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The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89 Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 ( t 1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [ 89 Zr]Zr-DFO-PEG 5 -Tz ([ 89 Zr]Zr- 3 ) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans -cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89 Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Introduction: Monoclonal antibodies (mAbs) are widely used in nuclear imaging. Radiolabelling with positron emitting radionuclides, typically radiometals, requires the incorporation of a bifunctional chelator for the formation of the radiometal-mAb complex. Additionally, mAbs can be conjugated with small molecules capable to undergo bioorthogonal click reactions in vivo, enabling pre-targeting strategies. The determination of the number of functionalities attached to the mAb is critically important to ensure a good labelling yield or to guarantee pre-targeting efficacy. In this work, we compare three different analytical methods for the assessment of average functionalisation and heterogeneity of the conjugated mAbs., Methods: Two selected mAbs (Trastuzumab and Bevacizumab) were randomly conjugated through lysine residues with 3-10 equivalents p-isothiocyanatobenzyl-desferrioxamine (p-NCS-Bz-DFO) or 20-200 equivalents trans-cyclooctene-N-hydroxysuccinimide ester (TCO-NHS). The DFO- or TCO-to-mAb ratio were determined using three different methods: direct titration (radiometric for DFO-conjugated mAbs, photometric for TCO-conjugated mAbs), MALDI/TOF MS mass analysis (Matrix-Assisted Laser Desorption-Ionization/Time of Flight Mass Spectrometry), and UPLC/ESI-TOF MS mass analysis (Ultra High Performance Liquid Chromatography/Electrospray Ionization-Time of Flight Mass Spectrometry)., Results: Radiometric and photometric titrations provided information on the average number of DFO and TCO functionalities per mAb respectively. MALDI/TOF MS provided equivalent results to those obtained by titration, although investigation of the heterogeneity of the resulting mixture was challenging and inaccurate. UPLC/ESI-TOF MS resulted in good peak resolution in the case of DFO-conjugated mAbs, where an accurate discrimination of the contribution of mono-, di- and tri-substituted mAbs could be achieved by mathematical fitting of the spectra. However, UPLC/ESI-TOF MS was unable to discriminate between different conjugates when the smaller TCO moiety was attached to the mAbs., Conclusions: The three techniques offered comparable results in terms of determining the average number of conjugates per mAb. Additionally, UPLC/ESI-TOF MS was able to shed a light on the heterogeneity of the resulting functionalised mAbs, especially in the case of DFO-conjugated mAbs. Finally, while using a single analytical method might not be a reliable way to determine the average functionalisation and assess the heterogeneity of the sample, a combination of these methods could substantially improve the characterization of mAb conjugates., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Inert and stable radiolabeling of monoclonal antibodies (mAb), antibody fragments, or antibody mimetics with radiometals is a prerequisite for immuno-PET. While radiolabeling is preferably fast, mild, efficient, and reproducible, especially when applied for human use in a current Good Manufacturing Practice compliant way, it is crucial that the obtained radioimmunoconjugate is stable and shows preserved immunoreactivity and in vivo behavior. Radiometals and chelators have extensively been evaluated to come to the most ideal radiometal-chelator pair for each type of antibody derivative. Although PET imaging of antibodies is a relatively recent tool, applications with 89 Zr, 64 Cu, and 68 Ga have greatly increased in recent years, especially in the clinical setting, while other less common radionuclides such as 52 Mn, 86 Y, 66 Ga, and 44 Sc, but also 18 F as in [ 18 F]AlF are emerging promising candidates for the radiolabeling of antibodies. This review presents a state of the art overview of the practical aspects of radiolabeling of antibodies, ranging from fast kinetic affibodies and nanobodies to slow kinetic intact mAbs. Herein, we focus on the most common approach which consists of first modification of the antibody with a chelator, and after eventual storage of the premodified molecule, radiolabeling as a second step. Other approaches are possible but have been excluded from this review. The review includes recent and representative examples from the literature highlighting which radiometal-chelator-antibody combinations are the most successful for in vivo application.

Introduction: The assessment of ex vivo biodistribution is the preferred method for quantification of radiotracers biodistribution in preclinical models, but is not in line with current ethics on animal research. PET imaging allows for noninvasive longitudinal evaluation of tracer distribution in the same animals, but systemic comparison with ex vivo biodistribution is lacking. Our aim was to evaluate the potential of preclinical PET imaging for accurate tracer quantification, especially in tumor models., Methods: NEMA NU 4-2008 phantoms were filled with 11 C, 68 Ga, 18 F, or 89 Zr solutions and scanned in Mediso nanoPET/CT and PET/MR scanners until decay. N87 tumor-bearing mice were i.v. injected with either [ 18 F]FDG (~ 14 MBq), kept 50 min under anesthesia followed by imaging for 20 min, or with [ 89 Zr]Zr-DFO-NCS-trastuzumab (~ 5 MBq) and imaged 3 days post-injection for 45 min. After PET acquisition, animals were killed and organs of interest were collected and measured in a γ-counter to determine tracer uptake levels. PET data were reconstructed using TeraTomo reconstruction algorithm with attenuation and scatter correction and regions of interest were drawn using Vivoquant software. PET imaging and ex vivo biodistribution were compared using Bland-Altman plots., Results: In phantoms, the highest recovery coefficient, thus the smallest partial volume effect, was obtained with 18 F for both PET/CT and PET/MR. Recovery was slightly lower for 11 C and 89 Zr, while the lowest recovery was obtained with 68 Ga in both scanners. In vivo, tumor uptake of the 18 F- or 89 Zr-labeled tracer proved to be similar irrespective whether quantified by either PET/CT and PET/MR or ex vivo biodistribution with average PET/ex vivo ratios of 0.8-0.9 and a deviation of 10% or less. Both methods appeared less congruent in the quantification of tracer uptake in healthy organs such as brain, kidney, and liver, and depended on the organ evaluated and the radionuclide used., Conclusions: Our study suggests that PET quantification of 18 F- and 89 Zr-labeled tracers is reliable for the evaluation of tumor uptake in preclinical models and a valuable alternative technique for ex vivo biodistribution. However, PET and ex vivo quantification require fully described experimental and analytical procedures for reliability and reproducibility.

Purpose: Almost all radiolabellings of antibodies with 89 Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89 Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89 Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents., Methods: Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89 Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [ 89 Zr]Zr-DFO*-NCS and [ 89 Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially., Results: [ 89 Zr]Zr-DFO*-NCS-trastuzumab and [ 89 Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [ 89 Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates., Conclusion: DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89 Zr-immuno-PET.

Probody ® therapeutics are recombinant masked monoclonal antibody (mAb) prodrugs that become activated by proteases present in the tumor microenvironment. This makes them attractive for use as Probody drug conjugates (PDCs). CX-2009 is a novel PDC with the toxic drug DM4 coupled to an anti-CD166 Probody therapeutic. CD166 is overexpressed in multiple tumor types and to a lesser extent by healthy tissue. Methods : The tumor targeting potential of CX-2009 was assessed by performing 89 Zr-immuno-PET/biodistribution/therapy studies in a CD166-positive H292 lung cancer mouse model. Head-to-head comparisons of CX-2009 with the Probody therapeutic without DM4 (CX-191), the unmasked antibody drug conjugate (ADC) CX-1031, and the parental mAb CX-090 were performed. All constructs were 89 Zr labeled in a GMP compliant way, administered at 10, 110, or 510 µg, and ex vivo biodistribution was assessed at 24, 72, and 168 hours post-injection. Results : Comparable biodistribution was observed for all constructs, confirmed with PET/CT. Tumors showed the highest uptake: 21.8 ± 2.3 ([ 89 Zr]Zr-CX-2009), 21.8 ± 5.0 ([ 89 Zr]Zr‑CX-191), 18.7 ± 2.5 ([ 89 Zr]Zr-CX-1031) and 20.8 ± 0.9 %ID/g ([ 89 Zr]Zr-CX-090) at 110 µg injected. Increasing the dose to 510 µg resulted in lower tumor uptake and higher blood levels for all constructs, suggesting receptor saturation. In addition, CX-2009 and CX-1031 showed similar therapeutic potential. Conclusions : CX-2009 is optimally capable of targeting CD166-expressing tumors when compared with its derivatives, implying that enzymatic activation inside the tumor, required to allow CD166 binding, does not limit tumor targeting . Because CX-2009 does not bind to mouse CD166, however, reduced targeting of healthy organs should be confirmed in ongoing clinical 89 Zr-immuno-PET studies., Competing Interests: Competing Interests: M.N., M.K., R.V., B.H., and O.V. are current paid employees and stockholders of CytomX Therapeutics, Inc. CytomX Therapeutics, Inc. has filed patent applications related to the work described herein. PROBODY, a registered U.S. trademark, and IHZ are the trademarks of CytomX Therapeutics, Inc. All other brands and trademarks referenced herein are the property of their respective owners., (© The author(s).)

The N-Methyl-d-Aspartate receptor (NMDAR) is involved in many neurological and psychiatric disorders including Alzheimer's disease and schizophrenia. The aim of this study was to develop a positron emission tomography (PET) ligand to assess the bio-availability of the NMDAR ion channel in vivo. A series of tri-N-substituted diarylguanidines was synthesized and their in vitro binding affinities for the NMDAR ion channel assessed in rat forebrain membrane fractions. Compounds 21, 23 and 26 were radiolabeled with either carbon-11 or fluorine-18 and ex vivo biodistribution and metabolite studies were performed in Wistar rats. Biodistribution studies showed high uptake especially in prefrontal cortex and lowest uptake in cerebellum. Pre-treatment with MK-801, however, did not decrease uptake of the radiolabeled ligands. In addition, all three ligands showed fast metabolism., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.