LILRB3 (ILT5) is a myeloid cell checkpoint that elicits profound immunomodulation.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor

A bispecific IgG format containing four independent antigen binding sites

Bispecific antibodies come in many different formats, including the particularly interesting two-in-one antibodies, where one conventional IgG binds two different antigens. The IgG format allows these antibodies to mediate Fc-related functionality, and their wild-type structure ensures low immunogenicity and enables standard methods to be used for development. It is however difficult, time-consuming and costly to generate two-in-one antibodies. Herein we demonstrate a new approach to create a similar type of antibody by combining two different variable heavy (VH) domains in each Fab arm of an IgG, a tetra-VH IgG format. The VHs are used as building blocks, where one VH is placed at its usual position, and the second VH replaces the variable light (VL) domain in a conventional IgG. VH domains, binding several different types of antigens, were discovered and could be rearranged in any combination, offering a convenient "plug and play" format. The tetra-VH IgGs were found to be functionally tetravalent, binding two antigens on each arm of the IgG molecule simultaneously. This offers a new strategy to also create monospecific, tetravalent IgGs that, depending on antigen architecture and mode-of-action, may have enhanced efficacy compared to traditional bivalent antibodies

A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca(2+) influx

Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a γ-carboxyglutamic acid-containing peptide, conotoxin ɛ-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca(2+) channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca(2+) in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional (1)H NMR spectroscopy, identifies an electronegative patch created by the side chains of two γ-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region

LILRB3 (ILT5) is a myeloid cell checkpoint that elicits profound immunomodulation

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor