Thiopalmitoylation of altered peptide ligands enhances their protective effects in an animal model of multiple sclerosis

Previously, we have shown that conjugation of a palmitic chain via a thioester bond to a cysteine residue in weakly or nonencephalitogenic or neuritogenic peptides markedly enhances their ability to induce autoimmune disease in an MHC class II–restricted manner. From those studies, however, it was not clear whether thiopalmitoylation of the peptides was merely enhancing their disease-inducing potential or whether the lipid was itself playing a pathogenic role. To investigate this further, we have now tested the effects of thiopalmitoylation on MHC class II–restricted altered peptide ligands (APLs), which are normally protective in experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. We hypothesized that if thiopalmitoylation of a peptide merely enhances its innate potential, then thiopalmitoylated APLs (S-palmAPLs) should show enhanced protective effects. Alternatively, if thiopalmitoylation itself can make a peptide pathogenic, then S-palmAPLs should have decreased therapeutic potential. We synthesized APLs and corresponding S-palmAPLs and showed that the S-palmAPLs were much more effective than the nonconjugated APL at inhibiting the development of experimental autoimmune encephalomyelitis. This was due to several features of the S-palmAPL:S-palmAPL–primed cells show an enhanced ability to proliferate and produce the anti-inflammatory cytokine, IL-10, in vitro. Furthermore, the bioavailability of S-palmAPL was greatly enhanced, compared with the nonpalmitoylated APL, and S-palm APL was taken up more rapidly into dendritic cells and channeled into the MHC class II processing pathway. These results show that thiopalmitoylation of MHC class II–restricted peptides is a simple way to enhance their effects in vivo and could have wide therapeutic application

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [Zr-89]Zr-DFO-PEG(5)-Tz

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-ofconcept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new Zr-89-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. [Zr-89]Zr-DFO-PEG S -Tz ([Zr-89]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 Zr-89-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.Peer reviewe

Epitopes thiopalmitoylés des protéines du système nerveux central et périphérique (Synthèse et propriétés immunologiques)

La protéine zéro (P0), protéine majeure de la myéline du système nerveux périphérique (SNP), est un candidat autoantigène dans l attaque autoimmune rencontrée dans le syndrome de Guillain-Barré (SGB), une maladie inflammatoire démyélinisante du SNP, et dans son modèle animal la neurite autoimmune expérimentale (NAE). P0 est modifiée d une façon post-traductionnelle par une chaine palmitique sur sa cystéine 153. L axe principal de ma thèse a été d étudier l effet de la thiopalmitoylation sur les propriétés neuritogènes et immunogènes de la protéine P0. La première partie de ce travail a été consacrée à la synthèse des épitopes thiopalmitoylés par synthèse peptidique en phase solide. La seconde partie concerne l étude des propriétés neuritogènes de ces épitopes chez le rat Lewis. Cette étude a permis de développer deux nouveaux modèles animaux : 1) un modèle NAE rémittent mimant la CIDP (chronic inflammatory demyelinating polyradiculoneuropathy), la forme chronique du SGB 2) un modèle NAE axonal mimant l AMAN (acute motor axonal neuropathy), la forme axonal du SGB. Cette étude a aussi permis de confirmer que la thiopalmitoylation était un bon moyen d induire et d augmenter la réponse immunitaire restreinte au CMH de classe II. Les APLs Altered Peptide Ligands sont des analogues du peptide antigène. Les APLs pouvaient être très utiles dans le traitement des maladies autoimmunes comme la sclérose en plaques (SEP) en agissant en tant que modulateurs spécifiques de la réponse inflammatoire. Une dernière partie de ma thèse a permis de valider l utilisation d un APL thiopalmitoylé du Protéolipide de la myéline (PLP) en tant que vaccin thérapeutique dans l EAE, modèle animal de la SEP, en montrant sa capacité à stimuler la maturation des cellules dendritiques.P0 protein, the major protein of peripheral nervous system (PNS) myelin, is a putative autoantigen in the autoimmune attack seen in Guillain-Barré syndrome (GBS), an inflammatory demyelinating disease of PNS, and in its animal model experimental autoimmune neuritis (EAN). P0 is post-transnationally modified by a palmitic acid on cysteine 153. The main axis of my thesis was to study the effect of the thiopalmitoylation on the neuritogenic and immunogenic properties of the P0 protein. The first part of this work was devoted to the synthesis of thiopalmitoylated épitopes by solid phase peptide synthesis. The second part concerns the study of the neuritogenic properties of this epitopes in Lewis rats. This study allowed the development of two new animal models: 1) relapsing-remitting EAN, mimicking the CIDP (chronic inflammatory demyelinating polyradiculoneuropathy), the chronic form of GBS 2) axonal EAN, mimicking AMAN (acute motor axonal neuropathy), the axonal form of GBS. Furthermore, this study confirmed that thiopalmitoylation of peptides may provide a simple mean to induce and/or increase MHC class II restricted responses. APLs Altered Peptide Ligands are analogues of the antigen peptide. APLs can be very useful in the treatment of autoimmune disease such multiple sclerosis (MS) by acting as a specific immunomodulator of the inflammatory reaction. The study in the last part of my thesis validate the use of a thiopalmitoylated APL of PLP (proteolipid protein of the central nervous system myelin) as a therapeutic vaccine in EAE, an animal model of MS, by showing its capacity to stimulate the maturation of dendritic cells.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Evaluation of ⁶⁸Ga- and ¹⁷⁷Lu-DOTA-PEG₄‑LLP2A for VLA-4-Targeted PET Imaging and Treatment of Metastatic Melanoma

