Novel compositions, methods of manufacture and uses thereof

Abstract

Provided herein are nanobody-conjugated nanoparticles and methods of use thereof, e.g., for targeted delivery or accumulation of the nanoparticles in selected tissues.

Description

[0001] Attorney Docket No.29618-0451WO1 NOVEL COMPOSITIONS, METHODS OF MANUFACTURE AND USES THEREOF CLAIM OF PRIORITY This application claims the benefit of U.S. Provisional Application Serial No. 63 / 541,833, filed on September 30, 2023. The entire contents of the foregoing are incorporated herein by reference. TECHNICAL FIELD Provided herein are nanobody-conjugated nanoparticles and methods of use thereof, e.g., for targeted delivery or accumulation of the nanoparticles in selected tissues. BACKGROUND In recent decades, inorganic or hybrid inorganic-organic nanoparticles (NPs) have demonstrated significant promise for oncologic indications. One noteworthy example is the AGuIX (Activation and Guidance of Irradiation X) NPs. AGuIX are ultra-small NPs composed of gadolinium (Gd) chelates on a polysiloxane core, displaying a mean hydrodynamic diameter of 4 ± 2 nm. The corresponding mean molecular weight (MW) has been estimated at around 20 kDa. These NPs are currently being investigated in several clinical trials targeting different types of cancer, including brain metastasis (Phase II: NCT03818386 and NCT04899908), cervical cancer (Phase I: NCT03308604), glioblastoma (Phase I / II: NCT04881032), and pancreatic and lung cancers (Phase I / II: NCT04789486). AGuIX NPs possess theranostic properties, providing dual functionality for imaging and radiotherapy. This is achieved through the presence of paramagnetic gadolinium ions (Gd3+) embedded within the NPs. These gadolinium ions contribute to positive contrast effects in T1 magnetic resonance imaging (MRI) and enhance the effectiveness of radiotherapy due to their high atomic number (Z=64). The small size of AGuIX NP enables preferential passive uptake in tumors while facilitating rapid elimination through the kidneys (in humans t1 / 2= 1.29 ± 0.27 h), thus minimizing the risk of toxicity. Additionally, AGuIX NPs demonstrate remarkable tumor uptake when compared to other organic Attorney Docket No.29618-0451WO1 or inorganic NPs and to marketed contrast agents, achieving approximately 1.19 ± 0.87% ID / g through passive targeting after 24 hours in large variety of animal model. SUMMARY Provided herein are nanoparticle conjugates comprising: 1) one or more nanoparticles, wherein the nanoparticles comprises a) a polysiloxane core; b) one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to metal ions; 2) one or more second linking groups; and 3) one or more nanobodies; wherein each second linking group is covalently bonded to one nanoparticle and one nanobody. In some embodiments, the ratio of nanoparticles to nanobodies is about 1:1, about 1.3:1, about 1.5:1, or about 2:1, optionally wherein the ratio of nanoparticles to nanobodies is about 1.3:1. In some embodiments, the ratio of metal ions to nanobodies is about 15:1, about 20:1, about 23:1, or about 30:1, optionally wherein the ratio of metal ions to nanobodies is about 20:1. In some embodiments, the hydrodynamic diameter of the nanoparticle conjugate is from about 2 to 20 nm, optionally wherein the hydrodynamic diameter of the nanoparticle conjugate is about 2.3 to 8.5 nm. In some embodiments, the molecular weight of the nanoparticle conjugate is from about 25 to 50 kDa, optionally wherein the molecular weight of the nanoparticle conjugate is from about 29 to 44 kDa. In some embodiments, the metal ions are lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, tin (stannum), palladium, silver, zirconium, gold, or bismuth, or combination thereof. In some embodiments, the metal ions are Gd3+ions, Bi3+ions, or a combination thereof, optionally wherein the metal ions are Gd3+ions. In some embodiments, the one or more first linking groups each comprise a C1-10 alkylamine. Attorney Docket No.29618-0451WO1 In some embodiments, the one or more first linking groups are each , wherein indicates the bond connecting the first linking group to core and indicates the bond connecting the first linking group to the ligand. In some the ligand is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1-(glutaric acid)-4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7- triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylene diamine tetra-acetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA), cyclohexyl- 1,2-diaminetetraacetic acid (CDTA), ethyleneglycol-O,O'-bis(2-aminoethyl)- N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'- diacetic acid (HBED), triethylene tetramine hexaacetic acid (TTHA), hydroxyethyidiamine triacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane- N,N',N",N"'-tetraacetic acid (TETA), or 1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (TCMC). In some embodiments, the ligand is or group. In some embodiments, the indicates the bond connecting the In some embodiments, greater than about 20% of the ligands are complexed to the metal ions. In some embodiments, the nanoparticle comprises 4 to 26 metal ions, 6 to 24 metal ions, 8 to 22 metal ions, 10 to 20 metal ions, 12 to 18 metal ions, or 14 to 16 metal ions, optionally wherein the nanoparticle comprises about 15 metal ions. Attorney Docket No.29618-0451WO1 In some embodiments, the hydrodynamic diameter of the nanoparticle is from about 0.5 to 8 nm, about 0.5 to 5.5 nm, about 1 to 5 nm, about 1.5 to 4.5 nm, or about 2 to 4 nm, optionally wherein the hydrodynamic diameter of the nanoparticle is from about 2 to 4 nm. In some embodiments, the one or more second linking groups are each independently or wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein indicates the bond connecting the second linking group to the nanoparticle, and indicates the bond connecting the second linking group to the nanobody. In some embodiments, the one or more second linking groups are each independently or

