LSRII Flow Cytometer (BD, Franklin Lakes, NJ, USA) at the UCLA Flow Cytometry Core Facility and analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

For immunoblot analysis, protein lysates were prepared in cold RIPA buffer supplemented with protease and phosphatase inhibitors, normalized using BCA assay, resolved on 4–12% Bis-Tris gels and electro-transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk in TBS + 0.1% Tween-20 (TBS-T), membranes were cut into relevant sections and incubated overnight with the respective primary antibody (vinculin: clone E1E9V, phospho-STAT1 Tyr701: clone 58D6, Cell Signaling Technology, Danvers, MA, USA) diluted 1:1000 in 5% BSA in TBS-T. Membranes were washed and incubated with HRP-linked secondary antibodies at 1:2500 dilution in 5% nonfat dry milk / TBS-T. HRP was activated by incubating membranes with a mixture of SuperSignal Pico and SuperSignal Femto ECL reagents (100:1 ratio, ThermoFisher, Waltham, MA, USA). Exposure of photo film was used for detection.

[68Ga]Ga-FAPI-46

[68Ga]Ga-FAPI-46 was synthesized as previously described [1, 2]. The final product had a radiochemical purity of > 98% by HPLC and thin-layer chromatography with 50 mM EDTA as a mobile phase and a molar activity of 130 MBq/µmol.

Cell binding and internalization studies

Cells were plated in 24-wells (7.5 × 104 / well) and incubated for 24 h. Binding studies were performed as previously described [2]. Briefly, after washing twice with PBS, 20 kBq [68Ga]Ga-FAPI-46 were added and incubated for 1 h at 37ºC and 5% CO2. Surface bound fractions were collected with ice-cold 1 M glycine-HCl (pH 2.2) and cells were lyzed subsequently using two sequential washes with aqueous 0.3 M NaOH. Samples were counted on a PerkinElmer 2480 Wizard2 Automatic Gamma Counter (PerkinElmer, Waltham, MA, USA). Data were decay-corrected, background-subtracted and resulting activities were normalized to 105 cells.

Animal models

In vivo studies were approved by the UCLA Institutional Animal Care and Use Committee (#2005-090) and conducted in three murine models: (1) tumors derived from the inoculation of parental FSA, (2) FSA clones transduced with murine FAP (low/medium/high) and (3) locally immunosuppressed FSA-Fmed tumors. For all cell lines, tumor growth was tested in syngeneic, immunocompetent C3H/Sed/Kam mice. Female 6–8 weeks old mice (Department of Radiation Oncology, UCLA) were housed under gnotobiotic conditions (12 h–12 h light-dark cycle; food and water ad libitum). For tumor cell transplantation, cells were trypsinized, filtered through a 70 μm cell strainer and washed twice with PBS (500 x g, 4 °C, 5 min). Cells (0.5 × 106 per animal) were resuspended in ice-cold PBS and mixed with an equal amount of matrigel (Corning Inc, Corning, NY, USA) to a total injection volume of 100 µL per animal. To generate locally immunosuppressed tumors, FSA-Fmed cells were co-inoculated with the immunosuppressive and human/mouse cross-reactive CTLA-4 fusion protein Abatacept [17], in order to block CD80/86-mediated co-stimulation of T cells in the TME and thereby render tumors non-responsive to PD-1 ICB. For this, the cell pellet was resuspended in cold PBS containing 10 mg/mL Abatacept (final concentration = 0.5 mg/inoculation) before mixing with matrigel. Cells were inoculated subcutaneously into the right shoulder region of mice. Tumors reached a volume of 50–100 mm3 within 10–14 days (FSA), approximately 18 days (FSA-Fmed), and 16 days (locally immunosuppressed FSA-Fmed).

