(E)-4-Hydroxy-3-methyl-but-2-enyl
diphosphate
(HMBPP) and its phosphonate analogs are potent phosphoantigens. HMBPP
contains an (E)-allylic alcohol which interacts with
the molecular target BTN3A1 giving an antigenic signal to activate
Vγ9Vδ2 T cells. As probes of BTN3A1 function, we prepared
prodrug derivatives of the HMBPP analog C-HMBP that lack the (E)-allylic alcohol or have modified it to an aldehyde or
aldoxime and evaluated their biological activity. Removal of the alcohol
completely abrogates phosphoantigenicity in these compounds while
the aldoxime modification decreases potency relative to the (E)-allylic alcohol form. However, homoprenyl derivatives
oxidized to an aldehyde stimulate Vγ9Vδ2 T cells at nanomolar
concentrations. Selection of phosphonate protecting groups (i.e.,
prodrug forms) impacts the potency of phosphoantigen aldehydes, with
mixed aryl acyloxyalkyl forms exhibiting superior activity relative
to aryl amidate forms. The activity correlates with the cellular reduction
of the aldehyde to the alcohol form. Thus, the functionality on this
ligand framework can be altered concurrently with phosphonate protection
to promote cellular transformation to highly potent phosphoantigens.
Phage display is a powerful platform for the discovery of novel biologics with high binding affinities to a specific target protein. Here, we describe methods to construct a phage display library containing diverse single-chain variable antibody fragments (scFvs). Specifically, updated methods for polymerase chain reaction (PCR) amplification and fusion of human antibody genes, their ligation into the pComb3X vector for transformation into 5αF I q competent bacterial cells, and their expression in M13KO7 helper phage are presented. Additionally, we describe how to amplify and quantify the phage library and to prepare it in various formats for short-and long-term storage.
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