Dissolution of arterial platelet thrombi in vivo with a bifunctional platelet GPIIIa49-66 ligand which specifically targets the platelet thrombus

Abstract Patients with HIV-1 immune-related thrombocytopenia have a unique antibody (Ab) against integrin GPIIIa49-66 capable of inducing oxidative platelet fragmentation via Ab activation of platelet nicotinamide adenine dinucleotide phosphate oxidase and 12-lipoxygenase releasing reactive oxygen species. Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11) capable of inducing fragmentation of activated platelets. In this study, we investigated the in vivo use of A11. We show that A11 does not induce significant thrombocytopenia or inhibit platelet function. A11 can prevent the cessation of carotid artery flow produced by induced artery injury and dissolve the induced thrombus 2 hours after cessation of blood flow. In addition, A11 can prevent, as well as ameliorate, murine middle cerebral artery stroke, without thrombocytopenia or brain hemorrhage. To further optimize the antithrombotic activity of A11, we produced a bifunctional A11-plasminogen first kringle agent (SLK), which homes to newly deposited fibrin strands within and surrounding the platelet thrombus, reducing effects on nonactivated circulating platelets. Indeed, SLK is able to completely reopen occluded carotid vessels 4 hours after cessation of blood flow, whereas A11 had no effect at 4 hours. Thus, a new antithrombotic agent was developed for platelet thrombus clearance.

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Synergistic Effects of Bifunctional Antibodies Against beta3 Integrin on Dissolution of Platelet Thrombus,

Blood ◽

10.1182/blood.v118.21.3350.3350 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3350-3350

Author(s):

Wei Zhang ◽

Suying Dang ◽

Thomas Wisniewski

Keyword(s):

Conflicts Of Interest ◽

Ex Vivo ◽

Synergistic Effects ◽

Fibrin Clot ◽

Clot Lysis ◽

Single Chain ◽

Single Chain Antibody ◽

Platelet Thrombus ◽

Platelet Aggregates

Abstract Abstract 3350 HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo. Disclosures: No relevant conflicts of interest to declare.

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A single-chain antibody generation system yielding CAR-T cells with superior antitumor function

Communications Biology ◽

10.1038/s42003-021-01791-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Toshiki Ochi ◽

Masaki Maruta ◽

Kazushi Tanimoto ◽

Fumitake Kondo ◽

Toshihiro Yamamoto ◽

...

Keyword(s):

T Cells ◽

Variable Region ◽

Target Cells ◽

Single Chain ◽

Generation System ◽

Single Chain Antibody ◽

Car T Cells ◽

Car T ◽

Scfv Library

AbstractCancer immunotherapy using T cells redirected with chimeric antigen receptor (CAR) has shown a lot of promise. We have established a single-chain antibody (scFv) generation system in which scFv library-expressing CAR-T cells can be screened appropriately based on their antitumor functions. A variable region library containing the variable and J regions of the human immunoglobulin light or heavy chain was fused with the variable region of a heavy or light chain encoded by an existing tumor-specific antibody to generate a new scFv library. Then, scFv library-expressing CAR-T cells were generated and stimulated with target cells to concentrate the antigen-specific population. Using this system, target-specific recognition of CAR-T cells appeared to be finely tuned by selecting a new variable region. Importantly, we have demonstrated that the newly optimized scFv-expressing CAR-T cells had better proliferation capacity and durable phenotypes, enabling superior reactivity against advanced tumors in vivo in comparison with the original CAR-T cells. Therefore, the optimization of an scFv is needed to maximize the in vivo antitumor functions of CAR-T cells. This system may allow us to adjust an immunological synapse formed by an scFv expressed by CAR-T cells and a target antigen, representing an ideal form of CAR-T-cell immunotherapy.

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Llama-Derived Single-Chain Antibody Fragments Directed to Rotavirus VP6 Protein Possess Broad Neutralizing Activity In Vitro and Confer Protection against Diarrhea in Mice

Journal of Virology ◽

10.1128/jvi.00436-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9753-9764 ◽

Cited By ~ 74

Author(s):

Lorena Garaicoechea ◽

Aurelien Olichon ◽

Gisela Marcoppido ◽

Andrés Wigdorovitz ◽

Marina Mozgovoj ◽

...

Keyword(s):

Capsid Protein ◽

Binding Capacity ◽

Antibody Fragments ◽

Target Antigen ◽

Single Chain ◽

Single Chain Antibody ◽

Group A Rotavirus ◽

Group A

ABSTRACT Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.