[0002] Attorney Docket No.29618-0451WO1 or a pharmaceutically acceptable salt thereof, wherein indicates the bond connecting the second linking group to the nanoparticle, and indicates the bond connecting the second linking group to the nanobody. In some embodiments, the nanobody is listed in Table A, optionally selected from the following nanobodies: KN044, HLX-53, 131I-GMIB-Anti-HER2-VHH1, CAM-FAP-(I-131 / AC-225),NM-01, NM-02, NM-03, NM-04, NM-05, JS014 (fusion protein), RAD204 (177Lu), RAD202 (177Lu), A12, A4, Z15-0-2, Nb 16, Nb 36, B3, Envafolimab (KN035), Nb 97 (MY2935), Nb 109, 3 / E2, sbAb K2, 3131 / 3206, Nb 16925, Nb 16988. In some embodiments, the nanoparticle conjugate is prepared by the process of reacting i) one or more nanoparticle A, wherein nanoparticle A comprises a) a polysiloxane core; b) one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to Gd3+ions; c) one or more of linker A: wherein x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and indicates the bond connecting linker A to the nanoparticle A; with ii) one or more nanobody B, wherein nanobody B comprises one or more of linker B: wherein z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indicates the bond connecting linker B to the nanobody. Attorney Docket No.29618-0451WO1 Methods for making the nanoparticles, e.g., using this process, are also provided herein. Additionally provided herein are methods of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of a nanoparticle conjugate as described herein, wherein the nanobody binds to a cancer cell target or a checkpoint inhibitor or other immunotherapeutic target. In some embodiments, the cancer is a solid cancer. In some embodiments, the nanobody binds to ANGPT2, ANGPTL2, B7-H3, CLDN18.2, CTLA-4, EGFR, FAP, HER-2, MSLN, MUC1, PD-1, PD-L1, TROP-2, TIGIT, VEGFA, VEGF, VEGFR, BCMA, CD19, CD20, CD7, CD47, c-KIT, c-Met Nectin-4, IL-21,ODFR, or RANKL. In some embodiments, the nanobody is listed in Table A, optionally selected from the following nanobodies: KN044, HLX-53, 131I-GMIB-Anti-HER2-VHH1, CAM-FAP-(I-131 / AC-225),NM-01, NM-02, NM-03, NM-04, NM-05, JS014 (fusion protein), RAD204 (177Lu), RAD202 (177Lu), A12, A4, Z15-0-2, Nb 16, Nb 36, B3, Envafolimab (KN035), Nb 97 (MY2935), Nb 109, 3 / E2, sbAb K2, 3131 / 3206, Nb 16925, or Nb 16988. In some embodiments, the methods further comprise treating the subject with a radiotherapy modality, optionally either external beam radiotherapy (EBRT) or targeted radionuclide therapy, e.g., as described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. Attorney Docket No.29618-0451WO1 DESCRIPTION OF DRAWINGS FIGs.1A-C. Synthesis of AGuIX-VHH by sortagging reaction: method and characterizations. A. Scheme of the sortagging reaction, created with Biorender.com. B. Size Exclusion Chromatography (SEC) chromatogram in the fluorescence intensity (λexc=280 nm; λem=340 nm) of the purified bioconjugate AGuIX@A12, AGuIX@GGG, and A12. C. Measurement of hydrodynamic diameter by dynamic light scattering (DLS) of AGuIX@A12, AGuIX@GGG, and AGuIX. FIGs.2A-C. Synthesis of AGuIX-VHH by click chemistry: method and characterizations. A. Scheme of the click chemistry reaction, created with Biorender.com. B. SEC chromatogram in the fluorescence intensity (λexc=280 nm; λem=340 nm) of the purified bioconjugate AGuIX@A12, AGuIX-DBCO, and A12- azide. C. Comparison table of hydrodynamic diameters measured in Taylor dispersion analysis (TDA) and DLS. Data presented as mean + / - standard deviation. FIG.3. Reproducibility of the bioconjugation by click chemistry protocol on the bioconjugation of AGuIX with A4 VHH. The SEC chromatogram in the fluorescence intensity (λexc = 280 nm; λem = 340 nm) shows the purified bioconjugates AGuIX@A4, AGuIX-DBCO, and A4-azide. FIGs.4A-I. Binding affinity of AGuIX@VHH conjugates. A-B. ELISA plates were coated with either PD-L1 (FIG.4A) or CD47 (FIG.4B) proteins and affinity of free and AGuIX@VHH bound VHH was assessed. No difference in binding affinity for A12 or A4 free VHH compared to AGuIX validating that functional targeting capabilities of VHH are not impacted by conjugation. Control of AGuIX further confirmed that binding is due to presence of VHH conjugates rather than non-specific NP and protein interactions (n = 3). C. Table of binding affinity constants for AGuIX@VHH conjugates. All characteristic interaction constants (KD, kAand kD) between the A12 and A4 to their respectively PDL1 and CD47 receptors were determined by Langmuir 1:1 binding model. Triplicates were made for all measurements except A4@AGuIX only one measurement was made. D. Internalization of AGuIX@VHH in high PD-L1 expressing melanoma cells. Metastatic hiPD-L1 B16F10 cells were incubated with either Cy5.5 conjugated AGuIX or AGuIX@VHH for 1 hour prior to staining with CellMask plasma membrane and DAPI nuclear stain. For a subset of samples, PD-L1 and CD47 receptors were partially blocked with commercial anti-PD-L1 and anti-CD47, Attorney Docket No.29618-0451WO1 respectively, prior to AGuIX@VHH incubation. E. AGuIX and AGuIX@VHH fluorescence was quantified using ImageJ to calculate corrected total cell fluorescence, CTCF (n = 11 – 40 cells, **** p < 0.001). F-G. Quantitative analysis of ex vivo biodistribution (1.92 ± 0.09 MBq) at 4 hours and 24 hours after intravenous injection (i.v.) of AGuIX@A12 (FIG.4F) and AGuIX (FIG.4G) NPs which confirmed the specific active targeting of PD-L1 in B16F10 murine melanoma model. (n = 8; mean + / - SD). H-I. The tumor-to-blood (FIG.4H) and the tumor-to-muscle (FIG.4I) ratios of AGuIX and AGuIX@A12 at different time points. FIGs.5A-H. Characterization on the sortagging reaction. A-B. Monitoring of sortagging reaction between AGuIX-CGWWW and A12 using SEC (method A). All the reagents except StrA7m were introduced for FIG.5C, showing that no reaction happened in the absence of the enzyme. C-D. Chromatograms of AGuIX-A12 obtained by sortagging reaction between AGuIX-CGWWW and A12 in the presence (FIG.5C) and in the absence (FIG.5D) of StrA7m. E. Optimization of sortagging reaction between AGuIX-CTCCC and A12. F-G. Calibration curve for A12 quantification (fluorescence, 340 nm). H. Measurement of hydrodynamic diameter of AGuIX-CTGGG and AGuIX@A12 by DLS. FIG.6. Binding affinity of AGuIX@A12 conjugates obtained from sortagging and click chemistry. ELISA plates were coated with PD-L1 proteins and affinity of free A12, AGuIX and AGuIX@A12 were assessed. No difference in binding affinity for A12 free VHH compared to AGuIX bound regardless of chemistry (Click or Sortagging) validating that functional targeting capabilities of A12 are not impacted by the conjugation method. Control of AGuIX further confirmed that binding is due to presence of A12 rather than non-specific NP and protein interactions (n=3). FIGs.7A-J. Characterization of the click chemistry. The first step of the synthesis involves functionalizing the A12 VHH with the azide group. A. Functionalization was confirmed by SEC with a lower retention time suggesting an increase in size. B. This result was validated by Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry, revealing a distribution of 1 to 3 linkers per A12 VHH. The second step of the synthesis involves functionalizing the AGuIX with the DBCO group. C. Optimization of the DBCO / AGuIX ratio of the protocol to obtain more than one function per NP. D. Fluorescence area calibration curve of AF647-azide dye at 647nm depending on the Attorney Docket No.29618-0451WO1 dye concentration to quantify the DBCO grafted on AGuIX. The area was measured by SEC (Superdex 75 column). E-F. Following of the click reaction: The clear separation of the two initial products A12@azide and AGuIX@DBCO (FIG.7E), enables click bioconjugation to be followed up, and formation of the AGuIX@A12 product (FIG.7F). G. SEC chromatogram of the purified bioconjugate following the gadolinium 156 by HPLC-ICPMS, the fluorescence (λexc=280 nm; λem=340 nm) and absorbance (λ=295 nm) by HPLC UV-vis. H. Calibration curve of fluorescence area (λexc=280 nm; λem=340 nm) versus A12@azide-DBCO concentration was done to quantify the VHH in the final product. I-J. Analysis performed on Agilent HPLC on Superdex 7510 / 300 column and data processed on GraphPad 8.0.1 Representative Taylorgram and corresponding bimodal fit for gadolinium signal forAGuIX-DBCO nanoparticles (FIG.7I) and AGuIX@A12 (FIG.7J) after purification. The signal was fitted with the sum of two Gaussian curves of the two populations within the sample. FIGs.8A-E. Affinity measurement on PD-L1 using Biacore®. Curve fitting of the experimental data corresponding to phase of association and dissociation of samples at various concentration on the PD-L1 receptor immobilized on the surface of the sensor chip in the channel fc2. The signal corresponds to the fc2-fc1 channel as a function of time. Sensogram of SPR responses of compound AGuIX (0 to 915.7 nM in Gd3+) (FIG.8A), A12 (0 to 61.2 nM in VHH) (FIG.8B), AGuIX@A12 (0 to 61.2 nM in VHH) (FIG.8C), A4 (0 to 67nM in VHH) (FIG.8D), and AGuIX@A4 (0 to 67nM in VHH) (FIG.8E) with PD-L1. Triplicates were made for all measurements. FIGs.9A-B. Affinity measurement on CD47 using Biacore®. Curve fitting of the experimental data corresponding to phase of association and dissociation of samples at various concentration on the CD47 receptor immobilized on the surface of the sensor chip in the channel fc4. The signal corresponds to the fc4-fc3 channel as a function of time. Sensogram of SPR responses of compound A4 (0 to 67nM in VHH) (FIG.9A) and AGuIX@A4 (0 to 0-16.65nM in VHH) (FIG.9B) with CD47. Triplicates were made for A4. Due to a chip regeneration problem and the lack of product, only one analysis was carried out on A4@AGuIX. FIG.10. A4 modification with the linker azide-PEG4-NHS confirmed by MALDI-TOF analysis. The results revealed a distribution of 1 to 4 linkers per A4 VHH. It confirmed the complete grafting of all VHH in solution, as evidenced by the almost complete conversion of the initial A4 peak (14.85 kDa). Attorney Docket No.29618-0451WO1 FIGs.11A-B. Internalization of AGuIX-VHH in wild-type melanoma. A. Metastatic wild-type B16F10 cells were incubated with either Cy5.5 conjugated AGuIX or AGuIX-VHH for 1 hour prior to staining with CellMask plasma membrane and DAPI nuclear stain. B. AGuIX and AGuIX-VHH fluorescence was quantified using ImageJ software to calculate corrected total cell fluorescence, CTCF (n = 5 – 10 cells, * p < 0.05, ** p< 0.01, *** p < 0.001). DETAILED DESCRIPTION Preclinical studies employing bioconjugation of nanoparticles (NPs), including AGuIX NPs, with antibodies and peptides have showcased the effectiveness of active targeting, resulting in a minimum 1.43-fold increase in tumor accumulation.5,7–9Peptides have gained significant popularity as ligands for targeting cancer cells due to their compact size and ease of production. However, they often exhibit low affinity for the target compared to antibodies.10Conversely, antibodies have demonstrated remarkable efficacy as targeting molecules, but their larger size hampers tissue penetration and prevents access to certain areas in tumoral tissues.11The present disclosure focuses on bioconjugates based on nanobodies, otherwise known as Variable Heavy domain of Heavy chain (VHH). These targeting biomolecules have a molecular weight of approximatively 15 kDa and are roughly ten times smaller than monoclonal antibodies, and therefore offer enhanced efficiency in targeting.12Their reduced size enables them to readily penetrate tumor tissues and bind to a greater number of receptors with enhanced affinity and specificity. Furthermore, VHH are swiftly eliminated from the bloodstream, minimizing the risk of toxic accumulation. They possess a remarkable folding capacity and robust physicochemical properties, endowing them with superior stability and excellent solubility.12–16These distinctive features enable VHH to overcome some limitations associated with monoclonal antibody therapies.17,18It is worth highlighting that both AGuIX NPs and VHHs (single-domain antibodies) exhibit a similar compact size, approximately 15 to 20 kDa, endowing them with similar in vivo properties, particularly in terms of tissue penetration, circulation, and clearance. By bioconjugating them, it is possible to combine these benefits in a single product. The results presented herein demonstrate that bioconjugation of VHH with NPs, which could have doubled their effective size, didn’t negatively affect either biodistribution or renal clearance. Attorney Docket No.29618-0451WO1 Immunotherapeutic Targets Among cancer cell targets, some antigens expressed by tumor cells are involved in the interaction with immune cells and allow the cancer cells to block anti- tumor immunity. The PD-L1 ligand and the innate immune regulator CD47 are relevant immunotherapy targets. PD-L1 can be expressed on tumor cells and binds to PD-1 on the surface of T-cells, triggering immune cell exhaustion and inhibiting anti- tumor immunity. Interfering with this negative immune checkpoint, PD-L1 inhibitors have demonstrated efficacy across many different cancer types. Specifically, atezolizumab, an anti-PD-L1 therapy, was approved for treatment of lung cancer in the U.S. in 2016 after showing a 25.4% overall survival rate after 3 years in the treatment of metastatic non-squamous or squamous non-small cell lung cancer (NSCLC).22Four years later, in 2020, duvarlumab, a second anti-PD-L1 antibody, was also approved for lung cancer treatment, demonstrating a 5-year overall survival rate of 42.9%, when administered following definitive chemoradiation therapy.23These two antibodies have been approved as a second treatment after one or two doses of chemotherapy. However, despite these promising results, the majority of patients with solid tumors do not respond to PD-L1 therapy, highlighting the need for combination approaches and better patient stratification.24CD47 is an important receptor expressed on tumor infiltrating macrophages that interacts with signal regulatory protein alpha (SIRPα). This regulator is overexpressed in various malignancies and prevents phagocytosis by providing a “don’t eat me” signal to immune cells. Targeting and blocking CD47 on tumors restores this phagocytic response which can then upregulate secondary, adaptive responses.20,25–27Antibodies targeting the CD47 receptor are currently being evaluated in multiple clinical trials.28–30The limited efficacy of existing immunotherapy in treating most solid tumors has highlighted the need to better define the tumor immune microenvironment and develop combination treatment approaches. PD-L1 / CD47 AGuIX combinations could help achieve both aims. Three of the approved immune checkpoint inhibitors (ICI) that target the PD-1 / PD-L1 axis require biomarker confirmation of PD-L1, highlighting the need for PD-L1 in vivo diagnostic tools.31Similarly, visualization of CD47 expression may help identify tumors amenable to macrophage based therapies. Both PD-L1 and CD47 inhibitors have been demonstrated to synergize with Attorney Docket No.29618-0451WO1 radiotherapy in preclinical models32–35; enhancing the effectiveness of radiotherapy with AGuIX could further increase the potency of this combination. Therefore, our primary focus was on developing AGuIX NPs that could effectively target and delineate crucial immunomarkers, specifically PD-L1 and CD47. This research serves as a foundational demonstration of the chemical synthesis approach, aimed at creating a platform that harnesses both the diagnostic and therapeutic potential of AGuIX, in conjunction with the immunotherapeutic capabilities of immunocheckpoint targeting. Described herein are exemplary bioconjugates that target Programmed Death Ligand 1 (PD-L1) and the Cluster of Differentiation 47 (CD47) receptors by using the A12 and A4 VHH respectively.19–21A12 exhibits high affinity in the low nanomolar range for human and murine PD-L1, making it suitable for targeting its expression. Similarly, A4 exhibits a binding affinity of approximately 10 pM for murine CD47.21,20AGuIX NPs were grafted with these two different VHHs for targeted applications using two highly specific techniques: 1) sortagging36–38and 2) click chemistry.39–41Sortagging is based on an enzyme specific reaction and click on an azide-alkyne reaction.40,42–45A12 and A4-modified AGuIX were prepared to compare both conjugation techniques and to assess receptor targeting using various in vitro assays. For the selected coupling synthesis, the ex vivo approach was studied using A12 nanobody in a highly aggressive murine model of melanoma. Nanoparticle Conjugate Provided herein are nanoparticle conjugate, comprising 1) one or more nanoparticles, wherein the nanoparticles comprise a) a polysiloxane core; b) one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to metal ions; 2) one or more second linking groups; and 3) one or more nanobodies; wherein each second linking group is covalently bonded to one nanoparticle and one nanobody. In some embodiments, the ratio of nanoparticles to nanobodies is about 1:1, about 1.3:1, about 1.5:1, or about 2:1. In some embodiments, the ratio of nanoparticles to nanobodies is about 1.3:1. Attorney Docket No.29618-0451WO1 In some embodiments, the ratio of metal ions to nanobodies is about 15:1, about 20:1, about 23:1, or about 30:1. In some embodiments, the ratio of metal ions to nanobodies is about 20:1. In some embodiments, the ratio of Gd3+ions to nanobodies is about 15:1, about 20:1, about 23:1, or about 30:1. In some embodiments, the ratio of Gd3+ions to nanobodies is about 20:1. In some embodiments, the hydrodynamic diameter of the nanoparticle conjugate is from about 2 to 20 nm. In some embodiments, the hydrodynamic diameter of the nanoparticle conjugate is from about 2 to 15 nm, 2 to 10 nm, 5 to 20 nm, 10 to 20 nm, or 15 to 20 nm. In some embodiments, the hydrodynamic diameter of the nanoparticle conjugate is from about 2.3 to 8.5 nm. In some embodiments, the hydrodynamic diameter of the nanoparticle conjugate is about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, or about 9 nm. In some embodiments, the hydrodynamic diameter of the nanoparticle conjugate is about 5 nm. In some embodiments, the molecular weight of the nanoparticle conjugate is from about 25 to 50 kDa. In some embodiments, the molecular weight of the nanoparticle conjugate is from about 25 to 45 kDa, about 25 to 40 kDa, or about 35 to 45 kDa. In some embodiments, the molecular weight of the nanoparticle conjugate is from about 29 to 44 kDa. Nanoparticles The nanoparticles described herein can comprise a polysiloxane core and one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to metal ions. In some embodiments, the polysiloxane is a polyorganosiloxane. In some embodiments, the nanoparticle comprises at least 8% by weight of silicon. In some embodiments, the nanoparticle comprises 8% and 50% by weight of silicon. In some embodiments, the metal ions are high-Z elements, optoinally lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, tin (stannum), palladium, silver, zirconium, gold, or bismuth. In some embodiments, the metal ions are gadolinium or bismuth. In some embodiments, the metal ions have an atomic Attorney Docket No.29618-0451WO1 number of at least 20. In some embodiments, the metal ions have an atomic number of at least 50. In some embodiments, the metal ions have an atomic number of 50 to 83. In some embodiments, the metal ions are Gd3+ions, Bi3+ions, or a combination thereof. In some embodiments, the metal ions are Bi3+ions. In some embodiments, the metal ions are Gd3+ions. In some embodiments, the metal ions are Gd3+ions and Bi3+ions. In some embodiments, the metal ions are Gd3+ions and Bi3+ions and ratio of Gd3+ions : Bi3+ions is from about 1:1 to about 3:1. In some embodiments, the one or more first linking