A. Left-most panel: Coomassie staining of an SDS-PAGE gel showing the input cell lysates used in immunoprecipitation experiments depicted in the next panels. Middle panel: lysates as shown on left-side panel were incubated with nanobody coated paramagnetic beads overnight. Beads were then washed 5 times then boiled in SDS sample buffer before loading on SDS-PAGE. This panel shows Coomassie staining of SDS-PAGE of the bead eluates. In: input lysates. IP: Nanobody used for immunoprecipitation. Right panel: the same eluates as in middle panel were run on SDS-PAGE gel then transferred to a PVDF membrane. Membrane was blocked, stained using a mouse monoclonal anti-TfR (cross-reactive for human and mouse), washed then stained with an anti-mouse-HRP secondary monoclonal. See Methods section for more details. B. Cell-lines were incubated with 35S-labelled Met before performing the same immunoprecipitation procedure as described in A, with the exception that the beads were washed only twice before elution in Laemli buffer. SDS-PAGE was run with input cell lysate (in), unbound fraction (SN), washes (W1 and W2) and eluates for each condition before transfer to a PVDF membrane. Membrane was blocked, stained using a mouse monoclonal anti-TfR (cross-reactive for human and mouse), washed then stained with an anti-mouse-HRP secondary monoclonal. C. Flowcytometry characterization of the specificity of VHH123 and VHH188. CHO cells overexpressing either the mouse isoform of TfR (mTfr, left panel) or human isoform (hTfr, right panel) were labelled with serial dilutions of either Flag-tagged VHH123 or VHH188. I647-fluorescently labeled anti-FLAG IgG was used to detect the presence of either VHH at the cell-surface by flow cytometry.

Summary of peptides identified from LC/MS/MS analysis of the gel sections from figure 1A, middle panel. Transferrin receptor 1 is highlighted in red.

A. Schematic of sortase A and click-chemistry steps to generate each radio-conjugate used in this study. Capital letters denote amino-acids, unless if written in italic where they denote chemical groups or elements. Bold italic ‘R’ represents either DFO (deferoxamine) or NOTA (2,2′,2”-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid). DBCO: dibenzocyclooctyne, PEG20kDa: poly-ethylene-glycol (20kDa mw), TCO: trans-cyclooctene. Underlined is the LPETG Sortase A cleavage site consensus motif. See suppl. Fig 1 for detailed methods. B. Structures of GGG-nucleophiles used in sortase A mediated conjugations (GGG-DFO-N3, GGG-NOTA-N3 and GGG-tetrazine). The 18F-TCO click-chemistry partner of GGG-tetrazine is also depicted.

A-D: C57BL/6 and huTfR+/+ mice were injected with 3.7 MBq (100 µCi) of either VHH123-PEG(20kDa)-DFO-89Zr or VHH188-PEG(20kDa)-DFO-89Zr by retro-orbital injection. The mice were imaged by PET/CT at several timepoints post-injection. Shown here at the maximum intensity projection images acquired at 48 hours post injection of the conjugate. Each panel comprises maximum intensity projection (MIP) overlayed with CT signal on the left, and PET MIP alone on the right. PET intensity scale is displayed on the right (%ID/g). A. C57BL/6 mouse injected with the VHH123 based conjugate. B. C57BL/6 mouse injected with the VHH188-based conjugate. C. huTfR+/+ mouse injected with the VHH123-based conjugate. D. huTfR+/+ mouse injected with the VHH188-based conjugate. Experiment performed with 3 mice per condition, with one mouse shown as representative of each condition. E. Region Of Interest (ROI) analysis of images acquired from mice as shown in A and C and all repeats thereof. The mean ID%/g is plotted for each ROI and mouse repeat. F. Same as E, but for images acquired from mice as shown B and D and all repeats thereof. G. Left graph: Ex vivo activity measurement of flushed femurs (thus mineral bone) and the bone marrow they contained, 72 hours post radiotracer injection. Each dot represents measurement of one mouse on a scale of total count per minute (CPM). Right graph: ex vivo activity measurement of 20 µL of whole blood. Each dot represents the activity from one mouse on a scale of total CPM per 20µL of whole blood. Bars show SD. H. Ex vivo activity measurements from different tissues, performed at 72 hours post radiotracer injection. Each dot represents one measurement from one mouse on a scale of CPM per mg of tissue. Bars show SD.

A. C57BL/6 mice were injected with 3.7 MBq (100µCi) of different VHH-123-PEG(20kDa) based conjugates as indicated on the left side of the panels. PET/CT images were acquired for each condition at 1 hour post injection and at 24 hours post injection, if possible, as indicated below. Each panel comprises maximum intensity projection (MIP) overlayed with CT signal on the left, and PET MIP alone on the right. PET intensity scales are displayed on the right of each panel (%ID/g). This figure pools the representative pictures obtained from 3 independently performed sets of experiments where one specific VHH123-radiolabelled conjugate was tested per experiment. 3 mice were imaged in each experiment for each condition. B. ROI analysis of images acquired from mice as shown in all panels of A, and all repeats thereof. Top graph: each point represents the mean ID%/g for one mouse. Bottom graph: same data as in top graph, but each point represents the mean ID%/g of a specific tissue normalized to the average ID%/g values found in the kidneys of the same group.

