Creation and evaluation of a single‐chain antibody tetramer that targets brain endothelial cells

Zhang, Xiaobin; Wang, Xin Xiang; Shusta, Eric V.

doi:10.1002/aic.14348

Cited by 7 publications

(5 citation statements)

References 35 publications

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“…Through biotinylation in vivo using biotin ligase, we produced a 47VHH1H4B variant bearing single C-terminal biotin and assembled a tetravalent streptabody based on it. This molecule is anticipated to have an extended half-life due to its size exceeding the glomerular filtration barrier, and the presence of four nanobodies in its composition should enhance its affinity for CD47 owing to avidity [ 42 ]. Furthermore, the dissociation constant (Kd) measurements for 47VHH1H4 and 47VHH1H4B revealed that BAD-containing forms had slightly higher affinities for CD47 regardless of their biotinylation status.…”

Section: Discussionmentioning

confidence: 99%

A Novel Anti-CD47 Nanobody Tetramer for Cancer Therapy

Ratnikova,

Kravchenko,

Ivanova

et al. 2024

Antibodies

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CD47 acts as a defense mechanism for tumor cells by sending a “don’t eat me” signal via its bond with SIRPα. With CD47’s overexpression linked to poor cancer outcomes, its pathway has become a target in cancer immunotherapy. Though monoclonal antibodies offer specificity, they have limitations like the large size and production costs. Nanobodies, due to their small size and unique properties, present a promising therapeutic alternative. In our study, a high-affinity anti-CD47 nanobody was engineered from an immunized alpaca. We isolated a specific VHH from the phage library, which has nanomolar affinity to SIRPα, and constructed a streptavidin-based tetramer. The efficacy of the nanobody and its derivative was evaluated using various assays. The new nanobody demonstrated higher affinity than the monoclonal anti-CD47 antibody, B6H12.2. The nanobody and its derivatives also stimulated substantial phagocytosis of tumor cell lines and induced apoptosis in U937 cells, a response confirmed in both in vitro and in vivo settings. Our results underscore the potential of the engineered anti-CD47 nanobody as a promising candidate for cancer immunotherapy. The derived nanobody could offer a more effective, cost-efficient alternative to conventional antibodies in disrupting the CD47–SIRPα axis, opening doors for its standalone or combinatorial therapeutic applications in oncology.

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Section: Discussionmentioning

confidence: 99%

A Novel Anti-CD47 Nanobody Tetramer for Cancer Therapy

Ratnikova,

Kravchenko,

Ivanova

et al. 2024

Antibodies

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“…Although it is not clear why such a high tag to streptavidin ratio (30:1) is required to drive most of SAVSBPM32 into complexes with two SBP(A18C) tags bound per streptavidin tetramer, this requirement has also been shown in related studies. In a study to create tetramerized single-chain antibody (SCA) complexes using streptavidin as the tetramerization agent, mixing biotinylated single-chain antibodies with streptavidin in a 8:1 ratio only leads to a population of complexes where the majority of complexes have three single-chain antibodies per streptavidin [ 45 ]. When using biotinylated DNA as a binding ligand, the majority of streptavidin complexes have two to three DNA fragments per streptavidin even when the DNA:streptavidin ratio is five [ 46 ].…”

Section: Discussionmentioning

confidence: 99%

Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

Fogen

et al. 2015

PLoS ONE

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To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.

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“…Fusions of these scFv’s to the 202–08 intein in the pCTre vector, were transfected into EBY100 yeast cells as described previously . Transfected cells were grown at 30 °C in SD–CAA medium (20.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0 g/L casamino acids, 10.19 g/L Na 2 HPO 4 ·7 H 2 O, 8.56 g/L NaH 2 PO 4 ·H 2 O) as described previously. ,, Cultures were induced with SG–CAA medium, that is identical to SD-CAA medium except dextrose is substituted for galactose at an OD 600 nm of 0.8–0.9. Cultures were induced for 48 h at 20 °C before harvesting the scFv from the yeast surface.…”

Section: Methodsmentioning

confidence: 99%

Site-Specific Antibody Functionalization Using Tetrazine–Styrene Cycloaddition

et al. 2018

Self Cite

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Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.

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Creation and evaluation of a single‐chain antibody tetramer that targets brain endothelial cells

Cited by 7 publications

References 35 publications

A Novel Anti-CD47 Nanobody Tetramer for Cancer Therapy

A Novel Anti-CD47 Nanobody Tetramer for Cancer Therapy

Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

Site-Specific Antibody Functionalization Using Tetrazine–Styrene Cycloaddition

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