groups each comprise a C1-10alkylamine. In some embodiments, the one or more first linking groups each comprise a C2-8 alkylamine, C2-6 alkylamine, or C3-4 alkylamine. In some embodiments, the one or more first linking groups are each , wherein indicates the bond connecting the first linking group to core and indicates the bond connecting the first linking group to the ligand. In some embodiments, the ligand is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1-(glutaric acid)-4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7- triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylene diamine tetra-acetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA), cyclohexyl- 1,2-diaminetetraacetic acid (CDTA), ethyleneglycol-O,O'-bis(2-aminoethyl)- N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'- diacetic acid (HBED), triethylene tetramine hexaacetic acid (TTHA), hydroxyethyidiamine triacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane- N,N',N",N"'-tetraacetic acid (TETA), or 1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (TCMC), or combination thereof. In some embodiments, the ligand is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10- tetraazacyclododecane-1-(glutaric acid)-4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1- glutaric acid-4,7-diacetic acid (NODAGA), ethylene diamine tetra-acetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA), cyclohexyl-1,2- diaminetetraacetic acid (CDTA), ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'- Attorney Docket No.29618-0451WO1 tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexaacetic acid (TTHA), hydroxyethyidiamine triacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA), or 1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)- cyclododecane (TCMC). In some embodiments, the ligand is 1,4,7,10- tetraazacyclododecane-1-(glutaric acid)-4,7,10-tetraacetic acid (DOTAGA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the ligand is wherein In some embodiments, the , wherein indicates the bond connecting the In some embodiments, the nanoparticle comprises 4 to 200 ligands, 4 to 150 ligands, 4 to 125 ligands, 4 to 100 ligands, or 4 to 90 ligands. In some embodiments, the nanoparticle comprises 4 to 80 ligands. In some embodiments, greater than about 20% of the ligands are complexed to the metal ions. In some embodiments, greater than about 20% of the ligands are complexed to the Gd3+ions. In some embodiments, the nanoparticle comprises 4 to 26 metal ions, 6 to 24 metal ions, 8 to 22 metal ions, 10 to 20 metal ions, 12 to 18 metal ions, or 14 to 16 metal ions. In some embodiments, the nanoparticle comprises 4 to 26 Gd3+ions, 6 to 24 Gd3+ions, 8 to 22 Gd3+ions, 10 to 20 Gd3+ions, 12 to 18 Gd3+ions, or 14 to 16 Gd3+ions. In some embodiments, the nanoparticle comprises about 10 metal ions, about 13 metal ions, about 15 metal ions, about 18 metal ions, or about 20 metal ions. In some embodiments, the nanoparticle comprises about 10 Gd3+ions, about 13 Gd3+ions, about 15 Gd3+ions, about 18 Gd3+ions, or about 20 Gd3+ions. In some Attorney Docket No.29618-0451WO1 embodiments, the nanoparticle comprises about 15 metal ions. In some embodiments, the nanoparticle comprises about 15 Gd3+ions. In some embodiments, the hydrodynamic diameter of the nanoparticle is from about 0.5 to 8 nm, about 0.5 to 5.5 nm, about 1 to 5 nm, about 1.5 to 4.5 nm, or about 2 to 4 nm. In some embodiments, the hydrodynamic diameter of the nanoparticle is from about 2 to 4 nm. In some embodiments, the hydrodynamic diameter of the nanoparticle is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, or about 6 nm. In some embodiments, the one or more nanoparticles comprise: a) at least 8 % by weight of silicon; and b) 4 to 200 first linking groups covalently bonded a ligand wherein one or more of the ligands are complexed to metal ions with an atomic weight of at least 20. In some embodiments, the one or more nanoparticles comprise: a) 8% to 50% by weight of silicon; and b) 4 to 80 first linking groups covalently bonded a ligand wherein one or more of the ligands are complexed to metal ions with an atomic weight of at least 20. Linker In some embodiments, the one or more second linking groups are each independently O O O or or a Attorney Docket No.29618-0451WO1 wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein indicates the bond connecting the second linking group to the nanoparticle, and indicates the bond connecting the second linking group to the nanobody. In some embodiments, n and m are the same. In some embodiments n and m are different. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, n is 5 and m is 4. In some embodiments, the one or more second linking groups are O O O In some embodiments, the one or more second linking groups are In some embodiments, the one or more second linking groups are each independently or Attorney Docket No.29618-0451WO1 O O O O O N or a wherein group nanoparticle, and indicates the bond connecting the second linking group to the nanobody. In some embodiments, the one or more second linking groups are each or a In some embodiments, the one or more second linking groups are each O or a In some embodiments, the one or more second linking groups are each independently or Attorney Docket No.29618-0451WO1 O O O O O N . each Nanobodies By the term “nanobody” is meant a variable heavy domain of heavy chain (VHH) that is bound by a molecule (e.g., protein, sugar, or lipid, or combination thereof) present in or on the plasma membrane of a target cell (e.g., a cancer cell). As described herein, a nanobody can be covalently linked to a secondary molecule or composition (e.g., any of the therapeutic nanoparticles described herein) to target the secondary molecule or composition to a target cell (e.g., a cancer cell). In some embodiments, a nanobody that is covalently linked to a secondary molecule or composition (e.g., any of the therapeutic nanoparticles described herein) results in the uptake of the secondary molecule or composition by the targeted cell (e.g., cellular uptake by endocytosis or pinocytosis). In some embodiments, the one or more nanobodies is a VHH that targets a checkpoint inhibitor or other immunotherapeutic target, e.g., PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, or VISTA (see, e.g., Tison, A., Garaud, S., Chiche, L. et al. Immune-checkpoint inhibitor use in patients with cancer and pre-existing autoimmune diseases. Nat Rev Rheumatol 18, 641–656 (2022)., April 7, 2022; Sun et al. Immune checkpoint therapy for solid tumours: clinical dilemmas and future trends. Signal Transduction and Targeted Therapy 20238:18, 1–26 (2023); ElTanbouly et al., Semin Immunol 42, 101308 (2019)). In some embodiments, the one or more nanobodies are listed in Table A. Attorney Docket No.29618-0451WO1 Table A. Exemplary Nanobodies oduct Name Disea Clinical Pr se and / or Condition Targeted Target Antigen Trial Manufacturer Status & C, , l, m , , re , h e, ed Attorney Docket No.29618-0451WO1 cell lung cancer / Non- small cell lung cancer a b ls, s , Attorney Docket No.29618-0451WO1 Melbourne, Australia Radiopharm . l . l ) di & ll s 7 9 t In some embodiments, the one or more nanobodies are directed to ANGPT2, ANGPTL2, B7-H3, CLDN18.2, CTLA-4, EGFR, FAP, HER-2, MSLN, MUC1, PD- 1, PD-L1, TROP-2, TIGIT, VEGFA, VEGF, VEGFR, BCMA, CD19, CD20, CD7, CD47, c-KIT, c-Met Nectin-4, IL-21,ODFR, or RANKL (Arbabi-Ghahroudi, International Journal of Molecular Sciences 2022, Vol.23, Page 500923, 5009 (2022);. Yu S, Xiong G, Zhao S, Tang Y, Tang H, Wang K, Liu H, Lan K, Bi X, Duan S. Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review). Int J Mol Med.2021 Feb;47(2):444- Attorney Docket No.29618-0451WO1 454. doi: 10.3892 / ijmm.2020.4817. Epub 2020 Dec 14. PMID: 33416134; PMCID: PMC7797440.; Jinet al., NANOBODIES®: A Review of Generation, Diagnostics and Therapeutics. Int J Mol Sci 24(6): 5994 (2023). In some embodiments, the one or more nanobodies are directed to cancer cell targets: ANGPT2, ANGPTL2, B7-H3, CLDN18.2, CTLA-4, EGFR, FAP, HER-2, MSLN, MUC1, PD-1, PD-L1, TROP-2, TIGIT, VEGFA, VEGF, VEGFR. Cancer cell Targets For active tumor targeting using a nanobody construct, the nanobody should have the ability to specifically bind to antigens presented by cancer cells. Some antigens are specifically expressed on cancer cell surfaces and absent from non- malignant tissues, others are present in both malignant and non-malignant tissues but overexpressed on cancer cells. For example, the present conjugates can include a nanobody that targets 5T4, ADAM-9, ALPV, ANGPT2, ANGPTL2, Axl, B7-H3, B7- H4, BCMA, CAIX, CCR7, CD3, CD7, CD19, CD20, CD22, CD25, CD33, CD37, CD38, CD46, CD47, CD52, CD71, CD74, CD123, CD142, CD147, CD166, CD205, CD228, CDH6, CLDN-6, CLDN-18.2, CTLA-4, DLL3, EGFR, EGFR2, FAP, FOLR- 1, FRα, GD2, Globo-H, GPC3, GRP-R, HER-1, HER-2, HER2, HER-3, IGF-1R, Integrin αVβ3, Integrin β4, KAAG-1, LAG-3, LIV-1, MC1R, MET, MSLN, MUC-1, Nectin-4, Norepinephrine, NTSR1, ODFR, PD-1, PD-L1, PDGFR-α, PSMA, PTK7, RANKL, ROR1, ROR2, SEZ6, SSTR, SSTR2, STn, TIGIT, TIM-3, Tissue factor (TF), TROP-2, TROP2, transferrin receptor, VISTA, VEGF, VEGF-A, VEGFR, CXCR4. Bioconjugation Methods – Sortagging and Click Chemistry Two exemplary methods were used to create the bioconjugates described herein, but others could be used. The sortagging bioconjugation approach permits the selection of specific sites for modification, enabling more accurate predictions regarding the biological impact of a chemical alteration. Modifying a protein terminus is expected to have a smaller effect on the protein's folding and functionality as well as the added advantage of defining the orientation of the immobilized protein, which is anticipated to better preserve its function compared to other immobilization techniques. This enzymatic reaction relies on the enzyme Sortase A (SrtA), a transpeptidase produced by Gram-positive bacteria, which catalyzes the formation of Attorney Docket No.29618-0451WO1 a peptide bond between two peptides to attach specific proteins to the cell wall or pili assembly.56–58SrtA specifically recognizes the LPXTG sequence (with X representing any amino acid) and by nucleophilic attack makes it reactive to an N-terminal oligoglycine, forming a peptide bond between the threonine of LPXT and the oligoglycine. This natural ligation system has been repurposed for protein modification and has gained popularity as a research tool due to its high ligation selectivity, simplicity, robustness, and the availability of various SrtA variants (expressed in Escherichia coli or commercially available), as well as other required materials.37,59Sortagging reactions have been used to graft specific biomolecules, such as peptides or proteins, onto a protein or other biological targets of interest and has already proved efficacious in NP conjugation.60–62Click chemistry describes chemical reactions aimed at achieving high yield and high selectivity in the formation of carbon-heteroatom bond. Click chemistry reactions are based on the 1,3-dipolar cycloaddition of an azide and an alkyne to form a 1,2,3-triazole. This reaction has been widely utilized in various applications due to its simplicity in terms of preparation and purification steps, enabling rapid generation of new products with high reaction rates, such as bioconjugates.60,63–65The formation of the triazole linkage is irreversible and quantitative, providing excellent reaction stability. Azide and dibenzocyclooctyne (DBCO) were selected as strain-promoted azide-alkyne cycloaddition reactions (SPAAC) tools due to their relatively small functional groups with a favorable rate constant (k2= 0.2-0.5 M-1.s-1)39which have been shown to be effective. The crucial challenge of click chemistry is to preserve the integrity and functionality of the biomolecule after grafting. Also provided herein is a method or preparing a nanoparticle by reacting i) one or more nanoparticle A, wherein nanoparticle A comprises a) a polysiloxane core; b) one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to metal ions; c) one or more of linker A: Attorney Docket No.29618-0451WO1 indicates the bond connecting linker A to the nanoparticle A; with ii) one or more nanobody B, wherein nanobody B comprises one or more of linker B: wherein z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indicates the bond connecting linker B to the nanobody. Also provided herein is a nanoparticle prepared by the process of reacting i) one or more nanoparticle A, wherein nanoparticle A comprises a) a polysiloxane core; b) one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to metal ions; c) one or more of linker A: indicates the bond connecting linker A to the nanoparticle A; with ii) one or more nanobody B, wherein nanobody B comprises one or more of linker B: Attorney Docket No.29618-0451WO1 wherein z is 0, 1, 2, 3, 4, or the bond connecting linker B to the nanobody. In some embodiments, x and z are the same. In some embodiments x and z are different. In some embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, x is 4. In some embodiments, x is 5. In some embodiments, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, z is 4. In some embodiments, z is 5. In some embodiments, x is 5 and z is 4. Pharmaceutical Compositions When employed as pharmaceuticals, the compositions provided herein can be administered in the form of pharmaceutical formulations. These formulations can be prepared as described herein or elsewhere, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. In some embodiments, the administration is selected from the group consisting of pulmonary administration (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal administration, or intranasal administration), oral administration, or parenteral administration (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, intravesicular, intramuscular or injection or infusion, intracranial, intrathecal, intraventricular administration, and the like). In some embodiments, the administration is intravenous or nasal administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like, may be necessary or desirable. Also provided are pharmaceutical formulations which contain, as the active ingredient, a composition provided herein in combination with one or more pharmaceutically acceptable carriers (excipients). In making a pharmaceutical formulation provided herein, the nanoparticle composition may be, for example, mixed with an excipient or diluted by an excipient. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier, or medium for the nanoparticle composition. Thus, the pharmaceutical Attorney Docket No.29618-0451WO1 formulations can be in the form of powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), sterile injectable solutions, sterile packaged powders, and the like. Methods of Use Also provided herein is a method of treating a subject comprising administering to the subject a therapeutically effective amount of a nanoparticle conjugate described herein. In some embodiments, the subject has cancer, e.g., a solid cancer. In some embodiments, the cancer overexpresses a cancer cell target protein in the tumor microenvironment, and the nanobodies bind to the cancer cell target thereby improving the specificity and retention of the nanoparticle conjugates in the tumor. The subject can then be treated using a radiotherapy modality, e.g., using either External Beam RadioTherapy (EBRT) or the administration of a vectorized radioisotope-containing drug (Targeted Radionuclide Therapy). Combination with External Beam RadioTherapy (EBRT) The nanoparticle conjugate at the treatment phase when used in combination with radiotherapy preferably comprises an element with an atomic number Z higher than 50 for obtaining radiosensitizing properties. As used herein, the term “radiosensitizing” means the capacity to interact with ionizing radiation to enhance the generation of chemical reactive species and thus trigger biological damages and / or dysregulation. The nanoparticle conjugate can be administered to the subject using different possible routes such as local (intra-tumoral (IT), intra-arterial (IA)), subcutaneous, intravenous (IV), intradermic, airways (inhalation), intra-peritoneal, intramuscular, intra-thecal, intraocular or oral route. In preferred embodiments, said treatment phase comprises administering said nanoparticle conjugate, intravenously to the subject. In specific embodiments, the nanoparticle conjugate is administered the same day as the first irradiation of RadioTherapy, or 1 or 2 days before. Typically, when using fractionated radiation therapy, the nanoparticle conjugate may be further injected once every week during multiple sessions of radiation therapy. For example, in a specific embodiment, the treatment as disclosed herein further comprises at least one additional step of injecting a therapeutically Attorney Docket No.29618-0451WO1 effective amount of the nanoparticle conjugate within 5-10 days after one or more sessions of fractionated radiation therapy, for example 7 days after said injecting step for the first session of fractionated radiation therapy. The amount of radiation used in radiation therapy is measured in gray (Gy), and varies depending on the type and stage of cancer being treated. For standard of care radiotherapy, the typical total dose for a solid tumor ranges from 10 to 120 Gy. Many other factors are considered by radiation oncologists when selecting a dose, including whether the patient is receiving chemotherapy, patient co-morbidities, whether radiation therapy is being administered before or after surgery, and the degree of success of surgery. In some embodiments, the total dose is preferably fractionated (spread out over time). Amount and schedules (planning and delivery of ionizing radiations, fraction dose, fraction delivery schema, total dose alone or in combination with other anti-cancer agents etc) is defined for any disease / anatomical site / disease stage patient setting / age and constitutes the standard of care for any specific situation. The primary and / or the metastatic tumors may be targeted with the radiotherapy. In some embodiments, said standard-of-care radiotherapy at the treatment phase comprises exposing the subject with a total dose of radiations comprised between 15 and 80 Gy, for example at a dose per fraction comprised between 1.8 and 10 Gy, for example fractionated in 5 to 40 fractions, and for example, with 5 fractions per week. A typical conventional fractionation schedule for adults for the methods of the present disclosure may be 2.5 to 3.5 Gy per day, five days a week, for example for 2 to 3 consecutive weeks. In some embodiments, said radiotherapy consists of exposing the subject to a total dose of ionizing radiations between 20 and 35 Gy, for example 30 Gy. In some embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 2 to 8 Gy, and the total dose is administered preferably in a maximum of 10 fractions, for example 10 fractions of 3 Gy, 5 days a week for two weeks. This dosing may be suitable for example for whole brain radiotherapy (WBRT) for treating brain metastases. In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 4 to 10 Gy, and the total dose is ranging from 20-30 Gy, Attorney Docket No.29618-0451WO1 administered preferably in a maximum of 5 fractions, for example in 5 or 6 days. This dosing may be suitable for example for stereotactic radiotherapy for treating brain metastases. In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1,5 to 2 Gy, for example 1,8 Gy, and the total dose is ranging from 40-60 Gy, administered preferably in 25 fractions, for example in 4-6 weeks. This dosing may be suitable for example for stereotactic radiotherapy for treating cervical cancer. In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1,5 to 4 Gy, for