Generation of glycan-specific nanobodies

Thiol-mediated Uptake of a Cysteine-containing Nanobody for Anti-Cancer Drug Delivery

“…CB2 recognizes several B cell lymphoma cell lines but not healthy lymphocytes As part of a previous study for Nb generation 20 , we screened several Nb candidates for binding to antigens we had used to immunize an alpaca. In parallel, binding to several B cell lymphoma cell lines was tested by flow cytometry.…”

“…We show that CB2 binding and internalization correlate with higher surface levels of reduced cysteines on lymphoma cells compared to healthy lymphocytes and that CB2 can be easily functionalized for different applications. As part of a previous study for Nb generation 20 , we screened several Nb candidates for binding to antigens we had used to immunize an alpaca. In parallel, binding to several B cell lymphoma cell lines was tested by flow cytometry.…”

Microarray-guided evaluation of the frequency, B-cell origins, and selectivity of human glycan-binding antibodies reveals new insights and novel antibodies

“…Recent examples include the development of smart anti-glycan reagents (SAGRs) from lamprey lymphocyte receptors, nanobodies from alpacas, and phage-display derived anti-heparan sulfate single chain antibody fragment (scFv) and anti-Tn scFv. (21)(22)(23)(24) While these are promising tools, the complexities of glycan structure, inherently weak protein-glycan monovalent interactions, and limited availability to high purity glycans for selection procedures present unmet challenges in the field. To complicate matters, many of the commercially available anti-glycan monoclonal antibodies are proprietary clones or hybridoma supernatants, lacking both sequence information and affinity and selectivity characterization.…”

“…Alternative routes, such as in vitro selection platforms, have had some success in expanding the glycobiology toolbox with synthetic antibodies. Recent examples include the development of smart antiglycan reagents from lamprey lymphocyte receptors, nanobodies from alpacas, and phage display–derived antiheparan sulfate single-chain variable antibody fragment and anti-Tn single-chain variable antibody fragment ( 21 , 22 , 23 , 24 ). While these are promising tools, the complexities of glycan structure, inherently weak protein–glycan monovalent interactions, and limited availability to high-purity glycans for selection procedures present unmet challenges in the field.…”

Engineered Glycan-Binding Proteins for Recognition of the Thomsen–Friedenreich Antigen and Structurally Related Disaccharides

“…Due to the drawbacks to antibody elicitation and lectin discovery, there have been numerous attempts to generate novel GBPs with higher affinities or different specificities by engineering existing GBPs. , These attempts have employed techniques such as mutation of a single amino acid to increase affinity to the native ligand or more complicated directed-evolution approaches to pivot the specificity of the GBP to structurally distinct sugars. − Additionally, type B lamprey variable lymphocyte receptors (VLRBs) have shown promise as glycan recognition reagents. , This scaffold, referred to as a “lambody” (Figure ), is a leucine-rich repeat protein that is part of the lamprey adaptive immune response, and glycan-binding properties have been engineered by directed evolution of naïve libraries of VLRBs using yeast surface display or by elicitation in lampreys. − Recently, nanobody (see Figure ) generation using a camelid immunization strategy in alpaca to elicit GBPs against the Globo-H glycan antigen has also established a complementary approach for the development of GBPs …”