RESUMEN
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.
Asunto(s)
Antígenos CD/genética , Epítopos/inmunología , Proteínas de Punto de Control Inmunitario/genética , Tolerancia Inmunológica , Linfoma/genética , Monocitos/inmunología , Receptores Inmunológicos/genética , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Antígenos CD/inmunología , Línea Celular Tumoral , Proliferación Celular , Mapeo Epitopo , Epítopos/química , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Xenoinjertos , Humanos , Proteínas de Punto de Control Inmunitario/inmunología , Linfoma/inmunología , Linfoma/mortalidad , Linfoma/patología , Ratones , Monocitos/citología , Biblioteca de Péptidos , Cultivo Primario de Células , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/inmunología , Análisis de Supervivencia , Linfocitos T/citología , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Linfocitos B/inmunología , Inmunoglobulina G/metabolismo , Animales , Anticuerpos Biespecíficos/genética , Sitios de Unión/genética , Antígenos CD40/inmunología , Proliferación Celular , Células Cultivadas , Humanos , Inmunoglobulina G/genética , Ligando OX40/inmunología , Unión Proteica , Ingeniería de Proteínas , Transducción de Señal , Anticuerpos de Cadena Única/genéticaRESUMEN
The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.