example 1,8 Gy, and the total dose is ranging from 60-90 Gy, for example 80 Gy, administered preferably in 30-50 fractions, for example in 6-10 weeks. This dosing may be suitable for example for External Beam Radiation Therapy (EBRT) for treating prostate cancer. In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1,5 to 4 Gy, for example 2 Gy, and the total dose is ranging from 35-70 Gy, for example 60 Gy, administered preferably in 10-35 fractions, for example in 2-6 weeks. This dosing may be suitable for example for External Beam Radiation Therapy for treating glioblastoma. In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 6 to 10 Gy, for example 8 Gy, and the total dose is ranging from 30-50 Gy, for example 40 Gy, administered preferably in 5-6 fractions, for example in 5 consecutive days. This dosing may be suitable for example for Stereotactic Magnetic resonance-guided Adaptive Radiation Therapy (SMART) for treating pancreatic cancer. In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1.5 to 3 Gy, for example 2 Gy, and the total dose is ranging from 30-60 Gy, for example 54 Gy, administered preferably in 5 to 6 weeks. This dosing may be suitable for example for Stereotactic Body Radiation Therapy (SBRT) for treating pancreatic cancer. In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 8 to 12 Gy, for example 10 Gy, and the total dose is ranging from 40-60 Gy, for example 50 Gy, administered preferably in 5-6 fractions, for Attorney Docket No.29618-0451WO1 example in 5 consecutive days. This dosing may be suitable for example for SMART for treating non-small lung cell cancer. In some embodiments, magnetic resonance image-guided radiation therapy may be used in the standard of care radiotherapy. As used herein, the term “magnetic resonance image-guided radiation therapy” refers to the combined use of a magnetic resonance imaging unit with a radiation therapy unit, allowing real-time imaging of target volumes and organs at risk before and during treatment delivery with replanning as necessary. Magnetic resonance image-guided radiation therapy is particularly useful in sites affected by inter- and intrafraction motion such as the thorax, abdomen and pelvis. Typically, organ at risk and target visualization can be improved with magnetic resonance image-guided radiation therapy compared to cone beam computerized tomography, which can permit plan adaptation and reduction of toxicity. This technique may use automated beam gating for precise and accurate dosing. When the tumor moves, the beam automatically stops. Clinicians can therefore shrink margins with confidence while escalating dose. Any MR-Linac for image-guided radiation therapy may be used in the methods of treatment of the present disclosure. The MR-Linac as currently used includes perpendicular beam-field systems (e.g. Elekta and Viewray) which are now commercial products. Other systems include inline orientation (Aurora-RT) and both perpendicular and inline orientation (Australian). The field strength of the current MR-Linac systems vary from 0.35 T; for example the MRIdian (Viewray), 0.5T (Aurora-RT, MagneTx), and 1.5T (Unity, Elekta). Further details of the MR-Linac systems and their mode of use are described in Liney et al Clinical Oncology 30 (2018) 686-691. In preferred embodiment, a MR-Linac used in the treatment phase of the present disclosure has a magnetic field strength of 0.5 T or lower, for example 0.35 T. Such embodiment is particularly preferred with AGuIX nanoparticles as described in the previous sections. Examples of such method of image-guided radiation therapy in combination with nanoparticles (e.g. AGuIX) are also disclosed in WO2021 / 228867. Combination with Targeted Radionuclide Therapy (TRT) Attorney Docket No.29618-0451WO1 The present disclosure also relates to high-Z element containing nanoparticle conjugate for use in a method of treating a tumor by radiopharmaceutical therapy, in a subject in need thereof, the method comprising a combined administration of an efficient amount high-Z element containing nanoparticle conjugate and of an efficient amount of radionuclide containing therapeutic radiopharmaceutical. As used herein, the terms “high-Z element containing nanoparticles” refer to the nanoparticles described in the previous sections. According to the present disclosure, the term “radiopharmaceutical therapy”, also referred as “targeted radionuclide therapy” (abbreviated TRT), "“targeted radiopharmaceutical therapy"” or “molecular radiotherapy” or “radioimmunotherapy” or “targeted radiotherapy” refers to the treatment of diseases of oncological nature with a radionuclide containing therapeutic radiopharmaceutical. In radiopharmaceutical therapy, radiation is systemically or locally delivered to the tumor using a therapeutic radiopharmaceutical comprising a radionuclide able to deliver ionizing radiation directly to tumor cells. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue and the so called “targeted effects”) by damaging their genetic material, making it impossible for these cells to continue to grow. Typically, said ionizing radiations are alpha, beta particles and Auger electrons. Examples of such method of combine therapy with nanoparticles (e.g. AGuIX) and radiopharmaceuticals are also disclosed in WO2024 / 013272. EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1. Materials Characterizations Chemical Products Phosphate-Buffered Saline (PBS), dimethyl sulfoxide (DMSO,>99.5%), ammonium acetate (CH3CO2NH4,>99.9%), and acetic acid glacial (C2H4O2, > 99.9%) were purchased from Sigma Aldrich (Germany). The gadolinium-chelated polysiloxane nanoparticles (AGuIX) were provided by NH TherAguix (Grenoble, France) as a lyophilized powder. The VHH A12 A4 and a heptamutant variant of S. Attorney Docket No.29618-0451WO1 aureus Sortase (StrA7m) were provided by the Massachusetts General Hospital (USA). DBCO-PEG5-NHS ester and Azido-PEG4-NHS ester were purchased from BroadPharm (San Diego,CA). The fluorescent dyes, Lumiprobe CY5.5-NHS ester (Cy5.5-NHS) and Alexa FluorTM647 Azide (AF647-azide) and the reactif N-(γ- maleimidobutyryloxy) succinimide ester (GMBS) were purchased from Thermo Fischer Scientific. Only milli-Q water (ρ > 18 MΩ.cm) was used to prepare aqueous solutions of nanoparticles (NPs). Each AGuIX NPs concentration is stated in g.L-1of gadolinium element. Size Exclusion Chromatography (SEC) UltraViolet / Visible-Fluorescence The Superdex 75 increase 10 / 300 GL (Cytiva) was used for SEC, and the process was conducted using an isocratic system of elution in acetate buffer 100 mM (pH 4.7) at a flow rate of 0.8 mL min-1(25 μL injected). The G1311A pump (Agilent), Photodiode Array Detector G1315B (Agilent), and fluorescence detector G1321A (Agilent) were employed during the process to record absorbance at 295 nm and fluorescence at 280 nm excitation and 340 nm emission for the method A and fluorescence at 650 nm excitation and 665 nm emission for the method B. The data were acquired using on ChemStation B.04.03 SP1 and analyzed on GraphPad Prism 8.0.1. For preparative purification, the same column was employed within an AKTA PURE chromatography system (Cytiva) using an isocratic system of elution in phosphate-buffered saline (PBS) 100 mM at a flow rate of 0.8 mL min-1(5 mL injected). Induced Coupled Plasma Mass Spectroscopy (ICP-MS) To achieve optimal results, the ICP-MS was operated under specific conditions, including a nebulizer gas flow of 0.84 L min-1, plasma gas flow of 15 L min-1, an auxiliary gas flow of 1.2 L min-1, and a plasma radiofrequency power of 1600 W. The Syngistix 2.3 software was used to control the ICP-MS, and the tuning of all other parameters was carried out to optimize the Gd signal. High-Performance Liquid Chromatography (HPLC)-ICP-MS Flexar LC system (PerkinElmer) coupled with a Nexion 2000B (PerkinElmer) was used. The separation was executed using Superdex 75 increase 10 / 300 GL, and Attorney Docket No.29618-0451WO1 measurements were obtained through an isocratic mode of elution with acetate buffer 100 mM (pH 4.7) at a flow rate of 0.4 mL min-1. The Gd signal was monitored utilizing isotopes 156 and 158 and Empower software version 7.3 was utilized to acquire the Gd signal. Dynamic Light Scattering (DLS) The hydrodynamic diameter distribution of the purified product was measured with a Zetasizer NanoS DLS instrument (laser He–Ne 633 nm) from Malvern Instruments. Taylor Dispersion Analysis (TDA) TDA experiments were conducted using a TDA –ICP-MS hyphenation between a Sciex P / ACE MDQ instrument and a 7700 Agilent ICP-MS, described elsewhere. Fused silica capillaries with an inner diameter of 75 µm and outer diameter of 375 µm, and a total length of 64 cm, were coated with hydroxypropylcellulose (HPC) using a solution of 0.05 g / mL in water. The capillaries were then subjected to injection (5 psi for 3 seconds, equivalent to 15 nL), and the samples were mobilized by applying a pressure of 0.7 psi using a Tris 10 mM, NaCl 15 mM buffer at pH 7.4. Detection was carried out by ICP-MS at m / z=158 with a data acquisition rate of 500 ms / point. Between runs, the capillary was flushed at 10 psi for 5 minutes with the mobilization medium. The taylorgrams obtained by TDA can be assimilated to the sum of several Gaussians signals: ^ A^t − ^^^ = ^ ^ t^− ^^^where t0is the peak under the curve and temporal variance Equation 2 relates the molecular diffusion coefficient D to the hydrodynamic radius of the species under the specified experimental conditions. ^^ r^^t^ k^Twhere rcis the capillary T is the temperature, η is the viscosity, and Rh is the hydrodynamic radius of the species. The viscosity of a Attorney Docket No.29618-0451WO1 Tris 10 mM, NaCl 15 mM buffer was previously determined to be 0.908. Peak deconvolution was carried out using Origin 8.5 software. Matrix-Assisted Laser Desorption Ionization – Time of Flight mass spectrometry (MALDI-TOF MS) The mass spectrum was generated using a Voyager-DE PRO (Sciex, Framingham, Massachusetts) equipped with a nitrogen laser that emitted at 337 nm with a 2 ns pulse. To create the mass spectrum, the ions were accelerated to a final potential of 25 kV, and 300 laser shots were summed together. An external mass calibration was performed using a mixture of proteins from the Sequazyme™ standards kit (Sciex). The analysis was conducted in linear mode with an instrument mass accuracy of 0.05%. To prepare the samples, the protein solution (1 g L-1) was diluted 20-fold in a matrix of sinapinic acid (SA, Sigma-Aldrich) and dissolved in 0.1%TFA / acetonitrile (70 / 30, v / v). Finally, 1 μL of the mixture was utilized for analysis. Competition ELISA High-affinity 96-well plates were coated with either 2 µg mL-1PD-L1 or 1.25 µg mL-1CD47 and allowed to adhere overnight at 4°C. Plates were washed and blocked with a 10% FBS solution. Plates were incubated with sample VHH (A12 or A4) or NPs (AGuIX, AGuIX@A12, or AGuIX@A4) followed by incubation with 6.25 nM biotin-anti-PD-L1 or 50 nM biotin-anti-CD47. Plates were then incubated with avidin-HRP (Abcam ab7403, 1:40000 dilution) followed by TMB (ThermoFisher N301) to detect colorimetric changes. TMB conversation was stopped at 40 minutes and absorbance was read at 450 nm. The equilibrium inhibitory dissociation constant (Ki) was curve fitted using Graphpad 8.0.1. Top and Bottom are the plateaus in the units of the y-axis. LogKi is the log of the molar equilibrium dissociation constant of the unlabeled ligand (AGuIX, AGuIX@A12, or AGuIX@A4). RadioligandNM is the concentration of the labeled ligand (biotin-anti- PD-L1 or biotin-anti-CD47) and HotKdNM is the equilibrium dissociation constant of the labeled ligand (biotin-anti-PD-L1 or biotin-anti-CD47). Attorney Docket No.29618-0451WO1 Cell culture Murine melanoma (wild-type B16F10 or hiPDL1-B16F10) cells were cultured in 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% pen / strep (10,000 U / mL-1penicillin and 10,000 µg.mL-1streptomycin; Invitrogen, US) supplemented Roswell Park Memorial Institute medium (RPMI 1640; Gibco, Invitrogen, USA) at 37°C, 5% CO2, and optimal humidity. Fluorescence microscopy Cover slips were placed in 48-well plates and seeded with either hiPDL1-B16 or wild-type B16 cells (50,000 cells / well) and allowed to attached overnight at 37°C. Cells were incubated with Cy5.5 (683 / 703 nm) tagged NPs (0.2 mg.mL-1) for 1 hour. Cells were fixed, blocked, and permeabilizing with a solution of 10% FBS and 0.3% Triton X-100. Cells were stained with CellMask Orange Plasma Membrane stain (ThermoFisher, 554 / 567 nm) and mounted with DAPI (350 / 470 nm) and Flouromount-G. Images were taken using a Zeiss AxioObserver microscope 63x. Fluorescence images were analyzed using ImageJ (version 2.14.0 / 1.54f) and corrected total cell fluorescence was calculated. (CTCF = integrated densityCell– (areaCellx mean fluoresence background)). Animal tumor model C57BL / 6J mice (Janvier-Labs, France) were housed by 6 mice in each cage (bedding material: aspen wood) at room temperature 22°C, humidity 40%, under a regular 12-hour dark / light cycle. Food and water were available ad libitum.6 weeks old female C57BL / 6 mice were purchased from Janvier laboratory. B16F10 cells (ATCC, USA) were cultured in DMEM media (Life Technologies, France) supplemented with 10% FBS (Life Technologies, France) and 1% penicillin / streptomycin (100 mg / mL), and 1% l-Glutamine, and maintained at 37 °C with 5% CO2until confluence. Mice were subcutaneously injected with 1 x 106B16F10 cells suspended in DPBS (1× 100 µL) into both flanks while anesthetized with 2% isoflurane. Attorney Docket No.29618-0451WO1 Statistical analysis The results are reported as mean + / - SEM. Statistical analyses were conducted using GraphPad Prism (version 8.0.1). For comparisons involving three or more means, a one-way ANOVA followed by the Kruskal-Wallis nonparametric test (for non-Gaussian populations) was employed for in vitro internalization assays. All in vitro experiments were performed in triplicate. Mann-Whitney test for multiple comparisons was used to analyze ex vivo biodistribution data. Statistical significance was considered at p < 0.05. Example 2. Preparation of VHH A12 and A4 sequences were sub-cloned into the WK6 E. coli periplasmic expression vector pHEN6 to enable Gibson cloning and the inclusion of a C-terminal sortase motif and 6xHis tag. E. coli containing the plasmid were grown to mid-log phase at 37°C and VHH expression induced with 1 mM IPTG at 30°C overnight. Centrifugation (5000 x g, 15 mins, 4°C) was used to harvest the cells and resuspend them in 25 mL 1X TES buffer. Cells were submitted to osmotic shock in 1:40.25x TES buffer overnight at 4°C. The periplasmic fraction was separated by centrifugation (8000 rpm, 30 mins, 4°C) and loaded onto Ni-NTA beads (Qiagen) and eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole. Eluted protein was loaded onto a Superdex 7510 / 300 column. Recombinant VHH purity was assessed by SDS-PAGE and concentrated with an Amicon 10,000 kDa filtration unit (Millipore). VHH were stored at -80°C. The sequence of A12 is as follows: QVQLVESGGGLVQAGGSLRLSCTASGSTFSRNAMAWFRQAPGKEREFVSGIS RTGTNSYDADSVKGRFTISKDNAKNTVTLQMNSLKPEDTAIYYCALSQTASV ATTERLYPYWGQGTQVTVSSGGLPETGGHHHHHH (SEQ ID NO: 1) The sequence of A4 is as follows: MKYLLPTAAAGLLLLAAQPAMAQVQLVESGGLVEPGGSLRLSCAASGIIFKIN DMGWYRQAPGRREWVAASTGGDEAIYRDSVKDRFTISRDAKNSVFLQMNSL KPEDTAVYYCTAVISTDRDGTEWRRYWGQGTQVTVSSGGLPETGGHHHHH H (SEQ ID NO: 2) Attorney Docket No.29618-0451WO1 Example 3. Synthesis of Gd-chelated polysiloxane NPs (AGuIX) After the formation of a gadolinium oxide core Gd2O3in diethylene glycol (see e.g., Bridot et al., J. Am. Chem. Soc., 2007, 129:5047), a polysiloxane shell was grown using hydrolysis-condensation of tetraethyl orthosilicate (TEOS) and aminopropyl triethoxysilane (APTES) to form a SiGd nanoparticle. 1033 µmol of SiGd nanoparticles (SiGdNP) were dispersed in 12.7 mL of water for 1 hour at a pH of 7.4 ([Gd3+] = 81 mM). Then 15 mL of water were added. 591 mg of the derivative DOTA chelate (776 µmol; ratio Gd / DOTA = 1.3) were dissolved in 2.36 mL of DMSO ([DOTA] = 250 mg / mL). This solution was then gradually added to the SiGdNP solution under stirring at room temperature and the pH adjusted to 7.4 by addition of NaOH solution. The particles solution was stirred for 5 h. The mixture was diluted in water to [Gd3+] = 22.5 mM (V = 59 mL) in order to have a solution containing less than 4% DMSO and the pH was decreased to 1 to avoid ionic interactions between ammonium of SiGdNP and carboxylate of DOTA- NHS. Particles were then purified by tangential filtration trough Vivaspin® membranes (MWCO = 5 kDa) purchased from Sartorius Stedim Biotech (France). The colloidal solution was introduced into Vivaspin® tubes and centrifuged. This step was repeated several times, by filling the tubes with water and centrifuging again, until the desired purification rate was reached (x26,000). The particles solution was concentrated to approximately [Gd3+] =100 mM, the pH adjusted to 7.4 and finally the solution was sterile filtered through a 0.2 μm syringe filter in order to remove the largest impurities. It was freeze-dried for storage, using a Christ Alpha 1-2 lyophilizer. The Gd yield for the synthesis was 41%. AGuIX NPs contained roughly between 10 and 20 Gd atoms per particle, which was be quantified using ICP-MS (Inductively Coupled Plasma-Mass Spectrometry). The presence of the gadolinium atoms confers to AGuIX NPs radiation dose amplification and MRI contrast properties. The AGuIX NPs concentration is stated in g L-1of AGuIX NP or M of Gd element. Within the polysiloxane structure AGuIX NPs contained (3-Aminopropyl)triethoxysilane (APTES) function. Each NP is estimated to possess a similar number of amine functions as Gd chelates. Attorney Docket No.29618-0451WO1 Example 4. Synthesis of AGUIX@VHH by sortagging Synthesis of AGuIX@Mal AGuIX NP (10 g, 13.28 w% Gd, ~15 Gd / NP) were dispersed in MilliQ water (200 g.L-1) and stirred for 30 min. Then, a solution of N-(γ-maleimidobutyryloxy) succinimide ester (GMBS, 50.0 mg, 178 µmol) in DMSO (420 µL) was added and the mixture was stirred for another 1 h. The AGuIX-Mal NP was purified by filtration (5 kDa MWCOft turbo vivaspin, dilution x81) until no more maleimide (Mal) moiety could be detected in the filtrate and was finally dispersed into 4.4 mL of MilliQ water before lyophilisation. Mal quantification was performed by incubation with L-cystein (20 min, room temperature (RT)) and determination of the unreacted L-cystein with the photometric Ellman's test (Thermo Scientific, absorbance: 412 nm). The number of Mal moieties introduced on the surface of AGuIX NP could be modified in the range 0.3-2 Mal / NP by adjusting the amount of GMBS added. Synthesis of AGuIX@C(W / T)GGG AGuIX-Mal (1.3 g, 15.0 w% Gd, 17.98 nmol Mal / mg AGuIX-Mal, 0.283 Mal / NP (~15 Gd / NP), 100 mg AGuIX-Mal / mL final concentration in reaction, redispersed in MilliQ H2O) and GGG(W / T)C (26 µmol, 20 nmol GGG(W / T)C / mg AGuIX-Mal, 2.0 mM final concentration in reaction, dispersed in PBS) were introduced in PBS (final volume 13 mL). The mixture was stirred (orbital) at RT for 6 h. The final product AGuIX-C(W / T) GGG was finally filtered (5 kDa MWCO turbo vivaspin, dilution x320) and stored at -20 °C (18.1 mL, 2.79 mg Gd / mL, 17.8 mM Gd). Quantification of the coupling reaction yield was determined for the synthesis of AGuIX-CWGGG using SEC method A, taking advantage of the fluorescence of tryptophan residues. A similar coupling efficiency was supposed for the synthesis of AGuIX-CTGGG as replacement of the W residue by T is not expected to change the reaction yield. Synthesis of AGuIX@Cy5.5- CTGGG AGuIX-Mal NP (600 mg, 13.51 w% Gd, 50 mg AGuIX-Mal / mL final concentration in reaction, redispersed in MilliQ H2O) and Cy5.5-NHS ester (1.03 µmol, 85.9 µM final concentration in reaction) were introduced in H2O (final volume Attorney Docket No.29618-0451WO1 12 mL). The mixture was stirred at RT for 30 min. After x2 dilution in H2O, the AGuIX-Cy5.5 was purified by filtration (5 kDa MWCO vivaspin, dilution x2). AGuIX-Cy5.5 (600 mg, 13.51 w% Gd, 123 nmolMal / mgAGuIX, 2.1 Mal / NP (~15 Gd / NP), 46 mg AGuIX-Cy5.5 / mL final concentration in reaction, redispersed in MilliQ H2O) and GGGTC (78 µmol, 130 nmol GGGTC / mg AGuIX-Cy5.5, 6.0 mM final concentration in reaction, dispersed in PBS) were introduced in PBS (final volume 13.0 mL). The mixture was stirred (orbital) at RT for 4.5 h and left overnight at 4 °C. The final product AGuIX-Cy5.5-CTGGG was finally filtered (5 kDa MWCO vivaspin, dilution x3125) and stored at -20 °C. Synthesize of AGuIX@VHH by sortagging AGuIX-Cy5.5-CTGGG (600 µM GGGTC final concentration) and VHH (30 or 40 µM final concentration) were introduced in buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Ligation was triggered (t0) by the addition of StrA7m (2.5 µM final concentration). The mixture was stirred (orbital) at room temperature for 3 h. After reaction, the crude was negatively selected using Ni-NTA bead (400 µL slurry for 1 mg VHH, spin at 800 G for 1 min) to remove 6xHis-tag starting materials (StrA7m, VHH and released VHH residue after ligation), and filtered (MWCO 50 kDa vivaspin, dilution x100,000). AGuIX@VHH was purified by SEC (size exclusion chromatography, method B detailed in ESI) and stored at -20 °C. AGuIX-Cy5.5- CTGGG preparations and synthesis optimizations are detailed in ESI. A similar reaction procedure was used to prepare AGuIX@VHH from the starting materials AGuIX-C(W / T)GGG. Optimization of sortagging reaction The sortagging reaction is based on enzymatic synthesis involving three primary steps: (1) introduction of maleimide (Mal) moieties on AGuIX NPs, (2) introduction of the peptide GGG(W / T)C using the Mal moieties, and (3) introduction of VHH through the transpeptidation enzymatic reaction (Fig.1A). AGuIX NPs were modified to mimic the functionality of the N-terminal oligoglycine residue typically employed in sortagging. Initially, a Mal functional group was introduced onto the surface of AGuIX NPs using a bifunctional NHS / Mal linker. AGuIX-Mal was further modified with a short peptide linker that incorporates Attorney Docket No.29618-0451WO1 a cysteine amino acid at the C-terminal and a triglycine residue (GGG(W / T)C) at the N-terminal. To enable the use of sortagging, the VHH was engineered by incorporating a C-terminal StrA motif LPETGG and a 6xHis tag. The addition of the 6xHis tag motif facilitated protein purification and removal of the C-terminal residue released during sortagging.20The transpeptidation reaction between A12 and AGuIX- C(W / T)GGG was verified using SEC monitoring. After purification, the final product exhibited a retention time (tR) of 13.2 minutes, corresponding to a product size ranging from 29 to 44 kDa (Fig.1B, Fig.5C). The equilibrium for the reaction was reached within 2-3 hours at room temperature. Following isolation using Ni-beads to capture and remove the 6xHis-containing reagents and by-products (StrA7m, residual peptides from VHH C-terminal, and unreacted VHH), AGuIX@A12 was purified via filtration and dispersed in PBS (Fig.5C). A VHH conversion of 32-33% was achieved during the sortagging process, resulting in an isolated yield of AGuIX@A12 of 5.3% (relative to A12) with a Gd / A12 ratio of 20 (Fig.1B, Fig.5E). The hydrodynamic diameter (DH) of AGuIX@A12, as assessed by DLS, was 6.1 nm, slightly larger than that of AGuIX-C(W / T)GGG (4.1 nm) (Fig.1C). This outcome confirms the modification of the particle surface and hints at the possibility of VHH grafting. After optimization, sortagging proved successful in grafting VHH onto the surface of AGuIX NPs. The key parameter to increase the grafting yield of VHH was to increase the equivalence of the oligoglycine in the reaction. However, this strategy also resulted in a higher NP / A12 ratio, which ideally should be around 1 for imaging applications. The NP / A12 ratio of 1 could be achieved after optimization of several parameters (Fig.5E), while VHH conversion decreased to 15%. The binding affinity, evaluated using the inhibitory constant (Ki) through a competitive enzyme-linked immunosorbent assay (ELISA), confirmed that the addition of AGuIX to A12 did not impact the competitive binding affinities (Fig.6). Furthermore, AGuIX’s lack of interaction with PD-L1 proteins also confirmed that the high affinity is solely attributed to the presence of the nanobody on AGuIX’s surface. Moreover, the similar logKi values of 12.8 ± 1.1 nM, and 5.5 ± 0.6 nM for A12 and AGuIX@A12 obtained, respectively, strongly reinforced this result (Fig.6). AGuIX NPs were modified to mimic the functionality of the N-terminal oligoglycine residue typically employed in sortagging. Initially, a Mal functional group was introduced onto the surface of AGuIX NP through the use of a bifunctional Attorney Docket No.29618-0451WO1 NHS / Mal linker. This linker takes advantage of the amino groups present on AGuIX NP. By adjusting the ratio of linker to AGuIX NP, the number of grafted Mal groups could be controlled. This allowed for the production of AGuIX NPs carrying from 0.3 to 2 Mal groups per AGuIX NP (considering 15 Gd / NP). The low Mal / NP ratio could be beneficial if low amount of blocking agent is required compared to AGuIX NP (for therapeutic purpose). However, for effective MRI monitoring, it is crucial to ensure that a significant portion of AGuIX NPs is modified with VHH to minimize background signal originating from nonspecific AGuIX NPs. Consequently, AGuIX- Mal was further modified with a short peptide linker that incorporates a cysteine amino acid at the C-terminal and a triglycine residue (GGG) at the N-terminal. This modification allows for reactions to occur on both AGuIX-Mal and during sortagging, respectively. An additional tryptophan residue was introduced to create the peptide GGGWC, facilitating reaction monitoring using fluorescence detection. AGuIX- CWGGG was obtained following the coupling of the C-terminated peptide to AGuIX- Mal and subsequently purified through filtration to remove any residual unreacted linkers. When fluorescence monitoring of reactions was not necessary, peptide GGGWC was substituted with peptide GGGTC, and the reaction was carried out in the same manner. To enable the use of sortagging, the VHH was engineered by incorporating a C-terminal StrA motif LPETGG and a 6xHis tag. The addition of the 6xHis tag motif facilitated protein purification and removal of the C-terminal residue released during sortagging.80In this study, the mutant StrA7m was employed as it displayed improved kinetics and allowed for calcium-independent ligation.83The transpeptidation reaction between A12 and AGuIX-CWGGG was confirmed using SEC monitoring, and the presence of StrA7m was demonstrated to be necessary for the reaction to occur (Fig. 5A-D). The equilibrium for the reaction was reached within 2-3 hours at room temperature. Following isolation using Ni-beads to capture and remove the 6xHis- containing reagents and by-products (StrA7m, residual peptides from VHH C- terminal, and unreacted VHH), AGuIX@A12 was purified via filtration and dispersed in PBS. To accurately determine the sortagging yield, the non-fluorescent starting material AGuIX-CTGGG was used to avoid interference with VHH fluorescence. A conversion rate of 43% for VHH was achieved during the sortagging process, Attorney Docket No.29618-0451WO1 resulting in an isolated yield of AGuIX@A12 of 5.3% (relative to A12) with a NP / A12 ratio of 20 (Fig.5E-F). The diameter of AGuIX@A12, as assessed by DLS, was measured to be 6.1 nm, only slightly larger than that of AGuIX-CTGGG (4.1 nm) (Fig.5H). The reaction was optimized to obtain a final product, AGuIX@A12, with an equimolar ratio of AGuIX NPs and A12. This was achieved by using AGuIX-Mal starting material with a high density of maleimide groups (2.1 Mal / NP). By using this starting material, a reduced amount of AGuIX NPs (and therefore Gd) could be employed to provide the same quantity of oligoglycine GGG, resulting in a lower final NP / A12 ratio from 20 to 6.0 (Fig.5E). Further reduction in the equivalence of GGG compared to A12, from 15 to 3 and 1, allowed for a decrease in the NP / A12 ratio in the final AGuIX@A12 product to 2.8 and 1.0, respectively. However, this reduction also led to a decrease in the conversion of A12, from 37% to 20% and 15%, respectively (Fig.5E). To mitigate the decrease in A12 grafting during the reaction, an option was to double the amount of StrA7m while maintaining 3 equivalents of GGG compared to A12 (Figure 5E). Attempts to improve both A12 grafting and the final NP / A12 ratio of AGuIX@A12 by conducting sortagging at higher concentrations did not yield the desired results, likely due to the multicomponent nature of the reaction (Figure 5E). In the end, sortagging proved successful in grafting VHH onto the surface of AGuIX NPs. The key parameter to increase the grafting yield of VHH was to increase the equivalence of the oligoglycine in the reaction. However, this strategy also resulted in a higher NP / A12 ratio, which ideally should be around 1 for imaging purposes. Despite increasing the GGG equivalence, achieving significantly different A12 conversion rates proved challenging, with a maximum conversion rate of less than 50%. This could be attributed to the higher rate of hydrolysis of the SrtA7m- VHH complex, influenced by the increased reactivity of StrA7m. Additionally, the residual peptide from the VHH C-terminal released during the final step of sortagging, which contains a diglycine residue at the N-terminal, may also contribute to reactivity. Ultimately, optimized conditions were able to achieve a grafting yield of 32-33% and an NP / A12 ratio of 1.2 in the final product AGuIX@A12. Attorney Docket No.29618-0451WO1 Example 5. Synthesize AGuIX@VHH through click chemistry Grafting azido-PEG4-NHS ester on VHH A12 VHH (1.3 µmol, 1 mg.mL-1final concentration in reaction) were mixed with azido–PEG4–NHS (0,14 µmol, 7 µM final concentration in reaction) at 4°C for 2 hours. There was no post-reaction purification step to minimize VHH losses on the purification membranes. Azide grafting was characterized by SEC and MALDI-TOF (Fig.7A-B). Grafting DBCO-PEG5-NHS ester on NPs AGuIX NP (1 g, 13.51 w%Gd, 50 mg AGuIX / mL final concentration in reaction, redispersed in MilliQ H2O) and Cy5.5-NHS ester (1.72 µmol, 85.9 µM final concentration in reaction) were introduced in H2O (final volume 20 mL). The mixture was stirred at RT for 30 min. After x2 dilution in H2O, the AGuIX-Cy5.5 was purified by filtration (5 kDa MWCO vivaspin, dilution x2). AGuIX-Cy5.5 NPs (774 µmol Gd, 50 mg AGuIX / mL final concentration in reaction) were dispersed in Milli-Q water. Dibenzylcyclooctyne-PEG5–NHS (DBCO) was dissolved in dry DMSO (155 µmol, 20 mM final concentration in reaction), added to the AGuIX-Cy5.5 solution (final volume 18 mL) and the mixture was stirred for another 2 h. The final product AGuIX-Cy5.5-DBCO was finally filtered (5 kDa MWCO vivaspin, dilution x5000) until no more DBCO moiety could be detected in the filtrate and was finally dispersed into 9 mL of MilliQ water before lyophilisation. DBCO quantification was performed by incubatation with the fluorescent dye AF647@azide (24 h, RT). The determination of reacted DBCO was done by using AF647 calibration curve with the SEC method B. (Fig.7D) (cf. method B, fluorescence detection ex / em: 650 / 665 nm). Synthesize AGuIX@VHH through click chemistry A12-azide (50 µM, final concentration in reaction) was added to AGuIX- DBCO at (100 AGuIX mg mL-1final concentration in reaction) in 10 mM Phosphate- Buffered Saline (PBS) solution. The solution was stirred for 24 h at 4 °C. A temporal follow-up of the click reaction is monitored by SEC using method A. After reaction, AGuIX@VHH was purified by SEC (method B) to remove sub-products (NHS- terminated, azide-PEG4-NHS and AGuIX-DBCO excess) and stored at -20 °C. Attorney Docket No.29618-0451WO1 Optimization of the click chemistry reaction The first step of the synthesis involves functionalizing the VHH with the azide group. A12, which contains 9 lysine residues, was used for this purpose. These residues were able to react with the azide-PEG4-NHS ester to introduce the azide group onto the VHH surface. Although VHH has a chemically and physically robust structure, a ratio higher than an equimolar ratio of NHS per amine function led to its precipitation. The instability can be attributed to the alteration in charge of amino acids following the reaction, resulting in reduced solubility of the VHH. A ratio slightly below equimolar solved this issue and the A12-azide was successfully obtained. Functionalization was confirmed by SEC with a lower retention time suggesting an increase in size (Fig.7A). These findings were validated using Matrix- assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry, a highly sensitive technique that enables reliable determination of biomolecule size. This analytical method enabled precise evaluation of azide linker grafting, revealing a distribution of 1 to 3 linkers per A12 VHH. Additionally, it confirmed the complete grafting of all VHH in solution, as evidenced by the almost complete conversion of the initial A12 peak (14.78 kDa) (Fig.7B.). A second surface modification was necessary to enable the reactivity of AGuIX NPs with A12. Within the polysiloxane structure AGuIX contain primary amines in the (3-Aminopropyl)triethoxysilane (APTES) function. Each NP was estimated to possess a similar number of amine functions as gadolinium chelate or gadolinium, averaging 15 gadolinium (Gd) atoms per particle. This provided approximately the same number of amine sites on the NP surface that can be accessed for modification. The AGuIX-DBCO was prepared by reacting the NHS group on the DBCO-PEG4-NHS with the primary amines on the nanoparticle surface. The objective was to achieve at least one functional group per nanoparticle. There were two limiting factors to consider for this reaction: 1) the concentration of DBCO in the solution should not exceed 20 mM to prevent linker precipitation and 2) the percentage of dimethyl sulfoxide (DMSO) in the aqueous solution should not exceed 60% to avoid the precipitation of AGuIX NPs. These factors needed to be carefully controlled to ensure successful synthesis of AGuIX-DBCO. Thus, ratios ranging from 0.15 to 5 DBCO per particle was tested to optimize the synthesis of AGuIX-DBCO Attorney Docket No.29618-0451WO1 (Fig.7C.). Considering the limiting factors, the maximum achievable ratio for the synthesis of AGuIX NPs was 5 DBCO molecules per particle. Based on the calibration curve analysis, the resulting product exhibited a ratio of 10 Gd / DBCO (Fig.7D). Given that there are 15 Gd per particle, this outcome indicated an average of at least one or two DBCO molecules per particle. This confirmed that the minimum target of one functional group per nanoparticle had been successfully achieved. The click reaction between the azide and DBCO moieties was primarily monitored through SEC (Fig.6E-F). Due to the distinct separation of the three products (AGuIX-DBCO, A12-azide, and AGuIX@A12) equally facilitated the selection of SEC as the preferred purification method. The purified product underwent concentration and analysis using SEC-HPLC and SEC-HPLC-ICPMS to compare the elution profiles of gadolinium (to verify the presence of AGuIX), fluorescence (indicative of A12), and absorbance (related to the two products). The precise alignment of these three chromatograms and the absence of compounds with retention times exceeding 15 minutes confirmed the successful attainment of a pure bioconjugate (Fig.7G). To characterize AGuIX@A12 and to determine the number of NPs attached per VHH, ICPMS analysis was conducted to measure the gadolinium concentration, which directly correlates with the nanoparticle concentration. The gadolinium concentration in AGuIX was determined to be 0.02 g L-1or 1.5 g L-1. Fluorescence analysis was employed to quantify the VHH concentration. The fluorescence signals of the AGuIX@A12 are originated from two sources: 1) the VHH component of A12 and 2) the click linkers (azide-DBCO). For better accuracy, a calibration curve was established using the fluorescence of A12@azide-DBCO in the presence of AGuIX (non-grafted) at a concentration of 1.5 g L-1(Fig.7H). The concentration of A12 was determined to be 63 µM in the presence of 1278 µM Gd (ICP / MS), corresponding to a ratio of approximately 20 Gd per VHH. Example 6. Nanoparticle Conjugate Characterization Biacore analyses Biomolecular interactions between immobilized receptor PD-L1 and CD47 and analyte AGuIX@A12 or AGuIX@A4 were assessed by Surface Plasmon Resonance (SPR) on Biacore 2000 instrument (Cytiva). For the kinetic assays, the Attorney Docket No.29618-0451WO1 A12 was injected up to a concentration of 61.2 nM, the A4 up to 67 nM, the AGuIX NPs at 0-915.7 nM in Gd3+, the AGuIX@A12 VHH at 0-61.2 nM, (0-915.7 nM equivalent in Gd3+) and the AGuIX@A4 VHH at 0-16.7 nM (0-240 nM equivalent in Gd3+). The chip was prepared as described in SI. All characteristic interaction constants (equilibrium dissociation (KD) and kinetic rate of association and dissociation (kAand kD)) were determined by curve fitting using the Langmuir 1:1 binding model implemented in Biaevaluation software 4.1.1 The instrument was equipped with a CM5 sensor chip carrying a carboxymethylated dextran matrix covalently attached to a gold surface (Cytiva). The chip is connected to a microfluidic system that allows the flow of samples on the chip surface. All SPR experiments were performed at room temperature in HEPES (HBS- P, GE Healthcare, 0.01 M HEPES, 0.15 M NaCl, 0.005% surfactant P20) as running buffer. The four channels of the chip (fc1, fc2, fc3 and fc4) were used for this study and two were used as reference (fc1 and fc3). First, each channel was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) / N- hydroxysuccinimide (NHS) (0.2 M EDC / 0.05 M NHS) during 7 minutes to generate reactive succinimide esters Following, PD-L1 and CD47 receptors were injected on the fc2 and fc4 channel, respectively, at 10 μg / ml in acetate buffer pH 4.5 for the PD- L1 and pH 5 for the CD47 at a flow rate of 5 µL min-1for 24 minutes (PD-L1) and 12 minutes (CD47). Covalent immobilization of the receptors (PD-L1 and CD47) on the surface of the sensor chip was confirmed by an increase of 1040 response units (RU) for fc2 and 1600 RU for fc4. The unreacted succinimide esters were deactivated by ethanolamine (pH 8.5) during 7 minutes at a flow rate of 5 µL min-1. The fc1 and fc3 were used as a control for nonspecific interaction and the succinimide esters were directly deactivated with ethanolamine without adding receptors. For the analysis of the different samples, they were injected on all channel by several cycles. A cycle corresponds to an injection of the sample for 3 minutes at a flow rate of 30 µL min-1followed by a stabilization period of 2.5 minutes under the running buffer (HBS-P) then followed by a regeneration solution (1 M NaCl and 5 mM NaOH) for 30-60 seconds at a flow rate of 30 µL min-1and finally another stabilization period of 2 minutes. For the kinetic assays, the A12 was injected at the 0 - 61.2 nM concentration range, the A4 at 0-67 nM, the AGuIX NPs at 0-915.7 nM in Gd3+, the AGuIX@A12 VHH at 0-61.2 nM, (0- 915.7 nM equivalent in Gd3+) and the AGuIX@A4 VHH at 0- Attorney Docket No.29618-0451WO1 16.65 nM (0-250 nM equivalent in Gd3+). Each experiment began with three cycles of operating buffer injection to stabilize the baseline of the instrument. Then, the concentration