Malignant melanoma is a highly aggressive cancer, and the incidence of this disease is increasing worldwide at an alarming rate. Despite advances in the treatment of melanoma, patients with metastatic disease still have a poor prognosis and low survival rate. New strategies, including targeted radiotherapy, would provide options for patients who become resistant to therapies such as BRAF inhibitors. Very late antigen-4 (VLA-4) is expressed on melanoma tumor cells in higher levels in more aggressive and metastatic disease and may provide an ideal target for drug delivery and targeted radiotherapy. In this study, we evaluated ¹⁷⁷Lu- and ⁶⁸Ga-labeled DOTA-PEG₄-LLP2A as a VLA-4-targeted radiotherapeutic with a companion PET agent for diagnosis and monitoring metastatic melanoma treatment. DOTA-PEG₄-LLP2A was synthesized by solid-phase synthesis. The affinity of ¹⁷⁷Lu- and ⁶⁸Ga-labeled DOTA-PEG₄-LLP2A to VLA-4 was determined in B16F10 melanoma cells by saturation binding and competitive binding assays, respectively. Biodistribution of the LLP2A conjugates was determined in C57BL/6 mice bearing B16F10 subcutaneous tumors, while PET/CT imaging was performed in subcutaneous and metastatic models. ¹⁷⁷Lu-DOTA-PEG₄-LLP2A showed high affinity to VLA-4 with a <i>K</i>_d of 4.1 ± 1.5 nM and demonstrated significant accumulation in the B16F10 melanoma tumor after 4 h (31.5 ± 7.8%ID/g). The tumor/blood ratio of ¹⁷⁷Lu-DOTA-PEG₄-LLP2A was highest at 24 h (185 ± 26). PET imaging of metastatic melanoma with ⁶⁸Ga-DOTA-PEG₄-LLP2A showed high uptake in sites of metastases and correlated with bioluminescence imaging of the tumors. These data demonstrate that ¹⁷⁷Lu-DOTA-PEG₄-LLP2A has potential as a targeted therapeutic for treating melanoma as well as other VLA-4-expressing tumors. In addition, ⁶⁸Ga-DOTA-PEG₄-LLP2A is a readily translatable companion PET tracer for imaging of metastatic melanoma