Top: cartoon depicting the experimental procedure: bone marrow was harvested from the femur of C57BL/6 (A, D and E) or huTfR+/+ (B and C) mice before transplantation into 3 lethally irradiated (10 Gy) huTfR+/+ (A and E) or C57BL/6 (B, C and D) mice. These mice were then injected with 3.7 MBq (100µCi) of VHH123-PEG(20kDa)-DFO-89Zr (A and E) or VHH188-PEG(20kDa)-DFO-89Zr (B, C and D) immediately (E) or 2 weeks post transplantation (A, B, C and D) and PET/CT images were acquired at several timepoints thereafter. Bottom: PET/CT maximum intensity projections (MIP) that were deemed the most representative of each condition are shown. Each panel comprises maximum intensity projection (MIP) overlayed with CT signal on the left, and PET MIP alone on the right. A: MIP of one huTfR+/+ recipient mouse acquired 2 weeks after C57BL/6 bone marrow transplantation and 24 hours after radiotracer injection. B: MIP of one C57BL/6 mouse acquired 2 weeks after huTfR+/+ bone marrow transplantation and 24 hours after radiotracer injection. C. Same as B, but imaged 1 hour after radiotracer injection. D. MIP of one C57BL/6 mouse acquired 2 weeks after C57BL/6 (isogenic) bone marrow transplantation and 24 hours after radiotracer injection. E. MIP of one huTfR+/+ recipient mouse injected with radiotracer immediately after C57BL/6 bone marrow and imaged 24 hours thereafter. Two cohorts were set up separately to perform the PET/CT imaging immediately after bone marrow transplantation or two weeks thereafter. PET intensity scales are displayed on the right of each panel (%ID/g). F. ROI analysis of images acquired from mice as shown in panel A, and all repeats and imaging timepoints thereof. Error bars show SD. G. ROI analysis of images acquired from mice as shown in panels B, C and D and all repeats and imaging timepoints thereof. Error bars show SD.

Top: cartoon depicting the experimental procedure: 5 x 104 B16.F10 mouse melanoma cells were transfused to huTfR+/+ mice by tail-vein injection. 2 and 4 weeks later, the mice were injected with 3.7 MBq (100µCi) of either VHH123-PEG(20kDa)-NOTA-64Cu or VHHEnh-PEG(20kDa)-NOTA-64Cu radiotracers before PET/CT imaging. A-E. Maximum Intensity Projections (MIP) and lung traverse sections of one representative mouse out of three for each experimental condition at the 2 weeks timepoint post B16.F10 cell transfusion (except for E: no B16.F10 cells were injected). Images were acquired 24 hours post radiotracer injection. PET intensity scale is displayed on the right (%ID/g). A. MIP of a huTfR+/+ mouse imaged with VHH123-PEG-NOTA-64Cu 2 weeks post B16.F10 cell transfusion. B. MIP of a huTfR+/+ mouse imaged with VHHEnh-PEG-NOTA-64Cu 2 weeks post B16.F10 cell transfusion. C. MIP of a huTfR+/+mouse imaged with VHH123-PEG-NOTA-64Cu 4 weeks post B16.F10 cell transfusion. D. MIP of a huTfR+/+mouse imaged with VHHEnh-PEG-NOTA-64Cu 4 weeks post B16.F10 cell transfusion. E. MIP of a huTfR+/+mouse imaged with VHH123-PEG-NOTA-64Cu that did not receive any tumor cells. F. Photographs of dissected organs from huTfR+/+ mice euthanized at 4 weeks post B16.F10 cell infusion and 96 hours post radio-tracer injection (left) and from control mice that received no cells 96 hours post radio-tracer injection (right). RL: right lung, LL: left lung, H: heart, Li: liver, Sp: spleen, Ki, kidneys. Organs are from the same respective mice as shown in C. Red arrows delimit necrotic and hyperdense tumors growing out of the right and left lung. G. ROI analysis of images acquired from mice as shown in panels C, D and E. Each dot represents the mean ID%/g of a specific ROI for one mouse. Error bars show SD. No error bars are shown for the VHHEnh – tumor group as n=2 (one mouse died before imaging). H. ROI analysis of hyperdense lung tissue as visualized by CT on images acquired from mice as in panels C and D. Each point shows the mean ID%/g of one mouse. Error bars show SD. No error bars are shown for the VHHEnh – tumor group as n=2.