gradients of the samples (AGuIX, VHHs and bioconjugates) were analyzed 3 times. All characteristic interaction constants (equilibrium dissociation (KD), kinetic rate of association and dissociation (kA and kD)) were determined by curve fitting using the Langmuir 1:1 binding model implemented in Biaevaluation software 4.1.1.16Prior to the calculations, the signals were plotted on a sensogram by subtracting the nonspecific signal from the control channels (fc1 and fc3), from the signal obtained from the functionalized channels (respectively fc2 and fc4). Concerning the characteristic constants, the number of complexes analyte-receptor formed per second in a molar solution of VHH and receptor is described by the association rate constant ka (in M-1s-1)) defined by the formula: d[AB] / dt=ka [A][B with A the analyte (VHH or NPs) and B the ligand (receptor) and AB the final complex analyte-ligand. The number of complexes dissociated per second; they are determined by the dissociation rate constant kd (in s-1) given by the formula: -d[AB] / dt=kd [AB]. The association and the dissociation are two phenomena that happen at the same time, and at equilibrium they are equal. This equilibrium is defined by an equilibrium dissociation constant KD obtained by the formula: KD=[A][B] / [AB] =kd / ka. According to the KDformula, the lower the value, the stronger the interaction between the analyte and the receptor. The Langmuir 1:1 binding model implemented in Biaevaluation 4.1.1.1 software was used to analyze the sensograms and to determine the interaction constants (KD, kA and kD) associated to each sample. The association rate constants were assessed for the PD-L1 receptor, revealing that A12 exhibits remarkably high affinity, approximately 10 times greater than that of A4 (specifically, ka, A12 = (21.10 ± 2.21) x 105and ka, A4 = (1.15 ± 0.10) x 105) (Fig.8B and 8D). Conversely, the dissociation rate constant was four times faster for A4 than for A12 (namely, kd, A4 = (42.10 ± 1.64) x10-4and kd, A12 = (13.20 ± 1.10) x10-4s-1) (Fig.8B and 8D). These results confirmed that A12 possessed significantly higher affinity for PD-L1 compared to the nanobody A4, emphasizing the specificity of the interaction between the nanobody and the receptor. The same trends were observed for the bioconjugates, where AGuIX@A12 exhibited approximately 10 times higher affinity than AGuIX@A4 for the PD-L1 receptor (ka, AGuIX@A12 = Attorney Docket No.29618-0451WO1 (15.50 ± 2.55) x105 and ka, AGuIX@A4 = (1.37 ± 0.05) x105M-1.s-1)(Fig.8C and 8E). Dissociation rates were also faster for AGuIX@A4, consistent with the reference nanobodies (kd, AGuIX@A12 = 6.0x10-4and kd, AGuIX@A4 = 11.8x10-4s-1) (Fig. 8C and 8E). These findings highlighted A12's strong specificity for the PD-L1 receptor, whether grafted onto AGuIX or not. For the CD47 receptor, specificity was observed only with A4. Neither A12 nor AGuiX alone showed any affinity for CD47. Similar orders of magnitude for association and dissociation rates were obtained between the conjugate and the reference VHH (ka, A4 = 11.4x105 and ka, AGuIX@A4 = 6.7x105M-1s-1, and kd, A4 = 7.9x10-4and kd, AGuIX@A4 = 4.9x10-4s-1) (Fig.9), confirming once again the preservation of VHH functionality even after functionalization with AGuIX. Ex vivo biodistribution The animals were anaesthetized with isoflurane (induction: 3%, maintenance: 1.5-2.0%) in a mixture of 100% O2 (flow rate = 1.0-1.5 L min-1). At day 8 post- inoculation, 1.92 ± 0.09 MBq (mean ± SD), corresponding to the dose of 7.12 ± 0.35 and 2.29 ± 0.07 µg of AGuIX (mean ± SD) for AGuIX-[89Zr]- and for AGuIX- [89Zr]@A12 respectively, were intravenously injected into tumor-bearing mice (n = 8 for both AGuIX and AGuIX-A12, 22.0 ± 1.9 g per mouse). The mice were euthanized at 4 hours and 24 hours post-injection (n = 4 per time points) and tissue activity was determined for several organs of interest after harvesting (blood pool, intestines, kidneys, spleen, pancreas, liver, muscle, bone, brain, tumor). Activity in various organs of interest is represented in percentage of injected dose per gram (%ID per g) (Table 1). AGuIX-A12 have been radiolabeled with zirconium-89 (89Zr) according to the protocol described by Tran et al.96Briefly, 40 µg of AGuIX and 40 µg of AGuIX- A12, pH adjusted to 7.2, were incubated in a solution containing neutralized [89Zr][Zr-(oxalate)4]4- for 15 minutes at 37°C, 300 rpm. The oxalic acid of the89Zr oxalate solution from Perkin Elmer (200 µL, 430 MBq) was neutralized with 90 µL of Na2CO3(2 M) before adding them to the AGuIX-based solutions. The radiochemical purity of [89Zr]-AGuIX and [89Zr]-AGuIX-A12 was assessed by Thin Layer Chromatography (TLC).5 µL of samples from the reaction were taken after 15 minutes incubation to deposit on a chromatography paper (1 × 12 cm). These TLC Attorney Docket No.29618-0451WO1 papers were migrated in citrate sodium buffer, pH 5.5, and the activity was measured using a Mini-Scan TLC Imaging Scanner with gamma detector B-FC-3600 (Eckert & Ziegler, Germany) at 1.0 mm.s−1. High radiolabeling yield was obtained reaching 99% and 94% for [89Zr]-AGuIX and [89Zr]-AGuIX-A12 respectively (Table 1). [89Zr]-AGuIX and [89Zr]-AGuIX-A12 were purified and concentrated by Vivaspin®ultrafiltration tubes (Sartorius, 5 kDa MWCO). Specific activities were 0.29 and 0.32 MBq / µg AGuIX for [89Zr]-AGuIX and [89Zr]-AGuIX-A12 respectively. Table 1. 89Zr Labeled Specific Activity Injected Activity Number of Mice Radiolabeling AGuIX (µCi / µg Gd) (µCi) / Time Point Yield by iTLC . To evaluate the best approach for biofunctionalization of AGuIX NPs with VHH, we compared sortagging and click chemistry. The sortagging bioconjugation approach permits for the selection of specific sites for modification, enabling more accurate predictions regarding the biological impact of the chemical alteration. Modifying a protein terminus is expected to have a smaller effect on the protein's folding and functionality as well as the added advantage of defining the orientation of the immobilized protein, which is anticipated to better preserve its function compared to other immobilization techniques. This enzymatic reaction relies on the enzyme Sortase A (SrtA), a transpeptidase produced by Gram-positive bacteria, which catalyzes the formation of a peptide bond between two peptides to attach specific proteins to the cell wall or pili assembly. SrtA specifically recognizes the LPXTG sequence (with X representing any amino acid) and by nucleophilic attack makes it reactive to an N-terminal oligoglycine, forming a peptide bond between the threonine of LPXT and the oligoglycine. This natural ligation system has been repurposed for protein modification and has gained popularity as a research tool due to its high ligation selectivity, simplicity, robustness, and the availability of various SrtA variants (expressed in Escherichia coli or commercially available), as well as other required materials. Sortagging reactions have been used to graft specific biomolecules, such as peptides or proteins, onto a protein or other biological targets of interest and has already proved efficacious in NP conjugation. Attorney Docket No.29618-0451WO1 Click chemistry describes chemical reactions aimed at achieving high yield and high selectivity in the formation of carbon-heteroatom bond. Click chemistry reactions are based on the 1,3-dipolar cycloaddition of an azide and an alkyne to form a 1,2,3-triazole. This reaction has been widely utilized in various applications due to its simplicity in terms of preparation and purification steps, enabling rapid generation of new products with high reaction rates, such as bioconjugates. The formation of the triazole linkage is irreversible and quantitative, providing excellent reaction stability. Azide and dibenzocyclooctyne (DBCO) are selected as strain-promoted azide-alkyne cycloaddition reactions (SPAAC) tools due to their relatively small functional groups with a favourable rate constant (k2= 0.2-0.5 M-1.s-1) which have been shown to be effective. The crucial challenge of click chemistry is to preserve the integrity and functionality of the biomolecule after grafting. Synthesis of AGuIX-VHH by sortagging The sortagging reaction is based on enzymatic synthesis involving three primary steps: (1) introduction of maleimide (Mal) moieties on AGuIX NPs, (2) introduction of the peptide GGG(W / T)C using the Mal moieties, and (3) introduction of VHH through the transpeptidation enzymatic reaction (Fig.1A). AGuIX NPs were modified to mimic the functionality of the N-terminal oligoglycine residue typically employed in sortagging. Initially, a Mal functional group was introduced onto the surface of AGuIX NPs using a bifunctional NHS / Mal linker. AGuIX-Mal was further modified with a short peptide linker that incorporates a cysteine amino acid at the C-terminal and a triglycine residue (GGG(W / T)C) at the N-terminal. To enable the use of sortagging, the VHH was engineered by incorporating a C-terminal StrA motif LPETGG and a 6xHis tag. The addition of the 6xHis tag motif facilitated protein purification and removal of the C-terminal residue released during sortagging. The transpeptidation reaction between A12 and AGuIX- C(W / T)GGG was verified using SEC monitoring. After purification, the final product exhibited a retention time (tR) of 13.2 minutes, corresponding to a product size ranging from 29 to 44 kDa (Fig.1B, Fig.5C). The equilibrium for the reaction was reached within 2-3 hours at room temperature. Following isolation using Ni-beads to capture and remove the 6xHis-containing reagents and by-products (StrA7m, residual peptides from VHH C-terminal, and unreacted VHH), AGuIX@A12 was purified via Attorney Docket No.29618-0451WO1 filtration and dispersed in PBS (Fig.5C). A VHH conversion of 32-33% was achieved during the sortagging process, resulting in an isolated yield of AGuIX@A12 of 5.3% (relative to A12) with a Gd / A12 ratio of 20 (Fig.1B, Fig.5E). The hydrodynamic diameter (DH) of AGuIX@A12, as assessed by DLS, was 6.1 nm, slightly larger than that of AGuIX-C(W / T)GGG (4.1 nm) (Fig.1C). This outcome confirms the modification of the particle surface and hints at the possibility of VHH grafting. After optimization, sortagging proved successful in grafting VHH onto the surface of AGuIX NPs. The key parameter to increase the grafting yield of VHH was to increase the equivalence of the oligoglycine in the reaction. However, this strategy also resulted in a higher NP / A12 ratio, which ideally should be around 1 for imaging applications. The NP / A12 ratio of 1 could be achieved after optimization of several parameters (Figs.5A-E), while VHH conversion decreased to 15%. The binding affinity, evaluated using the inhibitory constant (Ki) through a competitive enzyme- linked immunosorbent assay (ELISA), confirmed that the addition of AGuIX to A12 did not impact the competitive binding affinities (Fig.6). Furthermore, AGuIX’s lack of interaction with PD-L1 proteins also confirmed that the high affinity is solely attributed to the presence of the nanobody on AGuIX’s surface. Moreover, the similar logKi values of 12.8 ± 1.1 nM, and 5.5 ± 0.6 nM for A12 and AGuIX@A12 obtained, respectively, strongly reinforced this result (Fig.6). Synthesis of AGuIX@VHH by click chemistry Click chemistry based on strain-promoted azide-alkyne cycloaddition involves three primary steps: (1) introduction of the azide group on the VHH, (2) introduction of the DBCO group on the AGuIX NP, and (3) introduction of the VHH through the click chemistry reaction (Fig.2A). The first step of the synthesis involves functionalizing the VHH with the azide group based on NHS-ester reaction with lysine residues. Functionalization was confirmed by SEC (Fig.7A) and matrix- assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry (Fig.7B). The AGuIX-DBCO was prepared by the same reaction consisting in NHS reaction with the primary amines on the NP surface. After synthesis and purification, the modified NP presented a ratio of 10 Gd / DBCO, i.e.1 to 2 DBCO functions per NP (Fig.7C-D). AGuIX-DBCO high excess ratio (450Gd / A12) was chosen for the Attorney Docket No.29618-0451WO1 click chemistry to minimize any reactant rate limitations and ensure complete reaction (Fig.2B). At least 95% of the VHH in solution successfully reacted with AGuIX, resulting in the formation of AGuIX@A12 with a size ranging from approximately 29 to 44 kDa, as determined by Superdex 75 protein calibration (Fig.2D; Fig.7E-F). Purification by preparative SEC was effective in removing the unreacted AGuIX- DBCO and other by-products, isolating the AGuIX@A12 bioconjugate. DLS and TDA size measurements of the bioconjugate were consistent (Fig.3C, Fig.7I). The analyses of hydrodynamic diameters using DLS and TDA exhibit coherence and complementarity. DLS offers a standard resolution, providing an average value, but it is limited in its ability to differentiate between species as such ultrasmall sizes. On the other hand, application of TDA to nanoparticles is a recent method that has proven to be effective in distinguishing various populations of ultrasmall size. An increase in hydrodynamic diameter was observed following biofunctionalization (in DLS, DH, AGuIX = 3.0 ± 1.1 nm vs DH, AGuIX@A12 = 5.4 ± 3.1 nm). TDA is a highly accurate and absolute method based on deconvolution methods, enabling the hydrodynamic diameters of AGuIX NPs to be determined via their diffusion coefficients. This analysis revealed the presence of two populations within the AGuIX@A12 sample in which 75 ± 2% of AGuIX NPs were effectively functionalized with the VHH, while 25 ± 2% remained unfunctionalized. The minority population (25 ± 2%) are the remaining AGuIX-DBCO (Fig.7J). In summary, AGuIX@A12 was obtained with a 20Gd / A12 (approx.1 NP / A12) and a reaction yield close to 20% (based on VHH). Just as with the sortagging product, the affinity of AGuIX@A12 was assessed using ELISA, resulting in a logKi value of 13.0 ± 0.4 nM, similar to A12. The addition of AGuIX to the A12 nanobody through click chemistry did not influence competitive binding affinities; therefore, click chemistry did not compromise functional targeting properties (Fig.6). Comparison of “sortagging reaction” and “click chemistry” methods First, it was observed that both sortagging and click chemistry methods yielded AGuIX@VHH bioconjugates with relatively similar characteristics. The hydrodynamic size measurements (DH, sortagging: 6.1 ± 3.7 nm and DH, click: 5.4 ± 3.1 nm) and SEC chromatograms (tRsortagging: 13.2 min and tRclick: 12.9 min) were consistent between the two methods. Furthermore, a crucial factor determining the Attorney Docket No.29618-0451WO1 reliability of the synthesis is the maintenance of the VHH's affinity with the PD-L1 after its grafting to AGuIX. Evaluation of this affinity through a competitive ELISA demonstrated that, in both synthesis approaches, the binding affinity remained robust and similar to the reference VHH (logKisortagging: 5.5 ± 0.6 nM and logKiclick: 13.0 ± 0.4 nM). Although both conjugation products maintained similar binding affinities for the PD-L1 ligand, the reactivity of VHH upon click reaction conditions was higher, with a grafting yield before purification ηclick > 95% (vs ηsortagging < 50%). This observation aligns with existing literature, where sortagging reactivity efficacy may be limited due to equilibrium parameters (Fig 5E). Moreover, the AGuIX-DBCO created during the click chemistry process could be separated from the final AGuIX@VHH bioconjugate, unlike the AGuIX-C(W / T)GGG used for sortagging. This separation provided a larger amount of purified AGuIX NP to be used for the click chemistry, reaching reaction equilibrium. Moreover, economic considerations are crucial when strategizing the scale-up of a process. It is noteworthy that biologics as used in the sortagging reaction are more expensive and less stable compared to chemical compounds. In conclusion, both sortagging and click chemistry methods afforded the AGuIX@VHH bioconjugates, however, the higher yield and lower cost associated with click chemistry have encouraged its adoption for further investigations (Table 2). Table 2. Summary of the comparison between the two methods, "sortagging reaction" and "click chemistry," for grafting AGuIX with a VHH. Method Sortagging reaction Click chemistry Number of at ) Attorney Docket No.29618-0451WO1 (no elimination of excess AGuIX possible) Total yield 15% 20% To demonstrate the reproducibility and robustness of the click chemistry approach, conjugated a second VHH was conjugated to AGuIX, this one specific to the CD47 receptor (A4. Mw =14.8 kDa). The first functionalization of the VHH with azide-PEG4-NHS demonstrated comparable efficacy to that of A12 (mean size of MWA12-azide: 15.33 kDa (2 azide grafted) and MWA4-azide:15.5 kDa (2-3 azide grafted)) (Fig.10). The SEC indicate that the AGuIX@A4 (tR: 13 min) product has a size comparable to AGuIX@A12 (tR: 12.9 min) (Fig.3). AGuIX@A4 was synthesized with a >95% conversion yield, similar to AGuIX@A12. After purification, the final purified product showed a ratio of 17Gd / VHH ratio, with a final yield of around 26%. In Vitro Characterizations of AGuIX@VHH A competitive ELISA confirmed that the addition of AGuIX to either VHH, A12 (Fig.4A) or A4 (Fig.4B), did not impact competition binding affinities. We demonstrated similar logKi values of 12.8 + / - 1.1 nM and 13.0 + / - 0.4 nM for A12, and AGuIX@A12, respectively, and 2.9 + / - 0.2 nM and 3.3 + / - 0.4 nM for A4 and AGuIX-A4, respectively. Moreover, AGuIX on its own exhibited no interaction with PD-L1 or CD47 ligands, providing additional support for the notion that the robust affinity observed primarily results from the incorporation of functional VHH on the AGuIX surface. These results were further validated using a second method: Biacore analysis which is based on a surface plasmon resonance (SPR) technique that which quantifies the association and dissociation phenomena. The affinity analyses conducted on the VHHs, and their associated receptors confirmed a strong binding affinity between these VHHs and their respective receptors, as shown by the equilibrium dissociation constants (KD) (0.63 ± 0.01 nM for A12 on PD-L1 and 0.70 ± 0.13 nM for A4 on CD47) (Fig.4C, Fig.8, Fig.9). Attorney Docket No.29618-0451WO1 These results were supported by the literature, particularly in the case of A4, which has been extensively documented.73Comparing the KD values of AGuIX@VHH with their unmodified counterparts (0.39 ± 0.07 nM for AGuIX@A12 and 0.74 ± 0.01 nM for AGuIX@A4), similar binding affinities were observed, further confirming that conjugation of AGuIX did not impact VHH binding. This similarity in KD values between AGuIX@VHH and unmodified VHH provided confirmation of the relevance of click chemistry in preserving the function of the VHHs. The A4, specific for the CD47 receptor, was selected to verify the specificity of the AGuIX@A12 to the PD- L1 receptor. The characteristic constants (equilibrium dissociation (KD) and kinetic rate of association and dissociation (kA and kD)) indicated a 10-fold stronger and faster binding affinity for A12 towards PD-L1 compared to A4, regardless of the bioconjugation status. These results confirm the specificity of the interaction between the nanobodies and their respective receptors. To confirm ligand-receptor interaction on cells, internalization and receptor blocking assays on melanoma B16F10 tumor cells, modified for high PD-L1 expression, were conducted to further validate the functionality of VHH bound to AGuIX (Fig.4D-E). Melanoma hiPDL1-B16F10 murine cells are highly metastatic, aggressive models mimicking patient phenotypes, expressing both PD-L1 and CD47.51 The conjugation of A12 and A4 significantly increased internalization of AGuIX as seen in the