Human Antibody Inhibitor Research Articles

A humanized antibody inhibitor for cathepsin L

Cathepsin L (CTSL) is a cysteine protease involved in a variety of physiological and pathological processes. Potent inhibitors against CTSL have long been sought for drug development. Due to insufficient specificity and suboptimal pharmacological properties for current CTSL inhibitors, novel agents are still required for selectively blocking CTSL activity. Here we generated a humanized antibody inhibitor of CTSL by genetically fusing the inhibitory propeptide of procathepsin L to the N-terminus of the light chain of a humanized antibody. The resulting antibody fusion could be stably expressed and displays highly potent inhibition activity and specificity toward CTSL. This work demonstrates a new approach for the rapid generation of antibody inhibitors of CTSL. It can possibly be extended to create inhibitory antibodies targeting other cathepsin proteases, providing novel research and therapeutic tools.

Rational Design of a Humanized Antibody Inhibitor of Cathepsin B.

Cathepsin B (CTSB) is an abundant cysteine protease that functions in both endolysosomal compartments and extracellular regions. A considerable number of preclinical and clinical studies indicate that CTSB is implicated in many human diseases. Expression levels and activity of CTSB significantly correlate with disease progression and severity. Current inhibitors of CTSB are lack of adequate specificity and pharmacological activities. Through structure-guided rational design, we hereby designed and generated a humanized antibody inhibitor targeting human CTSB. This was achieved by genetically fusing the propeptide of procathepsin B, a naturally occurring inhibitor of CTSB, into heavy chain complementarity-determining region 3 (CDR3H) of Herceptin that is used in the clinic for the treatment of breast cancer. The resulting antibody-propeptide fusion displayed high specificity for inhibiting CTSB proteolytic activity at nanomolar levels. Pharmacokinetic studies in mice revealed a plasma half-life of approximately 42 h for this anti-CTSB antibody inhibitor, comparable to that of the parental Herceptin scaffold. This study demonstrates a new approach for the efficient generation of humanized antibody inhibitors with high potency and specificity for human CTSB, which may be extended to develop antibody inhibitors against other disease relevant cathepsin proteases.

Discovery and Characterization Of a Highly Specific Antibody Inhibitor Of Plasma Kallikrein

The plasma-kallikrein kinin (contact) system contributes to the physiological and pathophysiological reactions of vascular biology. Activation of this pathway causes the release of the potent nonapeptide vasodilator bradykinin following proteolytic cleavage of high-molecular weight kininogen (HMWK) by the serine protease plasma kallikrein (pKal). Normal vascular homeostasis requires regulation of pKal activity by interactions with the C1-inhibitor (C1-INH). This is most apparent in individuals with hereditary angioedema (HAE), a disease characterized by a genetic deficiency in C1-INH that results in persistent pKal activity and consequent bradykinin release. These events can ultimately manifest as unpredictable and potentially fatal attacks of subcutaneous and mucosal edema. Inhibition of pKal proteolytic activity has proven to be a viable therapeutic option for HAE, however there remains an unmet medical need for a long-lasting prophylactic treatment for this disease. Given the potential for target specificity and long serum half-life with antibody therapeutics, we used phage display to select a fully human antibody inhibitor (DX-2930) specific for pKal. In vitro enzyme inhibition and affinity assays demonstrate that DX-2930 is a potent antibody inhibitor of pKal (Ki = 125 pM) that binds the active form of pKal, but not the proenzyme form (prekallikrein) or any other serine protease tested. DX-2930 binding consequently prohibits pKal from cleaving bradykinin out of HMWK and thereby prevents the activation of the bradykinin receptor B2. A 2.1Å resolution X-ray crystallographic structure of pKal complexed to a DX-2930 Fab construct supports these findings, demonstrating that the pKal proteolytic active site is intimately bound - and thereby occluded - by the Fab. This structural analysis provides both a rationale for the potency and specificity of DX-2930, and demonstrates the utility of using antibodies to specifically target an antigen among a family of related proteins (e.g. serine proteases). To further address the functional activity of DX-2930, we demonstrate that subcutaneous dosing of DX-2930 effectively reduces carrageenan-induced paw edema in vivo in rats when injected 24 hours prior to challenge. Combined with our finding that DX-2930 has a prolonged serum residence time in cynomolgus monkeys (t1/2 = 301 hours, SC), the data presented here demonstrates the potential of DX-2930 for the prophylactic inhibition of pKal-mediated diseases, such as HAE. Disclosures:Kenniston:Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Martik:Dyax Corp: Employment, former employee of Dyax Corp Other. Faucette:Dyax Corp: Employment. Viswanathan:Dyax Corp: Employment. Kastrapeli:Dyax: Employment. Kopacz:Dyax Corp: Employment. Conley:Dyax Corp: Employment. Lindberg:Dyax Corp: Employment. Cosic:Dyax Corp: Employment. Comeau:Dyax Corp: Employment. Mason:Dyax Corp: Employment. DiLeo:Dyax Corp: Employment. Chen:Dyax Corp: Employment. Ladner:Dyax Corp: Employment. Edwards:Emerald Biostructures: Employment. TenHoor:Dyax Corp: Employment. Nixon:Dyax Corp: Employment. Adelman:Dyax Corp: Employment.

