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Background Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo. Methods In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody’s therapeutic potential for targeting IL25 in inflammation. Results A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule. Conclusions The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.

Receptor for advanced glycation end-products (RAGE) detects nucleic acids and promotes DNA uptake into endosomes, which in turn lowers the immune recognition threshold for TLR9 activation., Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.

The increasing incidence of Klebsiella pneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K. pneumoniae Several monoclonal antibodies were isolated from phage display and hybridoma platforms by functional screening for opsonophagocytic killing activity. We further identified their common target antigen to be MrkA, a major protein in the type III fimbriae complex, and showed that these serotype-independent anti-MrkA antibodies reduced biofilm formation in vitro and conferred protection in multiple murine pneumonia models. Importantly, mice immunized with purified MrkA proteins also showed reduced bacterial burden following K. pneumoniae challenge. Taken together, these results support MrkA as a promising target for K. pneumoniae antibody therapeutics and vaccines.

Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the β4–β5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I MHC recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49GB6 and a Ly-49GB6-like receptor, Ly-49Mnonobese diabetic, but not Ly-49GBALB/c. By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the β4–β5 loop and the β1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49GB6 ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the β4–β5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd α1/α2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.

Classical class I major histocompatibility complex (MHC) molecules, as well as the nonclassical class I histocompatibility leukocyte antigen (HLA)-E molecule, can negatively regulate natural killer (NK) cell cytotoxicity through engagement of NK inhibitory receptors. We show that expression of murine (m)CD1.1, a nonpolymorphic nonclassical MHC class I–like molecule encoded outside the MHC, protects NK-sensitive RMA/S target cells from adherent lymphokine-activated killer cell (A-LAK) cytotoxicity. Passage of effector cells in recombinant interleukin (rIL)-2 enhanced protection by mCD1.1, suggesting an expansion of relevant A-LAK population(s) or modulation of A-LAK receptor expression. Murine CD1.1 conferred protection from lysis by rIL-2–activated spleen cells of recombination activating gene (Rag)-1−/− mice, which lack B and T cells, demonstrating that mCD1.1 can protect RMA/S cells from lysis by NK cells. An antibody specific for mCD1.1 partially restored A-LAK lysis of RMA/S.CD1.1 transfectants, indicating that cell surface mCD1.1 can confer protection from lysis; therefore, mCD1.1 possibly acts through interaction with an NK inhibitory receptor. CD1.1 is by far the most divergent class I molecule capable of regulating NK cell activity. Finally, mCD1.1 expression rendered RMA/S cells resistant to lysis by A-LAK of multiple mouse strains. The conserved structure of mCD1.1 and pattern of mCD1.1 resistance from A-LAK lysis suggest that mCD1.1 may be a ligand for a conserved NK inhibitory receptor.

Murine class I molecules are ligands for Ly-49 molecules, a family of regulatory receptors expressed on murine NK cells. Since soluble sulfated mono- and polysaccharides interfere with the interaction of Ly-49A, a C-type lectin, and its class I ligand, Dd, it is possible that the oligosaccharides on class I molecules are sulfated and participate in Ly-49A binding. In this report, we show that H-2Dd expressed by activated T cells and various tumor cell lines is sulfated, as demonstrated by immunoprecipitation of Dd following Na235SO4 labeling. The 35SO4−2 label on Dd expressed by a representative tumor cell, NZB1.1, is removed by peptide N-glycosidase F, but is resistant to endoglycosidase H treatment, indicating that the sulfate group is located on mature N-linked oligosaccharides. Two-dimensional SDS-PAGE analysis revealed that all major mature glycosylation variants of the Dd expressed by NZB1.1 are sulfated. Sodium chlorate, a potent inhibitor of ATP-sulfurylase, which prevents the formation of the sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate, inhibited metabolic sulfation of Dd. NZB1.1 binds isolated Ly-49A immobilized on solid phase through an interaction by cell surface Dd, since cell adhesion was blocked by Abs directed against Dd or Ly-49A. Treatment of the Dd-expressing NZB1.1 tumor cells with sodium chlorate reduced their ability to bind immobilized Ly-49A, particularly when Ly-49A density was limiting. These results provide evidence for sulfation of H-2Dd oligosaccharide moieties, and suggest a role for this posttranslational modification in the interaction of Dd with Ly-49A.

Ly-49A molecules negatively regulate a subset of mouse natural killer (NK) cells, preventing lysis of H-2Dd-expressing target cells. In the present report, we immunoaffinity-purified Ly-49A from the EL4 lymphoma using the A1 monoclonal antibody (mAb) and examined cell adhesion to immobilized Ly-49A. Adhesion was observed by cells expressing relatively high levels of H-2Dd, but not cells expressing very low or no cell surface Dd, while antibodies specific for Dd or Ly-49A inhibited the cell binding, indicating that Dd and Ly-49A mediate the observed adhesion. The density of immobilized Ly-49A was varied and confirmed by ELISA. Cell binding exhibited a threshold Ly-49A density requirement, and above this threshold, increases in Ly-49A density resulted in substantial increases in cell adhesion to a high maximum cell binding. The density of Ly-49A homodimers required to mediate cell adhesion was found to be quite low: 140-250 molecules/microm2. These results suggest that the avidity of Ly-49A for Dd is relatively high and indicate that small changes in Ly-49A density near the threshold result in large changes in stable Ly-49A receptor engagement. The relatively sharp threshold and marked density dependence presented here for Ly-49A receptor engagement may explain the observation that relatively small differences in Ly-49A expression level on NK cells result in significant differences in functional outcome, i.e. whether a target cell expressing a low level of Dd is spared from lysis or not.