In addition to their mismatch recognition activities, bacterial and eukaryotic MutS activities have an associated ATPase activity that is required for function of the proteins in mismatch repair (1-5). Two distinct functions have been proposed for nucleotide binding and hydrolysis by MutS homologs, both of which are based on the effects of ATP on MutS-heteroduplex interaction. The presence of ATP greatly reduces the efficiency of specific complex formation between bacterial MutS or eukaryotic MutS␣ and heteroduplex DNA (5-10), and ATP challenge of preformed MutS⅐heteroduplex complexes has been shown to result in departure of the protein from the mismatch (11). Available information indicates that some of these effects are attributable to ATP binding. Thus, ATP␥S has been shown to promote departure of MutS from the mismatch in heteroduplex DNA (11), while ATP␥S or ATP (in the absence of a divalent cation) reduce the binding efficiency human MutS␣ (hMutS␣) to synthetic heteroduplexes (5, 10).Electron microscopy of complexes between bacterial MutS and large heteroduplexes prepared from natural DNAs has demonstrated that ATP-promoted release of MutS from a mismatch is associated with efficient conversion of protein⅐DNA complexes to ␣-shaped loop structures stabilized by MutS at the base (11). Loop formation requires a mismatch, loop size increases linearly with time, loop growth depends on continued ATP hydrolysis, and the mismatch usually ends up in the loop. These observations have been interpreted in terms of a mechanism in which ATP binding reduces affinity of the protein for a mispair and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (11). MutS movement in this manner has been postulated to be important for the coupling of mismatch recognition to loading of the excision system at the strand break that directs repair (12, 13), a site that can be located hundreds of base pairs from this mismatch.The finding that ATP binding reduces the efficiency of specific complex formation between hMutS␣ and oligonucleotide heteroduplexes has led to proposal of a molecular switch model for action of MutS activities. Like a G-protein, hMutS␣ is postulated to exist in two states, an ADP-bound form that binds with near irreversible affinity to a mismatch and an ATPbound form that does not (10). In this proposal hMutS␣⅐ADP binds to a mispair and recruits downstream activities to this site. After assembly of the excision system, ATP binding results in dissociation of hMutS␣ from the heteroduplex so that repair may proceed (10).To further clarify the role(s) of ATP binding and hydrolysis in hMutS␣ action, we have evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of hMutS␣⅐DNA complexes and have examined the effect of DNA topology on ATP-promoted dissociation of hMutS␣ complexes with small heteroduplexes. We demonstrate that ADP is not required for mismatch recognition by hMutS␣, but...

Background: Unregulated plasma kallikrein proteolytic activity can result from C1-inhibitor deficiency, causing excessive and potentially fatal edema.Results: The antibody DX-2930 potently and specifically inhibits plasma kallikrein and exhibits a long plasma half-life.Conclusion: An antibody protease inhibitor can lead to potent and specific bioactivity.Significance: DX-2930 could be an effective therapeutic for the prophylactic inhibition of plasma kallikrein-mediated diseases.

Mismatch repair corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination, and participates in the cellular response to certain types of DNA damage (1-6). Assembly of MutS and MutL homologues at a mismatch or at a DNA lesion is presumed to be an early event in each of these genetic stabilization functions. In human cells the MutS homologue MSH2 forms heterodimers with MSH6 (7-10) or MSH3 (10, 11), and these activities are responsible for mismatch recognition. The MutS␤ heterodimer (MSH2⅐MSH3) recognizes small insertion/deletion mispairs (9, 10, 12), but MutS␣ (MSH2⅐MSH6) recognizes and supports the repair of both basebase mismatches and insertion/deletion heterologies (7, 12, 13).In the human cell lines that have been examined, the majority of the MSH2 is associated with MSH6 (11,12,14), and the MutS␣ complex is present in a 6-to 10-fold excess over MutS␤.In addition to their mismatch recognition function, MutS homologues contain a highly conserved ATP hydrolytic center near the carboxyl terminus (15). The integrity of this site is required for function in mismatch repair (15-17), and there is extensive evidence indicating that adenine nucleotides modulate the interaction between MutS homologues and DNA. ATP and nonhydrolyzable ATP analogues reduce MutS homologue affinity for a mispair (7,8,(17)(18)(19)(20)(21)(22)(23), and nucleotide challenge of preformed protein⅐heteroduplex complexes provokes MutS homologue release from a mismatch (23-28).Several models for ATPase function in MutS homologue action have been suggested. Two of these invoke ATP-dependent movement of the protein along the helix, which is postulated to link mismatch recognition to activation of downstream events at the strand signal that directs repair (24 -26). Electron microscopic visualization of bacterial MutS⅐heteroduplex complexes has demonstrated the mismatch-and ATP-dependent extrusion of a DNA loop by the bacterial protein (24). Because nonhydrolyzable ATP analogues do not support loop extrusion and because their addition terminates ongoing loop growth, this reaction has been attributed to directional translocation along the helix in a reaction dependent on ATP hydrolysis by the DNA-bound protein. The use of streptavidin end-blocked linear DNAs has also suggested that MutS and MutS␣ leave a mismatch in an ATP-dependent manner, observations that have led to the suggestion that the protein may form a mobile clamp about the helix (22,25,26,29). Although ATP⅐Mg 2ϩ supports the formation of stable complexes on end-blocked heteroduplexes, AMPPNP⅐Mg 2ϩ , 1 ATP␥S⅐Mg 2ϩ , or ATP (no Mg 2ϩ ) do not, suggesting that formation of such intermediates may depend on ATP hydrolysis by heteroduplex-bound MutS␣ (25). The interaction with end-blocked DNA of a mutant form of MutS␣, which supports ATP binding but not hydrolysis, has led to a similar conclusion (27).A mechanism for MutS/MutS␣ movement along the helix that is independent of ATP hydrolysis by DNA-bound protein has also been proposed (26,29). This molecular switch model posi...

The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach – depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases.

The remarkable success of SARS CoV-2 mRNA-based vaccines and the ensuing interest in mRNA vaccines and therapeutics have highlighted the need for a scalable clinical-enabling manufacturing process to produce such products, and robust analytical methods to demonstrate safety, potency, and purity. To date, production processes have either not been disclosed or are bench-scale in nature and cannot be readily adapted to clinical and commercial scale production. To address these needs, we have advanced an aqueous-based scalable process that is readily adaptable to GMP-compliant manufacturing, and developed the required analytical methods for product characterization, quality control release and stability testing. We also have demonstrated the products produced at manufacturing scale under such approaches display good potency and protection in relevant animal models with mRNA products encoding both vaccine immunogens and antibodies. Finally, we discuss continued challenges in raw material identification, sourcing and supply, and the cold chain requirements for mRNA therapeutic and vaccine products. While ultimate solutions have yet to be elucidated, we discuss approaches that can be taken that are aligned with regulatory guidance.

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K D value of 49 and 10 pmol/L, respectively) compared with IGF

KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 microM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.

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