Discovery and Characterization of a Fully Human Monoclonal Antibody Inhibitor of Plasma Kallikrein for the Treatment of Plasma Kallikrein-Mediated Edema

Plasma kallikrein (pKal) is a serine protease responsible for generating the vasodilator bradykinin, and is mainly regulated by C1 inhibitor (C1-INH). Patients with hereditary angioedema (HAE) have deficient levels of functional C1-INH, leading to unpredictable attacks of debilitating edema via dysregulated pKal-mediated bradykinin generation. Inhibition of pKal is a viable therapeutic option for HAE, as demonstrated by the efficacy of ecallantide, a specific pKal inhibitor, for treatment of acute HAE attacks.

Antibody-mediates inhibition of human C1s and the classical complement pathway

Disregulation of complement activation plays a critical role in numerous inflammatory diseases and therefore, inhibition of the complement pathway is of great therapeutic interest. In the classical complement pathway, immune complexes formed by IgM, IgG1, IgG2 and IgG3 antibodies result in the activation of the C1s protease that in turn cleaves C4 and then C4-bound-C2 yielding the proteolytic fragments C4b and C2a which associate to form a C3 convertase enzyme. We report here the engineering of a potent human antibody inhibitor of C1s protease activity. Phage panning of a very large synthetic (FAB) antibody fragment library using a truncated version of C1s, comprising the second CCP domain and serine protease domain (CCP2-SP) and expressed in insect cells, resulted in the isolation of a FAB that inhibited the catalytic activity of C1s. An affinity matured variant of the FAB format antibody displaying subnanomolar KD for C1s was shown to exhibit >80% inhibition of C2 processing at a 5:1 antibody:C1s molar ratio. We show that this engineered antibody, D.35, displays potent inhibition of complement deposition and lysis of Ramos cells by the anti-CD20 therapeutic antibody rituximab relative to the approved, but less-specific, human plasma-derived C1-inhibitor (CINRYZE). C1s inhibitory antibodies should be useful for delineating the role of the classical pathway in disease models and may hold promise as therapeutic agents.

Quantitation of von Willebrand factor and an antibody inhibitor

The role of the von Willebrand factor (vWF) in platelet thrombosis is poorly defined, partly due to the limited time that methods for the assay of plasma vWF have been available. A brief review of some of the recent findings that contributed to the development and application of a simple specific assay procedure for vWF is presented. The assay is based on the demonstration that the rate of formation of platelet aggregates observed macroscopically is dependent on the concentration of vWF (“snowstorm test”). Formaldehyde-fixed human platelets are used. Platelet aggregation due to vWF is induced in human plasma with ristocetin, while in bovine and porcine plasma no cofactor is required for the platelet aggregating factor (PAF) to be manifest. The effects of pH and temperature on the test were investigated. Shortest platelet aggregation times were obtained in the pH range of about 7.1–7.3 for both the ristocetin-induced human vWF and the bovine platelet aggregating activity on human platelets. Aggregation times were prolonged at pH 6.8 and 8.0. The aggregating times became progressively shorter as the temperature was raised from 10° to 35°. The need for control of both pH and temperature in the assay procedure for this platelet aggregating activity is pointed out. The macroscopic aggregating test has been used for the recognition and titering of a human antibody inhibitor which neutralizes vWF. The efficacy of the inhibitor in neutralizing the platelet aggregating factor activity of several species of plasma was investigated. The inhibitor was most effective against human vWF, somewhat less effective against bovine vWF and had little neutralizing capacity for porcine platelet aggregating factor.