representative images (Fig.4D) and corrected total cell fluorescence (CTCF, Fig.4E). Blocking with either anti-PD-L1 or anti-CD47 decreased internalization confirming specificity and functionality of the AGuIX@VHH. Studies were repeated in wildtype B16F10 cells (lower PD-L1 expression)74(Fig.11) with similar increase in internalization with the conjugation of A12 and A4 to AGuIX. Although complete loss of internalization was not observed, this may be an indication of only partial blocking of PD-L1 and CD47 receptors allowing for some receptor mediated internalization. Ex vivo AGuIX@A12 Targeting Given the widespread use of PD-1 / PD-L1 inhibitors in clinical practice, the AGuIX@A12 product was evaluated in proof-of-concept ex vivo studies. Targeting of PD-L1 was investigated in a murine melanoma model to confirm that AGuIX@A12 binding increases tumor accumulation. An ex vivo biodistribution study using Attorney Docket No.29618-0451WO1 zirconium-89 (89Zr) radiolabeling was performed for accurate quantification of AGuIX and AGuIX@A12 in different organs at 4 hours and 24 hours post- intravenous injection (Fig.4F-I). A short and long time point was selected based on previous preclinical biodistribution studies and current clinical trials involving AGuIX. Currently, in clinical trials, radiotherapy is delivered 4 hours after AGuIX administration, justifying the clinical relevance of a 4-hour biodistribution time point and multiple preclinical trials showed that tumor accumulation was increased up to 4h, while the 24-hour time point was chosen to study the expected longer persistence of targeted nanoparticles in the tumor. As expected from previous work, both NPs accumulate within a few hours into the kidneys, leading to fast washout from the blood pool due to their ultrasmall size; lower accumulation of NPs in the liver can be detected. Accumulation in the bone was attributed to the free [89Zr] (non-coupled on the NPs). Both NPs can also accumulate in the tumor area due to the enhanced permeability and retention (EPR) effect, previously shown in several rodent models and in human studies. However, a significantly higher accumulation of AGuIX- [89Zr]@A12 was present in the tumor area compared to untargeted AGuIX-[89Zr] (at 4 hours, AGuIX: 2.10 ± 0.15 %ID.g-1vs. AGuIX@A12: 3.90 ± 0.67 %ID.g-1) (Fig. 4F-I). This difference was observed 4 hours post-injection and was more pronounced 24 hours post-injection (at 24hours, AGuIX: 1.34 ± 0.06 %ID.g-1vs AGuIX@A12: 3.10 ± 0.15 %ID.g-1). This higher accumulation and retention of AGuIX@VHH suggests targeting of the PD-L1 receptors at the surface of cancer cells, that is expected to increase active accumulation and slow the AGuIX-VHH washout within the tumor area. This ex vivo validation of the higher targeting of AGuIX@A12 vs. AGuIX is evidence of the in vivo targeting of PD-L1 by AGuIX@A12. AGuIX@A12 is thus a promising candidate for future work involving MRI-detection of PD-L1 or for more targeted radiosensitization of PD-L1 expressing tumors. We report the preparation of AGuIX@VHH bioconjugates using two distinct methods, sortagging and click chemistry. The two methods resulted in NPs with similar physicochemical characteristics using the A12 VHH. However, click chemistry exhibited higher VHH conversion while using lower-cost reagents, making it a more favorable approach for further investigations. Interestingly, the two Attorney Docket No.29618-0451WO1 bioconjugation methods yielded AGuIX@VHH with strong binding affinities, suggesting that site-specific VHH modification, such as sortagging, may not be required when coupling to relatively small NP such as AGuIX NP. PD-L1 targeting of AGuIX-A12 prepared by click chemistry was validated by ex vivo autoradiography demonstrating substantially greater uptake and persistence than untargeted NP. The A4 nanobody was used to the reliability and reproducibility of click synthesis for these constructs. Overall, click chemistry emerges as a favorable and promising approach for preparing a broad array of potential AGuIX@VHH bioconjugates that can be applied to multiple nanobodies, with diverse applications in targeted therapeutic interventions and imaging. REFERENCES 1 C. Verry, S. Dufort, J. Villa, M. Gavard, C. Iriart, S. Grand, J. Charles, B. Chovelon, J.-L. Cracowski, J.-L. Quesada, C. Mendoza, L. Sancey, A. Lehmann, F. Jover, J.-Y. Giraud, F. Lux, Y. Crémillieux, S. McMahon, P. J. Pauwels, D. Cagney, R. Berbeco, A. Aizer, E. Deutsch, M. Loeffler, G. Le Duc, O. Tillement and J. Balosso, Radiother. Oncol., 2021, 160, 159–165. 2 E. Thivat, M. Casile, J. Moreau, I. Molnar, S. Dufort, K. Seddik, G. Le Duc, O. De Beaumont, M. Loeffler, X. Durando and J. Biau, BMC Cancer, 2023, 23, 344. 3 F. Lux, V. L. Tran, E. Thomas, S. Dufort, F. Rossetti, M. Martini, C. Truillet, T. Doussineau, G. Bort, F. Denat, F. Boschetti, G. Angelovski, A. Detappe, Y. Crémillieux, N. Mignet, B.-T. Doan, B. Larrat, S. Meriaux, E. Barbier, S. Roux, P. Fries, A. Müller, M.-C. Abadjian, C. Anderson, E. Canet-Soulas, P. Bouziotis, M. Barberi-Heyob, C. Frochot, C. Verry, J. Balosso, M. Evans, J. Sidi-Boumedine, M. Janier, K. Butterworth, S. McMahon, K. Prise, M.-T. Aloy, D. Ardail, C. Rodriguez- Lafrasse, E. Porcel, S. Lacombe, R. Berbeco, A. Allouch, J.-L. Perfettini, C. Chargari, E. Deutsch, G. Le Duc and O. Tillement, Br. J. Radiol., 2019, 92, 20180365. 4 F. Lux, L. Sancey, A. Bianchi, Y. Crémillieux, S. Roux and O. Tillement, Nanomed., 2015, 10, 1801–1815. 5 L. Carmès, M. Banerjee, P. Coliat, S. Harlepp, X. Pivot, O. Tillement, F. Lux and A. Detappe, Adv. Ther., 2023, n / a, 2300019. Attorney Docket No.29618-0451WO1 6 G. Bort, F. Lux, S. Dufort, Y. Crémillieux, C. Verry and O. Tillement, Theranostics, 2020, 10, 1319–1331. 7 A. Detappe, C. Mathieu, C. Jin, M. P. Agius, M.-C. Diringer, V.-L. Tran, X. Pivot, F. Lux, O. Tillement, D. Kufe and P. P. Ghoroghchian, Int. J. Radiat. Oncol., 2020, 108, 1380–1389. 8 A. Detappe, M. Reidy, Y. Yu, C. Mathieu, H. V.-T. Nguyen, T. P. Coroller, F. Lam, P. Jarolim, P. Harvey, A. Protti, Q.-D. Nguyen, J. A. Johnson, Y. Cremillieux, O. Tillement, I. M. Ghobrial and P. P. Ghoroghchian, Nanoscale, 2019, 11, 20485–20496. 9 E. Thomas, C. Mathieu, P. Moreno-Gaona, V. Mittelheisser, F. Lux, O. Tillement, X. Pivot, P. P. Ghoroghchian and A. Detappe, Adv. Healthc. Mater., 2022, 11, 2101565. 10 M. M. Gomari, S. Abkhiz, T. G. Pour, E. Lotfi, N. Rostami, F. N. Monfared, B. Ghobari, M. Mosavi, B. Alipour and N. V. Dokholyan, Mol. Med., 2022, 28, 146. 11 P. Chames, M. Van Regenmortel, E. Weiss and D. Baty, Br. J. Pharmacol., 2009, 157, 220–233. 12 S. Sun, Z. Ding, X. Yang, X. Zhao, M. Zhao, L. Gao, Q. Chen, S. Xie, A. Liu, S. Yin, Z. Xu and X. Lu, Int. J. Nanomedicine, 2021, 16, 2337–2356. 13 A. Muruganandam, J. Tanha, S. Narang and D. Stanimirovic, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol., 2002, 16, 240–242. 14 J. Wesolowski, V. Alzogaray, J. Reyelt, M. Unger, K. Juarez, M. Urrutia, A. Cauerhff, W. Danquah, B. Rissiek, F. Scheuplein, N. Schwarz, S. Adriouch, O. Boyer, M. Seman, A. Licea, D. V. Serreze, F. A. Goldbaum, F. Haag and F. Koch-Nolte, Med. Microbiol. Immunol. (Berl.), 2009, 198, 157–174. 15 M. Liu, Y. Zhu, T. Wu, J. Cheng and Y. Liu, Chem. – Eur. J., 2020, 26, 7442–7450. 16 E. Y. Yang and K. Shah, Front. Oncol., 2020, 10, 1182. 17 T. De Meyer, S. Muyldermans and A. Depicker, Trends Biotechnol., 2014, 32, 263–270. 18 P. Bannas, J. Hambach and F. Koch-Nolte, Front. Immunol. 19 M. Dougan, J. R. Ingram, H.-J. Jeong, M. M. Mosaheb, P. T. Bruck, L. Ali, N. Pishesha, O. Blomberg, P. M. Tyler, M. M. Servos, M. Rashidian, Q.-D. Attorney Docket No.29618-0451WO1 Nguyen, U. H. von Andrian, H. L. Ploegh and S. K. Dougan, Cancer Immunol. Res., 2018, 6, 389–401. 20 J. R. Ingram, O. S. Blomberg, J. T. Sockolosky, L. Ali, F. I. Schmidt, N. Pishesha, C. Espinosa, S. K. Dougan, K. C. Garcia, H. L. Ploegh and M. Dougan, Proc. Natl. Acad. Sci., 2017, 114, 10184–10189. 21 J. R. Ingram, M. Dougan, M. Rashidian, M. Knoll, E. J. Keliher, S. Garrett, S. Garforth, O. S. Blomberg, C. Espinosa, A. Bhan, S. C. Almo, R. Weissleder, H. Lodish, S. K. Dougan and H. L. Ploegh, Nat. Commun., 2017, 8, 647. 22 L. Fehrenbacher, A. Spira, M. Ballinger, M. Kowanetz, J. Vansteenkiste, J. Mazieres, K. Park, D. Smith, A. Artal-Cortes, C. Lewanski, F. Braiteh, D. Waterkamp, P. He, W. Zou, D. S. Chen, J. Yi, A. Sandler and A. Rittmeyer, The Lancet, 2016, 387, 1837–1846. 23 D. R. Spigel, C. Faivre-Finn, J. E. Gray, D. Vicente, D. Planchard, L. Paz-Ares, J. F. Vansteenkiste, M. C. Garassino, R. Hui, X. Quantin, A. Rimner, Y.-L. Wu, M. Özgüroğlu, K. H. Lee, T. Kato, M. de Wit, T. Kurata, M. Reck, B. C. Cho, S. Senan, J. Naidoo, H. Mann, M. Newton, P. Thiyagarajah and S. J. Antonia, J. Clin. Oncol., 2022, 40, 1301–1311. 24 A. Bouleau, V. Lebon and C. Truillet, Pharmacol. Ther., 2021, 222, 107786. 25 Y.-C. Chen, W. Shi, J.-J. Shi and J.-J. Lu, J. Cancer Res. Clin. Oncol., 2022, 148, 1–14. 26 E. C. Piccione, S. Juarez, J. Liu, S. Tseng, C. E. Ryan, C. Narayanan, L. Wang, K. Weiskopf and R. Majeti, mAbs, 2015, 7, 946–956. 27 M. P. Chao, C. H. Takimoto, D. D. Feng, K. McKenna, P. Gip, J. Liu, J.-P. Volkmer, I. L. Weissman and R. Majeti, Front. Oncol., 2019, 9, 1380. 28 H. A. Burris III, A. I. Spira, M. H. Taylor, O. O. Yeku, J. F. Liu, P. N. Munster, E. P. Hamilton, J. S. Thomas, F. Gatlin, R. T. Penson, T. A. Abrams, M. S. Dhawan, J. M. Walling, J. W. Frye, K. Romanko, V. Sung, C. Brachmann and A. B. El-Khoueiry, J. Clin. Oncol., 2021, 39, 2516–2516. 29 Z. Jiang, H. Sun, J. Yu, W. Tian and Y. Song, J. Hematol. Oncol.J Hematol Oncol, 2021, 14, 180. 30 I. E. Krop, N. Masuda, T. Mukohara, S. Takahashi, T. Nakayama, K. Inoue, H. Iwata, T. Toyama, Y. Yamamoto, D. M. Hansra, M. Takahashi, A. Osaki, Attorney Docket No.29618-0451WO1 K. Koyama, T. Inoue, T. Yonekura, J. Mostillo, S. Ohwada, Y. Tanaka, D. W. Sternberg and K. Yonemori, J. Clin. Oncol., 2022, 40, 1002–1002. 31 R. K. Vaddepally, P. Kharel, R. Pandey, R. Garje and A. B. Chandra, Cancers, 2020, 12, 738. 32 H. Sato, N. Okonogi and T. Nakano, Int. J. Clin. Oncol., 2020, 25, 801–809. 33 L. Wen, F. Tong, R. Zhang, L. Chen, Y. Huang and X. Dong, Front. Oncol. 34 Y. Nishiga, A. P. Drainas, M. Baron, D. Bhattacharya, A. A. Barkal, Y. Ahrari, R. Mancusi, J. B. Ross, N. Takahashi, A. Thomas, M. Diehn, I. L. Weissman, E. E. Graves and J. Sage, Nat. Cancer, 2022, 3, 1351–1366. 35 E. Rostami, M. Bakhshandeh, H. Ghaffari-Nazari, M. Alinezhad, M. Alimohammadi, R. Alimohammadi, G. Mahmoodi Chalbatani, E. Hejazi, T. J. Webster, J. Tavakkol-Afshari and S. A. Jalali, PLoS ONE, 2022, 17, e0273547. 36 Y. Zhang, K.-Y. Park, K. F. Suazo and M. D. Distefano, Chem. Soc. Rev., 2018, 47, 9106–9136. 37 X. Dai, A. Böker and U. Glebe, RSC Adv., 2019, 9, 4700–4721. 38 M. Ritzefeld, Chem. – Eur. J., 2014, 20, 8516–8529. 39 L. Taiariol, C. Chaix, C. Farre and E. Moreau, Chem. Rev., 2021, acs.chemrev.1c00484. 40 J. M. Eeftens, J. van der Torre, D. R. Burnham and C. Dekker, BMC Biophys., 2015, 8, 9. 41 T. I. Chio and S. L. Bane, Methods Mol. Biol. Clifton NJ, 2020, 2078, 83–97. 42 J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo, J. A. Codelli and C. R. Bertozzi, Proc. Natl. Acad. Sci., 2007, 104, 16793–16797. 43 E. Kim and H. Koo, Chem. Sci., 2019, 10, 7835–7851. 44 B. Stump, ChemBioChem, 2022, 23, e202200016. 45 Explaining Nobel Prize-winning chemistry techniques, https: / / www.buffalo.edu / ubnow / stories / 2022 / 10 / nobel-prize-chemistry.html, (accessed March 9, 2023). Attorney Docket No.29618-0451WO1 46 G. Le Duc, S. Roux, A. Paruta-Tuarez, S. Dufort, E. Brauer, A. Marais, C. Truillet, L. Sancey, P. Perriat, F. Lux and O. Tillement, Cancer Nanotechnol., 2014, 5, 4. 47 A. Mignot, C. Truillet, F. Lux, L. Sancey, C. Louis, F. Denat, F. Boschetti, L. Bocher, A. Gloter, O. Stéphan, R. Antoine, P. Dugourd, D. Luneau, G. Novitchi, L. C. Figueiredo, P. C. de Morais, L. Bonneviot, B. Albela, F. Ribot, L. Van Lokeren, I. Déchamps-Olivier, F. Chuburu, G. Lemercier, C. Villiers, P. N. Marche, G. Le Duc, S. Roux, O. Tillement and P. Perriat, Chem. – Eur. J., 2013, 19, 6122– 6136. 48 N. Brown, P. Rocchi, L. Carmès, R. Guthier, M. Iyer, L. Seban, T. Morris, S. Bennett, M. Lavelle, J. Penailillo, R. Carrasco, C. Williams, E. Huynh, Z. Han, E. Kaza, T. Doussineau, S. M. Toprani, X. Qin, Z. D. Nagel, K. A. Sarosiek, A. Hagège, S. Dufort, G. Bort, F. Lux, O. Tillement and R. Berbeco, Theranostics, 2023, 13, 4711–4729. 49 E. Thomas, L. Colombeau, M. Gries, T. Peterlini, C. Mathieu, N. Thomas, C. Boura, C. Frochot, R. Vanderesse, F. Lux, M. Barberi-Heyob and O. Tillement, Int. J. Nanomedicine, 2017, Volume 12, 7075–7088. 50 C. Truillet, E. Thomas, F. Lux, L. T. Huynh, O. Tillement and M. J. Evans, Mol. Pharm., 2016, 13, 2596–2601. 51 E. Thomas, C. Mathieu, P. Moreno-Gaona, V. Mittelheisser, F. Lux, O. Tillement, X. Pivot, P. P. Ghoroghchian and A. Detappe, Adv. Healthc. Mater., 2022, 11, 2101565. 52 E. Thomas, Cancer, Université de Lyon, 2017. 53 L. Labied, P. Rocchi, T. Doussineau, J. Randon, O. Tillement, H. Cottet, F. Lux and A. Hagège, Anal. Chim. Acta, 2021, 1185, 339081. 54 L. Heinrich, N. Tissot, D. J. Hartmann and R. Cohen, J. Immunol. Methods, 2010, 352, 13–22. 55 V.-L. Tran, F. Lux, N. Tournier, B. Jego, X. Maître, M. Anisorac, C. Comtat, S. Jan, K. Selmeczi, M. J. Evans, O. Tillement, B. Kuhnast and C. Truillet, Adv. Healthc. Mater., 2021, 10, e2100656. 56 T. Spirig, E. M. Weiner and R. T. Clubb, Mol. Microbiol., 2011, 82, 1044–1059. Attorney Docket No.29618-0451WO1 57 H. E. Morgan, W. B. Turnbull and M. E. Webb, Chem. Soc. Rev., 2022, 51, 4121–4145. 58 J. M. Antos, M. C. Truttmann and H. L. Ploegh, Curr. Opin. Struct. Biol., 2016, 38, 111–118. 59 J. E. Glasgow, M. L. Salit and J. R. Cochran, J. Am. Chem. Soc., 2016, 138, 7496–7499. 60 S. A. M. van Lith, S. M. J. van Duijnhoven, A. C. Navis, W. P. J. Leenders, E. Dolk, J. W. H. Wennink, C. F. van Nostrum and J. C. M. van Hest, Bioconjug. Chem., 2017, 28, 539–548. 61 L. Liu, J. L. Gray, E. W. Tate and A. Yang, Trends Biotechnol., , DOI:10.1016 / j.tibtech.2023.05.004. 62 M. W. Popp, J. M. Antos, G. M. Grotenbreg, E. Spooner and H. L. Ploegh, Nat. Chem. Biol., 2007, 3, 707–708. 63 N. Kotagiri, Z. Li, X. Xu, S. Mondal, A. Nehorai and S. Achilefu, Bioconjug. Chem., 2014, 25, 1272–1281. 64 V. Aragon-Sanabria, A. Aditya, L. Zhang, F. Chen, B. Yoo, T. Cao, B. Madajewski, R. Lee, M. Z. Turker, K. Ma, S. Monette, P. Chen, J. Wu, S. Ruan, M. Overholtzer, P. Zanzonico, C. M. Rudin, C. Brennan, U. Wiesner and M. S. Bradbury, Clin. Cancer Res., 2022, 28, 2938–2952. 65 L. Williams, L. Li, P. J. Yazaki, P. Wong, A. Miller, T. Hong, E. K. Poku, S. Bhattacharya, J. E. Shively and M. Kujawski, Biotechnol. J., 2023, e2300115. 66 A. Debon, E. Siirola and R. Snajdrova, JACS Au, 2023, 3, 1267–1283. 67 C. J. Pickens, S. N. Johnson, M. M. Pressnall, M. A. Leon and C. J. Berkland, Bioconjug. Chem., 2018, 29, 686–701. 68 V. Solntceva, M. Kostrzewa and G. Larrouy-Maumus, Front. Cell. Infect. Microbiol., 2021, 10, 621452. 69 S. Mädler, C. Bich, D. Touboul and R. Zenobi, J. Mass Spectrom., 2009, 44, 694–706. 70 J. S. Nanda and J. R. Lorsch, in Methods in Enzymology, ed. J. Lorsch, Academic Press, 2014, vol.536, pp.87–94. 71 L. Labied, P. Rocchi, T. Doussineau, J. Randon, O. Tillement, H. Cottet, F. Lux and A. Hagège, Anal. Chim. Acta, 2021, 1185, 339081. Attorney Docket No.29618-0451WO1 72 Q. Wu, H. L. Ploegh and M. C. Truttmann, ACS Chem. Biol., 2017, 12, 664–673. 73 J. T. Sockolosky, M. Dougan, J. R. Ingram, C. C. M. Ho, M. J. Kauke, S. C. Almo, H. L. Ploegh and K. C. Garcia, Proc. Natl. Acad. Sci. U. S. A., 2016, 113, E2646–E2654. 74 A. Quijano-Rubio, A. M. Bhuiyan, H. Yang, I. Leung, E. Bello, L. R. Ali, K. Zhangxu, J. Perkins, J.-H. Chun, W. Wang, M. J. Lajoie, R. Ravichandran, Y.- H. Kuo, S. K. Dougan, S. R. Riddell, J. B. Spangler, M. Dougan, D.-A. Silva and D. Baker, Nat. Biotechnol., 2023, 41, 532–540. 75 S. Kotb, A. Detappe, F. Lux, F. Appaix, E. L. Barbier, V.-L. Tran, M. Plissonneau, H. Gehan, F. Lefranc, C. Rodriguez-Lafrasse, C. Verry, R. Berbeco, O. Tillement and L. Sancey, Theranostics, 2016, 6, 418–427. 76 E. Thivat, M. Casile, J. Moreau, I. Molnar, S. Dufort, K. Seddik, G. Le Duc, O. De Beaumont, M. Loeffler, X. Durando and J. Biau, BMC Cancer, 2023, 23, 344. 77 C. Verry, L. Sancey, S. Dufort, G. Le Duc, C. Mendoza, F. Lux, S. Grand, J. Arnaud, J. L. Quesada, J. Villa, O. Tillement and J. Balosso, BMJ Open, 2019, 9, e023591. 78 F. Lux, V. L. Tran, E. Thomas, S. Dufort, F. Rossetti, M. Martini, C. Truillet, T. Doussineau, G. Bort, F. Denat, F. Boschetti, G. Angelovski, A. Detappe, Y. Crémillieux, N. Mignet, B.-T. Doan, B. Larrat, S. Meriaux, E. Barbier, S. Roux, P. Fries, A. Müller, M.-C. Abadjian, C. Anderson, E. Canet-Soulas, P. Bouziotis, M. Barberi-Heyob, C. Frochot, C. Verry, J. Balosso, M. Evans, J. Sidi-Boumedine, M. Janier, K. Butterworth, S. McMahon, K. Prise, M.-T. Aloy, D. Ardail, C. Rodriguez- Lafrasse, E. Porcel, S. Lacombe, R. Berbeco, A. Allouch, J.-L. Perfettini, C. Chargari, E. Deutsch, G. Le Duc and O. Tillement, Br. J. Radiol., 2019, 92, 20180365. 79 D. S. Abou, T. Ku and P. M. Smith-Jones, Nucl. Med. Biol., 2011, 38, 675–681. 80 J. R. Ingram, M. Dougan, M. Rashidian, M. Knoll, E. J. Keliher, S. Garrett, S. Garforth, O. S. Blomberg, C. Espinosa, A. Bhan, S. C. Almo, R. Weissleder, H. Lodish, S. K. Dougan and H. L. Ploegh, Nat. Commun., 2017, 8, 647. 81 M. Dougan, J. R. Ingram, H.-J. Jeong, M. M. Mosaheb, P. T. Bruck, L. Ali, N. Pishesha, O. Blomberg, P. M. Tyler, M. M. Servos, M. Rashidian, Q.-D. Attorney Docket No.29618-0451WO1 Nguyen, U. H. von Andrian, H. L. Ploegh and S. K. Dougan, Cancer Immunol. Res., 2018, 6, 389–401. 82 J. T. Sockolosky, M. Dougan, J. R. Ingram, C. C. M. Ho, M. J. Kauke, S. C. Almo, H. L. Ploegh and K. C. Garcia, Proc. Natl. Acad. Sci. U. S. A., 2016, 113, E2646–E2654. 83 H. E. Morgan, W. B. Turnbull and M. E. Webb, Chem. Soc. Rev., 2022, 51, 4121–4145. 84 N. Asiimwe, M. F. Al Mazid, D. P. Murale, Y. K. Kim and J.-S. Lee, Pept. Sci., 2022, 114, e24235. 85 S. Mädler, C. Bich, D. Touboul and R. Zenobi, J. Mass Spectrom., 2009, 44, 694–706. 86 J. S. Nanda and J. R. Lorsch, in Methods in Enzymology, ed. J. Lorsch, Academic Press, 2014, vol.536, pp.87–94. 87 C. J. Pickens, S. N. Johnson, M. M. Pressnall, M. A. Leon and C. J. Berkland, Bioconjug. Chem., 2018, 29, 686–701. 88 V. Solntceva, M. Kostrzewa and G. Larrouy-Maumus, Front. Cell. Infect. Microbiol., 2021, 10, 621452. 89 C. Truillet, E. Thomas, F. Lux, L. T. Huynh, O. Tillement and M. J. Evans, Mol. Pharm., 2016, 13, 2596–2601. 90 E. Thomas, L. Colombeau, M. Gries, T. Peterlini, C. Mathieu, N. Thomas, C. Boura, C. Frochot, R. Vanderesse, F. Lux, M. Barberi-Heyob and O. Tillement, Int. J. Nanomedicine, 2017, Volume 12, 7075–7088. 91 N. Brown, P. Rocchi, L. Carmès, R. Guthier, M. Iyer, L. Seban, T. Morris, S. Bennett, M. Lavelle, J. Penailillo, R. Carrasco, C. Williams, E. Huynh, Z. Han, E. Kaza, T. Doussineau, S. M. Toprani, X. Qin, Z. D. Nagel, K. A. Sarosiek, A. Hagège, S. Dufort, G. Bort, F. Lux, O. Tillement and R. Berbeco, Theranostics, 2023, 13, 4711–4729. 92 E. Thomas, C. Mathieu, P. Moreno-Gaona, V. Mittelheisser, F. Lux, O. Tillement, X. Pivot, P. P. Ghoroghchian and A. Detappe, Adv. Healthc. Mater., 2022, 11, 2101565. 93 E. Thomas, Cancer, Université de Lyon, 2017. 94 L. Labied, P. Rocchi, T. Doussineau, J. Randon, O. Tillement, F. Lux and A. Hagège, Anal. Chem., 2021, 93, 1254–1259. Attorney Docket No.29618-0451WO1 95 L. Heinrich, N. Tissot, D. J. Hartmann and R. Cohen, J. Immunol. Methods, 2010, 352, 13–22. 96 V.-L. Tran, F. Lux, N. Tournier, B. Jego, X. Maître, M. Anisorac, C. Comtat, S. Jan, K. Selmeczi, M. J. Evans, O. Tillement, B. Kuhnast and C. Truillet, Adv. Healthc. Mater., 2021, 10, e2100656. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.29618-0451WO1 WHAT IS CLAIMED IS:

1. A nanoparticle conjugate, comprising 1) one or more nanoparticles, wherein the nanoparticles comprises a) a polysiloxane core; b) one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to metal ions; 2) one or more second linking groups; and 3) one or more nanobodies; wherein each second linking group is covalently bonded to one nanoparticle and one nanobody.

2. The nanoparticle conjugate of claim 1, wherein the ratio of nanoparticles to nanobodies is about 1:1, about 1.3:1, about 1.5:1, or about 2:1, optionally wherein the ratio of nanoparticles to nanobodies is about 1.3:

1.

3. The nanoparticle conjugate of claim 1 or 2, wherein the ratio of metal ions to nanobodies is about 15:1, about 20:1, about 23:1, or about 30:1, optionally wherein the ratio of metal ions to nanobodies is about 20:

1.

4. The nanoparticle conjugate of any one of claims 1 to 3, wherein the hydrodynamic diameter of the nanoparticle conjugate is from about 2 to 20 nm, optionally wherein the hydrodynamic diameter of the nanoparticle conjugate is about 2.3 to 8.5 nm.

5. The nanoparticle conjugate of any one of claims 1 to 4, wherein the molecular weight of the nanoparticle conjugate is from about 25 to 50 kDa, optionally wherein the molecular weight of the nanoparticle conjugate is from about 29 to 44 kDa.

6. The nanoparticle conjugate of any one of claims 1 to 5, wherein the metal ions are lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, tin (stannum), palladium, silver, zirconium, gold, or bismuth, or combination thereof.Attorney Docket No.29618-0451WO1 7. The nanoparticle conjugate of claim 6, wherein the metal ions are Gd3+ions, Bi3+ions, or a combination thereof, optionally wherein the metal ions are Gd3+ions.

8. The nanoparticle conjugate of any one of claims 1 to 7, wherein the one or more first linking groups each comprise a C1-10alkylamine.

9. The nanoparticle conjugate of any one of claims 1 to 8, wherein the one or more first linking groups are each , wherein indicates the bond connecting the first linking groupindicates the bond connecting the first linking group to the ligand.

10. The nanoparticle conjugate of any one of claims 1 to 9, wherein the ligand is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1- (glutaric acid)-4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylene diamine tetra-acetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N- bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexaacetic acid (TTHA), hydroxyethyidiamine triacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA), or 1,4,7,10- tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (TCMC).

11. The nanoparticle conjugate of any one of claims 1 to 10, wherein the ligand is whereinAttorney Docket No.29618-0451WO1 12. The nanoparticle conjugate of any one of claims 1 to 11, wherein the ligand is indicates the bond connecting the first13. The nanoparticle conjugate of any one of claims 1 to 12, wherein greater than about 20% of the ligands are complexed to the metal ions.

14. The nanoparticle conjugate of any one of claims 1 to 13, wherein the nanoparticle comprises 4 to 26 metal ions, 6 to 24 metal ions, 8 to 22 metal ions, 10 to 20 metal ions, 12 to 18 metal ions, or 14 to 16 metal ions, optionally wherein the nanoparticle comprises about 15 metal ions.

15. The nanoparticle conjugate of any one of claims 1 to 14, wherein the hydrodynamic diameter of the nanoparticle is from about 0.5 to 8 nm, about 0.5 to 5.5 nm, about 1 to 5 nm, about 1.5 to 4.5 nm, or about 2 to 4 nm, optionally wherein the hydrodynamic diameter of the nanoparticle is from about 2 to 4 nm.

16. The nanoparticle conjugate of any one of claims 1 to 15, wherein the one or more second linking groups are each independently O O O or or aAttorney Docket No.29618-0451WO1 wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein indicates the bond connecting the second linking group to the nanoparticle, and indicates the bond connecting the second linking group to the nanobody.

17. The nanoparticle conjugate of any one of claims 1 to 16, wherein the one or more second linking groups are each independently or or awherein indicates the bond connecting the second linking group to the nanoparticle, and indicates the bond connecting the second linking group to the nanobody.

18. The nanoparticle conjugate of any one of claims 1 to 17, wherein the nanobody is listed in Table A, optionally selected from the following nanobodies: KN044, HLX-53, 131I-GMIB-Anti-HER2-VHH1, CAM-FAP-(I-131 / AC-225),NM- 01, NM-02, NM-03, NM-04, NM-05, JS014 (fusion protein), RAD204 (177Lu), RAD202 (177Lu), A12, A4, Z15-0-2, Nb 16, Nb 36, B3, Envafolimab (KN035), Nb 97 (MY2935), Nb 109, 3 / E2, sbAb K2, 3131 / 3206, Nb 16925, Nb 16988.

19. The nanoparticle conjugate of any one of claims 1 to 18, prepared by the process of reacting i) one or more nanoparticle A, wherein nanoparticle A comprises a) a polysiloxane core;Attorney Docket No.29618-0451WO1 b) one or more of a first linking group covalently bonded to a ligand wherein one or more of the ligands are complexed to Gd3+ions; c) one or more of linker A:wherein x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and indicates the bond connecting linker A to the nanoparticle A; with ii) one or more nanobody B, wherein nanobody B comprises one or more of linker B:wherein z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indicates the bond connecting linker B to the nanobody.

20. A method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of the nanoparticle conjugate of any one of claims 1 to 19, wherein the nanobody binds to a cancer cell target or a checkpoint inhibitor or other immunotherapeutic target.

21. The method of claim 20, wherein the cancer is a solid cancer.

22. The method of claim 20 or 21, wherein the nanobody binds to ANGPT2, ANGPTL2, B7-H3, CLDN18.2, CTLA-4, EGFR, FAP, HER-2, MSLN, MUC1, PD-1, PD-L1, TROP-2, TIGIT, VEGFA, VEGF, VEGFR, BCMA, CD19, CD20, CD7, CD47, c-KIT, c-Met Nectin-4, IL-21,ODFR, or RANKL.

23. The method of any one of claims 20 to 22, wherein the nanobody is listed in Table A, optionally selected from the following nanobodies: KN044, HLX-53, 131I-Attorney Docket No.29618-0451WO1 GMIB-Anti-HER2-VHH1, CAM-FAP-(I-131 / AC-225),NM-01, NM-02, NM-03, NM-04, NM-05, JS014 (fusion protein), RAD204 (177Lu), RAD202 (177Lu), A12, A4, Z15-0-2, Nb 16, Nb 36, B3, Envafolimab (KN035), Nb 97 (MY2935), Nb 109, 3 / E2, sbAb K2, 3131 / 3206, Nb 16925, or Nb 16988.

24. The method of any one of claims 20 to 23, further comprising treating the subject with a radiotherapy modality, optionally either external beam radiotherapy (EBRT) or targeted radionuclide therapy.

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