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⁸⁹Zr-Labeled Anti-PD-L1 Antibody Fragment for Evaluating In Vivo PD-L1 Levels in Melanoma Mouse Model.

ABSTRACT: The rise of programmed death-1 (PD-1)/PD-L1 immune checkpoint inhibitor therapy has been one of the most promising developments in melanoma research. However, not all the melanoma patients respond to such immune checkpoint blockade. There is a great need of biomarkers for appropriate melanoma patient selection and therapeutic efficacy monitoring. The objective of this study is to develop a novel radiolabeled anti-PD-L1 antibody fragment, as an imaging biomarker, for evaluating the in vivo PD-L1 levels in melanoma. The Df-conjugated F(ab')₂ fragment of the anti-mouse PD-L1 antibody was successfully synthesized and radiolabeled with ⁸⁹Zr. Both Df-F(ab')₂ and ⁸⁹Zr-Df-F(ab')₂ maintained the nano-molar murine PD-L1 targeting specificity and affinity. ⁸⁹Zr-Df-F(ab')₂ showed less uptake in normal liver tissue in mice compared with its full antibody counterpart ⁸⁹Zr-Df-anti-PD-L1. Positron emission tomography (PET)/computed tomography images clearly showed that ⁸⁹Zr-Df-F(ab')₂ possessed superior pharmacokinetics and imaging contrast over the radiolabeled full antibody, with much earlier and higher tumor uptake (5.5 times more at 2 h post injection) and much lower liver background (51% reduction at 2 h post injection). The specific and high murine PD-L1-targeting uptake at tumor foci coupled with fast clearance of ⁸⁹Zr-Df-F(ab')₂ highlighted its potential for in vivo PET imaging of murine PD-L1 levels and future development of radiolabeled anti-human PD-L1 fragment for potential application in melanoma patients.

SUBMITTER: Bridgwater C

PROVIDER: S-EPMC7578182 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeTargeted delivery in vivo remains an immense roadblock for the translation of nanomaterials into the clinic. The greatest obstacle is the mononuclear phagocyte system (MPS), which sequesters foreign substances from general circulation and causes accumulation in organs such as the liver and spleen. The purpose of this study was to determine whether attaching an active targeting antibody, 5B1, to the surface of gold nanoparticles and using clodronate liposomes to deplete liver and splenic macrophages could help to minimize uptake by MPS organs, increase targeted delivery to CA19.9-positive pancreatic tumors, and enhance pancreatic tumor delineation.ProceduresTo produce the antibody-gold nanoparticle conjugate (Ab-AuNP), the Ab was conjugated to p-isothiocyanatobenzyl-desferrioxamine (p-SCN-DFO) and subsequently conjugated to NHS-activated gold nanoparticles. The Ab-AuNP was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modified Lindmo assay was performed to assess binding affinity and internalization potential in vitro. The Ab-AuNP was radiolabeled with 89Zr and injected into CA19.9-positive BxPc-3 pancreatic orthotopic tumor-bearing mice pretreated with or without clodronate liposomes for PET imaging and biodistribution studies. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was used to confirm delivery of gold nanoparticles to BxPc-3 pancreatic subcutaneous xenografts.ResultsMice pretreated with clodronate liposomes in an orthotopic setting demonstrated decreased liver uptake at early time points (12.2 ± 2.3 % ID/g vs. 22.8 ± 3.8 % ID/g at 24 h) and increased tumor uptake at 120 h (13.8 ± 8.0 % ID/g vs. 6.0 ± 1.2 % ID/g). This allowed for delineation of orthotopic pancreatic xenografts in significantly more mice treated with clodronate (6/6) than in mice not treated with clodronate (2/6) or mice injected with gold nanoparticles labeled with a nonspecific antibody (0/5).ConclusionsThe combination of clodronate liposomes and an active targeting antibody on the surface of gold nanoparticles allowed for PET/CT imaging of subcutaneous and orthotopic pancreatic xenografts in mice.

Project description:PurposeSite-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, 89Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue.ProceduresA variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue - Z(35BPA) - was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate - DFOZ(35BPA)-huA33 - was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFOZ(35BPA)-huA33 was optimized to produce [89Zr]Zr-DFOZ(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [89Zr]Zr-DFOZ(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [89Zr]Zr-DFO-huA33.ResultsHuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [89Zr]Zr-DFOZ(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~ 0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e., ~ 40%ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [89Zr]Zr-DFO-huA33.ConclusionsPhotoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method - DFOZ(35BPA)-huA33 - was well-defined and homogeneous, and the resulting radioimmunoconjugate ([89Zr]Zr-DFOZ(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.

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⁸⁹Zr-Labeled Anti-PD-L1 Antibody Fragment for Evaluating In Vivo PD-L1 Levels in Melanoma Mouse Model.

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89Zr-Labeled Anti-PD-L1 Antibody Fragment for Evaluating In Vivo PD-L1 Levels in Melanoma Mouse Model.

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OmicsDI is part of the ELIXIR infrastructure

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⁸⁹Zr-Labeled Anti-PD-L1 Antibody Fragment for Evaluating In Vivo PD-L1 Levels in Melanoma Mouse Model.