Top: cartoon depicting the experimental procedure: 8–12-week-old C57BL/6 females were mated in pairs with a single 12-week-old huTfR+/+ or C57BL/6 male. 2 weeks post fertilization (confirmed by observing vaginal plugs), the females are injected retro-orbitally with 3.7 MBq (100µCi) of VHH188-PEG(20kDa)-DFO-89Zr. A. PET/CT (left) and PET (right) maximum Intensity Projections (MIP) of one female carrying huTfR+/- embryos, imaged 24 hours after injection of the radiotracer. B. PET/CT (left) and PET (right) MIP of one female carrying C57BL/6 wild-type embryos, imaged 24 hours after injection of the radiotracer. PET intensity scale for A and B is displayed on the right (%ID/g). C. ROI analysis of images acquired from mice as shown in panels A and B. Each dot represents the mean ID%/g of the placenta for one mouse. Error bars show SD. No error bars are shown for the huTfR+/- condition as n=2 (2 out of 4 females had plugs 2 weeks prior but were not gestating at time of imaging).D. Left side panel: photograph of dissected embryos from the euthanized female carrying huTfR+/- embryos, 72 hours post radiotracer injection. Right side panel: medial PET/CT section of a 50 mL tube containing the dissected embryos as shown on the left-panel. The green arrows highlight the placenta of one embryo. E. Left side panel: photograph of dissected embryos from the euthanized female carrying wild-type C57BL/6 embryos, 72 hours post radiotracer injection. Right side panel: medial PET/CT section of a 50 mL tube containing the dissected embryos as shown on the left-panel. The green arrows highlight the placenta of one embryo. PET intensity scale for D and E is displayed on the right (%ID/g). F. PET/CT MIP of one female carrying huTfR+/- embryos, imaged 1 hour after injection of the radiotracer. PET intensity scale is displayed on the right (%ID/g). Experiment performed twice. Total of n=2 mice in B6 x huTfR+/+ group and n=3 in B6 x B6 group.

Schematic representation of each step required to generate 89Zr-based (blue), 64Cu-based (green) and 18F-based (orange) VHHconjugates. Capital letters denote amino-acids, unless written in italic where they denote chemical groups or elements. Each reaction condition is noted on the right hand of each reaction. DFO: deferoxamine, NOTA: 2,2′,2”-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid, PEG: poly-ethylene-glycol, TCO: trans cyclo-octene, DBCO: dibenzocyclooctyne. See methods for details.

185 MBq (5mCi) of 89Zr-oxalate stock solution was obtained from the Cyclotron Lab at UW Madison, USA. Stock solution was put at a neutral pH of 7.5 by successive addition of 90% of stock volume of 2.0 M Na2CO3 and 400% stock volume of HEPES 0.5M. VHH123-PEG-DFO was conjugated to 89Zr at a neutral pH of 7.4 by adding 44.4 MBq (1.2mCi) of pH neutralized 89Zr to 120µg of VHH123-PEG-DFO in chelexed PBS, put in a microcentrifuge tube for 1 hour at room temperature on an agitator. The mixture was then split into 6 tubes (200 µCi each), and diluted 1/15 (v/v) in citrate-sodium phosphate buffer of varying pH: 7.4, 6.6, 5.6, 4.6, 3.6 and 2.6. The conjugation mixture was then immediately passed onto a PD-10 gravity size exclusion chromatography column (Cytiva) pre-equilibrated with citrate-sodium phosphate buffer of same pH used to dilute the VHH conjugate. Fractions of 600µL were collected by addition of citrate-sodium phosphate buffer of same pH and each fraction was measured for radioactivity using a dosimeter (AtomLab 500, Biodex). Shown is a heat map of measured radioactivity from each fraction collected. ‘column loading’: flowthrough from column displaced by application of the reaction mixture to the column. ‘remain in column’: residual radioactivity measured from the whole column post-elution. Brackets on the left indicate the typical elution fraction range of a VHH-radiometal conjugate post successful conjugation.

PET/CT of one female C57BL/6 mouse that received 2.775 MBq (75 µCi) of 89Zr as a free element (un-chelated/un-conjugated) alongside 75 x 108 gold chiral nano-particles. This image exclusively shows the signal generated by the accumulation of free 89Zr in a mouse. The co-injection of gold nano-particles was done in relation to another work published by our group, but where this data was not included 48.

SDS-PAGE followed by Coomassie Blue stain, showing the individual constructs used throughout this work, as purified and concentrated post-sortase A transpeptidation and post DBCO-PEG20kDa click-chemistry conjugation when performed. Sortase A expected mw: 17.8 kDa. For expected mw of other constructs, see Suppl. Figure 10.

LC/MS mass measurements of each VHH construct used in this work. PEG20kDa modified constructs are not shown as their masses cannot be precisely measured due to the fact that the DBCO-PEG20kDa compound has an average mass of 20kDa.

Radio-Thin Layer Chromatography QC data for VHH-18F constructs.