Development and characterization of narsoplimab, a selective MASP-2 inhibitor, for the treatment of lectin-pathway-mediated disorders.

Introduction: Overactivation of the lectin pathway of complement plays a pathogenic role in a broad range of immune-mediated and inflammatory disorders; mannan-binding lectin-associated serine protease-2 (MASP-2) is the key effector enzyme of the lectin pathway. We developed a fully human monoclonal antibody, narsoplimab, to bind to MASP-2 and specifically inhibit lectin pathway activation. Herein, we describe the preclinical characterization of narsoplimab that supports its evaluation in clinical trials.

Mannan binding lectin-associated serine protease-2 (MASP-2) critically contributes to post-ischemic brain injury independent of MASP-1.

Background: Complement activation via the lectin activation pathway (LP) has been identified as the key mechanism behind post-ischemic tissue inflammation causing ischemia-reperfusion injury (IRI) which can significantly impact the clinical outcome of ischemic disease. This work defines the contributions of each of the three LP-associated enzymes-mannan-binding lectin-associated serine protease (MASP)-1, MASP-2, and MASP-3-to ischemic brain injury in experimental mouse models of stroke.

Methods: Focal cerebral ischemia was induced in wild-type (WT) mice or mice deficient for defined complement components by transient middle cerebral artery occlusion (tMCAO) or three-vessel occlusion (3VO).

Antibody discovery ex vivo accelerated by the LacO/LacI regulatory network.

Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo.

RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions.

Background: Gene conversion depends upon the same factors that carry out more general process of homologous recombination, including homologous gene targeting and recombinational repair. Among these are the RAD51 paralogs, conserved factors related to the key recombination factor, RAD51. In chicken and other fowl, gene conversion (templated mutation) diversifies immunoglobulin variable region sequences.

Genetic variation stimulated by epigenetic modification.

Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination.

E2A acts in cis in G1 phase of cell cycle to promote Ig gene diversification.

Rearranged Ig genes undergo diversification in sequence and structure initiated by the DNA deaminase, activation-induced deaminase. Ig genes must be transcribed for diversification to occur, but whether there are additional requirements for cis activation has not been established. Here we show, by chromatin immunoprecipitation, that the regulatory factor E2A associates with the rearranged Ig lambda(R) gene in the chicken DT40 B cell line, which performs constitutive Ig gene diversification.

Chromatin structure regulates gene conversion.

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors.

MRE11/RAD50 cleaves DNA in the AID/UNG-dependent pathway of immunoglobulin gene diversification.

MRE11/RAD50/NBS1 (MRN) is a ubiquitous complex that participates in the response to DNA damage and in immunoglobulin (Ig) gene diversification. Ig gene diversification is initiated by deamination of cytosine to uracil, followed by removal of uracil to create an abasic (AP) site. We find that MRE11 associates specifically with rearranged Ig genes in hypermutating B cells, whereas APE1, the major AP-endonuclease in faithful base excision repair, does not.

Formation of a large, complex domain of histone hyperacetylation at human 14q32.1 requires the serpin locus control region.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system to study cell-type-specific gene expression and chromatin structure. Activation of the serpin locus can be induced in vitro by transferring human chromosome 14 from non-expressing to expressing cells.

MutSalpha binds to and promotes synapsis of transcriptionally activated immunoglobulin switch regions.

Immunoglobulin class switch recombination joins a new constant (C) region to the rearranged and expressed heavy chain variable (VDJ) region in antigen-activated B cells (Figure 1A) (reviewed in [1, 2]). Switch recombination is activated by transcription of intronic, G-rich and repetitive switch (S) regions and produces junctions that are heterogeneous in sequence and position in the S regions. Switch recombination depends upon the B cell-specific cytidine deaminase, AID, and conserved DNA repair factors, including the mismatch repair heterodimer, MutSalpha (MSH2/MSH6).

The Coprinus cinereus adherin Rad9 functions in Mre11-dependent DNA repair, meiotic sister-chromatid cohesion, and meiotic homolog pairing.

Mitotic sister-chromatid cohesion (SCC) is known to depend in part on conserved proteins called adherins, which although necessary for SCC are not themselves localized between sister chromatids. We have examined mitotic DNA-repair and meiotic chromosome behavior in the Coprinus cinereus adherin mutant rad9-1. Genetic pathway analysis established that Rad9 functions in an Mre11-dependent pathway of DNA repair.

Evidence for a three-nucleon-force effect in proton-deuteron elastic scattering.

Developments in spin-polarized internal targets for storage rings have permitted measurements of 197 MeV polarized protons scattering from vector polarized deuterons. This work presents measurements of the polarization observables A(y), iT11, and C(y,y) in proton-deuteron elastic scattering. When compared to calculations with and without three-nucleon forces, the measurements provide further evidence that three-nucleon forces make a contribution to the observables.

Replication-dependent early meiotic requirement for Spo11 and Rad50.

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S.

Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants.

We have constructed a dominant selectable marker, PHT1, for transformation of the basidiomycete Coprinus cinereus. PHT1 consists of a bacterial hygromycin B resistance gene fused to the promoter and terminator regions of the C. cinereus beta-tubulin gene.

Functions of DNA repair genes during meiosis.

One of the most basic functions in any organism is DNA repair. In addition, programmed DNA "damage," in the form of DNA double-strand breaks (DSBs), is a regular part of the physiology of most organisms. There are three main types of DSB repair: homologous recombination; single-strand annealing; and nonhomologous end joining.

The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis.

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C.