In vivo high-affinity uptake and axonal transport of D-[2, 3-3H]aspartate in excitatory neurons

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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A High-Affinity Human Antibody That Targets Tumoral Blood Vessels

Blood ◽

10.1182/blood.v94.1.192.413k22_192_198 ◽

1999 ◽

Vol 94 (1) ◽

pp. 192-198 ◽

Cited By ~ 141

Author(s):

Lorenzo Tarli ◽

Enrica Balza ◽

Francesca Viti ◽

Laura Borsi ◽

Patrizia Castellani ◽

...

Keyword(s):

Blood Vessels ◽

Small Cell Lung Carcinoma ◽

Antibody Fragment ◽

Human Colon ◽

Experimental Models ◽

Human Antibody ◽

Cell Lung Carcinoma ◽

High Affinity ◽

Biodistribution Studies

Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 μg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of angiogenesis in patients.

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Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽

10.1182/blood-2003-07-2334 ◽

2004 ◽

Vol 103 (4) ◽

pp. 1356-1363 ◽

Cited By ~ 30

Author(s):

Barbara P. Schick ◽

David Maslow ◽

Adrianna Moshinski ◽

James D. San Antonio

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Tandem Repeats ◽

Factor Xa ◽

Low Molecular Weight ◽

Heparin Binding ◽

High Affinity ◽

Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

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112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0112 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A124-A124

Author(s):

Letizia Giardino ◽

Ryan Gilbreth ◽

Cui Chen ◽

Erin Sult ◽

Noel Monks ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Single Chain ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Animal Care ◽

Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

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C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

Science Advances ◽

10.1126/sciadv.abg3013 ◽

2021 ◽

Vol 7 (15) ◽

pp. eabg3013

Author(s):

Laura Fumagalli ◽

Florence L. Young ◽

Steven Boeynaems ◽

Mathias De Decker ◽

Arpan R. Mehta ◽

...

Keyword(s):

Axonal Transport ◽

Motor Neurons ◽

Single Molecule ◽

Repeat Expansion ◽

Physical Interaction ◽

Hexanucleotide Repeat ◽

Hexanucleotide Repeat Expansion ◽

Axonal Trafficking ◽

Inhibitory Interactions

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell–derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

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The structural basis for high‐affinity uptake of lignin‐derived aromatic compounds by proteobacterial TRAP transporters

FEBS Journal ◽

10.1111/febs.16156 ◽

2021 ◽

Author(s):

Claudine Bisson ◽

Robert C. Salmon ◽

Laura West ◽

John B. Rafferty ◽

Andrew Hitchcock ◽

...

Keyword(s):

Aromatic Compounds ◽

Structural Basis ◽

High Affinity ◽

High Affinity Uptake

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Design of PSMA ligands with modifications at the inhibitor part: an approach to reduce the salivary gland uptake of radiolabeled PSMA inhibitors?

EJNMMI Radiopharmacy and Chemistry ◽

10.1186/s41181-021-00124-1 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Veronika Barbara Felber ◽

Manuel Amando Valentin ◽

Hans-Jürgen Wester

Keyword(s):

Salivary Gland ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

In Vivo Studies ◽

Tumor Xenograft ◽

Tumor Uptake ◽

Lncap Cells ◽

High Affinity

Abstract Aim To investigate whether modifications of prostate-specific membrane antigen (PSMA)-targeted radiolabeled urea-based inhibitors could reduce salivary gland uptake and thus improve tumor-to-salivary gland ratios, several analogs of a high affinity PSMA ligand were synthesized and evaluated in in vitro and in vivo studies. Methods Binding motifs were synthesized ‘on-resin’ or, when not practicable, in solution. Peptide chain elongations were performed according to optimized standard protocols via solid-phase peptide synthesis. In vitro experiments were performed using PSMA+ LNCaP cells. In vivo studies as well as μSPECT/CT scans were conducted with male LNCaP tumor xenograft-bearing CB17-SCID mice. Results PSMA ligands with A) modifications within the central Zn2+-binding unit, B) proinhibitor motifs and C) substituents & bioisosteres of the P1′-γ-carboxylic acid were synthesized and evaluated. Modifications within the central Zn2+-binding unit of PSMA-10 (Glu-urea-Glu) provided three compounds. Thereof, only natLu-carbamate I (natLu-3) exhibited high affinity (IC50 = 7.1 ± 0.7 nM), but low tumor uptake (5.31 ± 0.94% ID/g, 1 h p.i. and 1.20 ± 0.55% ID/g, 24 h p.i.). All proinhibitor motif-based ligands (three in total) exhibited low binding affinities (> 1 μM), no notable internalization and very low tumor uptake (< 0.50% ID/g). In addition, four compounds with P1′-ɣ-carboxylate substituents were developed and evaluated. Thereof, only tetrazole derivative natLu-11 revealed high affinity (IC50 = 16.4 ± 3.8 nM), but also this inhibitor showed low tumor uptake (3.40 ± 0.63% ID/g, 1 h p.i. and 0.68 ± 0.16% ID/g, 24 h p.i.). Salivary gland uptake in mice remained at an equally low level for all compounds (between 0.02 ± 0.00% ID/g and 0.09 ± 0.03% ID/g), wherefore apparent tumor-to-submandibular gland and tumor-to-parotid gland ratios for the modified peptides were distinctly lower (factor 8–45) than for [177Lu]Lu-PSMA-10 at 24 h p.i. Conclusions The investigated compounds could not compete with the in vivo characteristics of the EuE-based PSMA inhibitor [177Lu]Lu-PSMA-10. Although two derivatives (3 and 11) were found to exhibit high affinities towards LNCaP cells, tumor uptake at 24 h p.i. was considerably low, while uptake in salivary glands remained unaffected. Optimization of the established animal model should be envisaged to enable a clear identification of PSMA-targeting radioligands with improved tumor-to-salivary gland ratios in future studies.

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