Micro positron emission tomography (PET)/computed tomography (CT)

For in vivo FAP imaging, animals were intravenously (i.v.) injected with 1.1 MBq [68Ga]Ga-FAPI-46. After 60 min, animals were anesthetized and kept under 2% isoflurane for static 10 min PET scans with subsequent CT. Mice underwent CT scans every 4–7 days to monitor tumor size. All scans were performed on a G8 benchtop PET/CT (SOFIE Biosciences, Culver City, CA, USA). OsiriXv.10.0.2 (Pixmeo, Bernex, Switzerland) [18] was used for PET and CT analysis. For analyzing PET data, the 3D ball isocontour function was used to define the maximal and mean standardized uptake values (SUVmax, SUVmean) of tumors. CT data were analyzed by delineating tumors on ≥ 7 CT slices and using the compute volume function to derive tumor volumes.

Immunohistochemistry (IHC)

Formalin-fixed paraffin-embedded tumor samples (4 μm) were stained with an anti-FAP antibody (EPR20021, 1:50, Abcam, Cambridge, UK) as described previously [19]. For staining of T cells, antigens were retrieved according to the manufacturer’s instructions and specimens were incubated overnight at 4 °C with either anti-CD4 (clone EPR19514, 1:1000, Abcam) or anti-CD8 (clone 4SM15, 1:500, ThermoFisher) antibodies. All samples were subsequently stained with DAB Chromogen reagent (#K3467, Dako, Glostrup, Denmark) and counterstained with hematoxylin before dehydration, mounting with Permount Mounting Media and digital scanning at 20x magnification using ScanScope AT (Leica Biosystems, Vista, CA, USA). Cell densities per area were estimated from the averaged CD4+ or CD8+ cell counts of five 200 × 200 μm squares (0.04 mm3).

[225Ac]Ac-FAPI-46

FAPI-46 precursor was kindly provided by the University of Heidelberg and Actinium-225 was obtained from the National Isotope Development Center (Oak Ridge, TN, USA). Unless specified otherwise, all reagents and chemicals used were purchased from ThermoFisher Scientific and/or Sigma-Aldrich and used as received. For radiolabeling, [225Ac]Ac(NO3)3 was dissolved in 0.1 M HCl and mixed with FAPi-46 in 1 M NaOAc containing 10 mg/mL gentisic acid resulting in a final reaction pH of ~ 5.5 [20]. Incubation for thirty minutes at 90 °C provided [225Ac]Ac-FAPi-46 in a purity of 97 ± 2% and at a molar activity of 45–55 MBq/µmol. Final product stability was confirmed by TLC up to 24 h after labelling (98.5% intact).

Therapy studies

For an initial activity escalation study, FSA-bearing mice were injected intravenously with 20, 40 or 60 kBq [225Ac]Ac-FAPi-46. For the combination study with one or three injections of 60 kBq [225Ac]Ac-FAPI-46 and anti-PD-1 therapy, FSA-bearing mice were treated with either a single injection of 60 kBq [225Ac]Ac-FAPI-46, or three consecutive injections of 60 kBq [225Ac]Ac-FAPI-46 in 24 h intervals. Anti-PD-1 antibody (intraperitoneal injection, 10 mg/kg in PBS; clone RMP1-14, #BE0146, BioXCell, Lebanon, NH, USA) or isotype-control (clone 2A3, #BE0089, BioXCell) treatment was started 24 h after the last [225Ac]Ac-FAPI-46 administration and injected every 3–4 days for a total of four doses.

Mice with locally immunosuppressed FSA-Fmed tumors were treated with three consecutive injections of 60 kBq [225Ac]Ac-FAPI-46 in 24 h intervals. Anti-PD-1 antibody or isotype was administered as mentioned above.

For all studies, 8–12 animals were randomized to each group based on tumor volume, and tumor volumes as well as body weights were monitored by (semi-)weekly CT. Animals were euthanized when reaching a humane endpoint according to ARC protocol (tumors > 2cm3, ulceration) or a study endpoint (survival, tumor regrowth), and overall survival (OS) was calculated.

Statistics

Unless stated otherwise, data are shown as mean ± SEM. Comparisons of two groups were evaluated using unpaired 2-tailed Student’s t test. Statistical analysis of more than two groups was performed using one-way analysis of variance (ANOVA) with Tukey’s post hoc correction. Median survival was analyzed using the log-rank test. P values of less than 0.05 were considered significant. GraphPad Prism software (version 9, GraphPad, San Diego, CA, USA) was used for all statistical calculations.