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Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors

Blood ◽

10.1182/blood-2002-04-1041 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3155-3163 ◽

Cited By ~ 107

Author(s):

Nicole M. Haynes ◽

Joseph A. Trapani ◽

Michèle W. L. Teng ◽

Jacob T. Jackson ◽

Loretta Cerruti ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Escape ◽

Single Chain ◽

Single Chain Antibody ◽

Practical Advantage ◽

Established Tumor ◽

Single Chain Fv ◽

Primary Mouse

Abstract Tumor cells are usually weakly immunogenic as they largely express self-antigens and can down-regulate major histocompatability complex/peptide molecules and critical costimulatory ligands. The challenge for immunotherapies has been to provide vigorous immune effector cells that circumvent these tumor escape mechanisms and eradicate established tumors. One promising approach is to engineer T cells with single-chain antibody receptors, and since T cells require 2 distinct signals for optimal activation, we have compared the therapeutic efficacy of erbB2-reactive chimeric receptors that contain either T-cell receptor zeta (TCR-ζ) or CD28/TCR-ζ signaling domains. We have demonstrated that primary mouse CD8+ T lymphocytes expressing the single-chain Fv (scFv)–CD28-ζ receptor have a greater capacity to secrete Tc1 cytokines, induce T-cell proliferation, and inhibit established tumor growth and metastases in vivo. The suppression of established tumor burden by cytotoxic T cells expressing the CD28/TCR-ζ chimera was critically dependent upon their interferon gamma (IFN-γ) secretion. Our study has illustrated the practical advantage of engineering a T-cell signaling complex that codelivers CD28 activation, dependent only upon the tumor's expression of the appropriate tumor associated antigen.

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The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo

Journal of Clinical Medicine ◽

10.3390/jcm10225349 ◽

2021 ◽

Vol 10 (22) ◽

pp. 5349

Author(s):

Lydie Crescence ◽

Markus Kramberg ◽

Martine Baumann ◽

Markus Rey ◽

Sebastien Roux ◽

...

Keyword(s):

Blood Flow ◽

Guinea Pigs ◽

Thrombus Formation ◽

P2y12 Receptor ◽

Early Application ◽

Acute Thrombosis ◽

Thrombosis Model ◽

Platelet Thrombus ◽

Platelet Thrombi

Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.

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Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

10.1101/2020.07.27.224089 ◽

2020 ◽

Author(s):

Matthew D. Beasley ◽

Sanja Aracic ◽

Fiona M. Gracey ◽

Ruban Kannan ◽

Avisa Masarati ◽

...

Keyword(s):

Vaccine Development ◽

Spike Protein ◽

Receptor Binding Domain ◽

Single Chain ◽

Single Chain Antibody ◽

High Affinity ◽

Scfv Library ◽

Antibody Libraries ◽

Prophylactic Use

AbstractAntibodies with high affinity against the receptor binding domain (RBD) of the SARS-CoV-2 S1 ectodomain were identified from screens using the Retained Display™ (ReD) platform employing a 1 × 1011 clone single-chain antibody (scFv) library. Numerous unique scFv clones capable of inhibiting binding of the viral S1 ectodomain to the ACE2 receptor in vitro were characterized. To maximize avidity, selected clones were reformatted as bivalent diabodies and monoclonal antibodies (mAb). The highest affinity mAb completely neutralized live SARS-CoV-2 virus in cell culture for four days at a concentration of 6.7 nM, suggesting potential therapeutic and/or prophylactic use. Furthermore, scFvs were identified that greatly increased the interaction of the viral S1 trimer with the ACE2 receptor, with potential implications for vaccine development.

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Effect of an anti-sulfatide single-chain antibody probe on platelet function

Thrombosis and Haemostasis ◽

10.1160/th07-05-0351 ◽

2008 ◽

Vol 99 (03) ◽

pp. 552-557 ◽

Cited By ~ 7

Author(s):

Corie Shrimpton ◽

Koichi Honke ◽

Rolando Rumbaut ◽

Jose Lopez ◽

Perumal Thiagarajan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Thrombus Formation ◽

Lag Phase ◽

Single Chain ◽

Single Chain Antibody ◽

Platelet Surface ◽

Occlusion Time ◽

Single Chain Fragment Variable

SummarySulfatide (galactocylceramide-3'-sulfate), a cell surface glycosphingolipid interacts with several cell adhesion molecules including fibrinogen, von Willebrand factor (VWF), P-selectin, thrombospondin (TSP) and laminin, which are involved in haemostasis.We have used a sulfatide-specific single-chain fragment variable (scFv) antibody probe PA38 and sulfatide-deficient mice to investigate the role of membrane sulfatide in platelet function. PA38 bound to platelets and binding increased following platelet activation. Sulfatide was localized as a large cluster towards the center of the platelet surface when examined in a confocal microscope. PA38 (20 μg/ml) inhibited the adhesion of activated platelets to fibrinogen,VWF, P-selectin,TSP1 and laminin by 30%, 30%,75%,20% and 35%,respectively,compared to a control scFv (p<0.05). Furthermore, PA38 inhibited collagen, ADP, thrombin and ristocetin-induced platelet aggregation in PRP by 25%, 30%, 18% and 20%, respectively, compared to the control scFv (p<0.05). In a PFA-100 platelet function assay, PA38 prolonged the occlusion time by 25% (p<0.05).Under flow PA38 decreased the thrombus formation on collagen by 31%, (p<0.01). Sulfatidedeficient mice displayed an extended lag-phase in collagen-induced platelet aggregation compared to wild type (p<0.05), though in-vivo haemostasis did not differ significantly.Thus, this study provides new evidence for a role for membrane sulfatide in platelet function.

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