Cu-64-Labeled Phosphonate Cross-Bridged Chelator Conjugates of c(RGDyK) for PET/CT Imaging of Osteolytic Bone Metastases

Objective: The goal of this research was to evaluate c(RGDyK) conjugated to phosphonate-based cross-bridged chelators using Cu-free click chemistry in the 4T1 mouse mammary tumor bone metastasis model in comparison with Cu-64-CB-TE2A-c(RGDyK), which previously showed selective binding to integrin v3 on osteoclasts. Experimental: Two phosphonate-based cross-bridged chelators (CB-TE1A1P and CB-TE1K1P) were conjugated to c(RGDyK) through bio-orthogonal strain-promoted alkyne-azide cycloaddition. In vitro and in vivo evaluation of the Cu-64-labeled TE1A1P-DBCO-c(RGDyK) (AP-c(RGDyK)), TE1K1P-PEG4-DBCO-c(RGDyK) (KP-c(RGDyK)), and CB-TE2A-c(RGDyK) were compared in the 4T1 mouse model of bone metastasis. The affinities of the unconjugated and chelator-c(RGDyK) analogs for v3 integrin were determined using a competitive-binding assay. For in vivo evaluation, BALB/c mice were injected with 1x10(5) 4T1/Luc cells in the left ventricle. Formation of metastases was monitored by bioluminescence imaging (BLI) followed by small-animal PET/CT 2 h postinjection of radiotracers. Results: The chelator-peptide conjugates showed similar affinity to integrin v3, in the low nM range. PET imaging demonstrated a higher uptake in bones having metastases for all Cu-64-labeled c(RGDyK) analogs compared with bones in nontumor-bearing mice. The correlation between uptake of Cu-64-AP-c(RGDyK) and Cu-64-KP-c(RGDyK) in bones with metastases based on PET/CT imaging, and osteoclast number based on histomorphometry, was improved over the previously investigated Cu-64-CB-TE2A-c(RGDyK). Conclusion: These data suggest that the phosphonate chelator conjugates of c(RDGyK) peptides are promising PET tracers suitable for imaging tumor-associated osteoclasts in bone metastases

α-Synuclein Radiotracer Development and In Vivo Imaging: Recent Advancements and New Perspectives

α-Synucleinopathies including idiopathic Parkinson's disease, dementia with Lewy bodies and multiple systems atrophy share overlapping symptoms and pathological hallmarks. Selective neurodegeneration and Lewy pathology are the main hallmarks of α-synucleinopathies. Currently, there is no imaging biomarker suitable for a definitive early diagnosis of α-synucleinopathies. Although dopaminergic deficits detected with single-photon emission computed tomography (SPECT) and positron emission tomography (PET) radiotracers can support clinical diagnosis by confirming the presence of dopaminergic neurodegeneration, dopaminergic imaging cannot visualize the preceding disease process, nor distinguish α-synucleinopathies from tauopathies with dopaminergic neurodegeneration, especially at early symptomatic disease stage when clinical presentation is often overlapping. Aggregated α-synuclein (αSyn) could be a suitable imaging biomarker in α-synucleinopathies, because αSyn aggregation and therefore, Lewy pathology is evidently an early driver of α-synucleinopathies pathogenesis. Additionally, several antibodies and small molecule compounds targeting aggregated αSyn are in development for therapy. However, there is no way to directly measure if or how much they lower the levels of aggregated αSyn in the brain. There is clearly a paramount diagnostic and therapeutic unmet medical need. To date, aggregated αSyn and Lewy pathology inclusion bodies cannot be assessed ante-mortem with SPECT or PET imaging because of the suboptimal binding characteristics and/or physicochemical properties of current radiotracers. The aim of this narrative review is to highlight the suitability of aggregated αSyn as an imaging biomarker in α-synucleinopathies, the current limitations with and lessons learned from αSyn radiotracer development, and finally to propose antibody-based ligands for imaging αSyn aggregates as a complementary tool rather than an alternative to small molecule ligands. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society