Engineering therapeutic antibodies for improved function.

The binding sites on human IgG1 for human Fc gamma receptor (Fc gamma R) I, Fc gamma RIIa, Fc gamma RIIb, Fc gamma RIIIa and neonatal FcR have been mapped. A common set of IgG1 residues is involved in binding to all Fc gamma Rs, while Fc gamma RII and Fc gamma RIII utilize distinct sites outside this common set. In addition to residues which abrogated binding to the Fc gamma R, several positions were found which improved binding only to specific Fc gamma Rs or simultaneously improved binding to one type of Fc gamma R and reduced binding to another type. Selected IgG1 variants with improved binding to Fc gamma RIIIa were then tested in an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay and showed an enhancement in ADCC when either peripheral blood mononuclear cells or natural killer cells were used.

Anti-IgE efficacy in murine asthma models is dependent on the method of allergen sensitization.

BACKGROUND: Murine models used to delineate mechanisms and key mediators of asthma have yielded conflicting results and suggest that the dominant mechanism and mediators required for disease induction differ depending on the model and method of allergen sensitization used. OBJECTIVE: The goal of this study was to determine whether the mode of allergen sensitization influenced the role that IgE had in allergen-induced pulmonary eosinophilic inflammation. METHODS: Mice were exposed to dust mite extract in 2 models of allergic inflammation that differed in the method of sensitization. We compared sensitization by aerosol exposure with and without concomitant human respiratory syncytial virus infection with sensitization by means of systemic (intraperitoneal) exposure with adjuvant. After sensitization, animals were similarly challenged with aerosolized allergen. Animals were treated with anti-IgE mAb to deplete IgE and to determine its role in the induction of allergic inflammation and mucosa pathology in these models. RESULTS: Concomitant respiratory syncytial virus infection significantly enhanced allergen sensitization by aerosol exposure and exacerbated eosinophilic inflammation and airway mucosa pathology. Depletion of IgE in this model significantly reduced lung eosinophilic inflammation and airway mucosa pathology. However, in the model in which animals were sensitized by means of systemic allergen exposure with adjuvant, depletion of IgE had no ameliorative effect on lung inflammation or pathology. CONCLUSION: We demonstrated that the method of antigen sensitization can delineate the role of IgE in allergen-induced lung inflammation. In a murine model that more closely resembles ambient allergen exposure in human subjects, IgE had a critical role in the pathogenesis of allergic asthma and mucosa pathology. The results parallel the results reported with anti-IgE efficacy in allergic asthmatic human subjects.

High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R.

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

Anti-immunoglobulin E antibody treatment blocks histamine release and tissue contraction in sensitized mice.

Inhibition of antigen-specific IgE response has been shown to lead to amelioration of allergic disease symptoms. In an effort to design a therapy aimed at decreasing IgE levels, we reported previously that treatment of mice with an anti-IgE antibody coincident with the primary antigen immunization resulted in significant decreases in antigen-specific IgE synthesis, without substantially altering IgG levels. In the present study, we employed this mouse model and a surrogate antibody to investigate the capacity of anti-IgE treatment to block an established IgE response in vivo. Results of these experiments suggest that anti-IgE treatment concomitant with an antigen boost results in removal of detectable circulating IgE for at least 7 weeks (the duration of the study). Moreover, tissues removed from mice following anti-IgE treatment failed to release histamine and contract in response to antigen challenge ex vivo. These findings demonstrate that reduction of circulating IgE correlates to an inhibition of tissue mast cell sensitization and mediator release in response to antigen challenge and further supports the concept of anti-IgE treatment as a promising therapy for the treatment of allergic disease.

Inhibition of allergic reactions with antibodies to IgE.

Numerous clinical studies show that direct interference with the IgE response leads to a decrease or elimination of allergic symptoms. The aim of these studies was to design a therapy aimed at decreasing IgE levels in order to ameliorate atopic disease. To this end, a murine monoclonal antibody, MAE11, directed against IgE was identified, which had all the properties necessary to interfere with IgE responses, but lacked the harmful side effects of inducing receptor cross-linking. The antibody was selected on the basis of its ability to bind circulating IgE at the same site as the high-affinity receptor, thus blocking the binding of IgE to mast cells and basophils. To allow for possible chronic administration and to avoid the problems of antigenicity, MAE11 was humanized. The best of several humanized variants, version 25 (rhumAb-E25) was selected since it possessed binding affinity and biological activity comparable to MAE11. Clinical studies are underway to determine the safety and efficacy of this treatment for allergic rhinitis and asthma.

Humanization of an antibody directed against IgE.

IgE antibodies bind to specific high-affinity receptors on mast cells, leading to mast cell degranulation and release of mediators, such as histamine, which produce symptoms associated with allergy. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies must also not bind to IgE once it is bound to the receptor because this would trigger histamine release. This study describes the humanization of a murine antibody, MaE11, with these characteristics. Variants of the humanized antibody were evaluated to probe the importance of framework residues on antibody binding and to determine which charged residues in the CDR interacted with IgE. We found that only five changes in human framework residues were required to provide for binding comparable to that of the original murine antibody.

Effect of inhaled ozone on lung histamine in conscious guinea pigs.

The effect of short-term ozone (O3) exposure on pulmonary mast cell function was examined. Guinea pigs were continuously exposed to 1.0 ppm O3 for 2, 4, and 8 hr. O3 exposure produced a significant decrease in lung histamine concentration. Two-hour exposure to O3 caused a 22.4 +/- 7.0% decrease in lung histamine concentration compared with controls. Ozone exposures of 4 and 8 hr caused lung histamine concentrations to decrease by 43.7 +/- 7.7 and 49.0 +/- 7.5% (P less than 0.05), respectively, without significant changes in lung water or protein, or evidence of cytotoxicity. These results suggest that O3 or its metabolites affect pulmonary mast cell function by stimulating the release of histamine from the lung.

Immunologic and nonimmunologic responsiveness in ragweed-sensitized dogs.

The relationship between airway responsiveness to inhaled antigen and histamine, immunologic release of lung histamine, immunologic responsiveness of skin, and specific immunoglobulin E (IgE) antibodies were examined in 11 inbred allergic dogs immunized with extracts of ragweed and grass and 5 nonimmunized control dogs from the same colony. Airway responsiveness to antigen and histamine was characterized by the doses that increased the airflow resistance of the total respiratory system to twice the control values (ED200). Highly significant correlations were found between airway responsiveness and cutaneous responsiveness to antigen and other immunologic characteristics (e.g., IgE and histamine released from lung by inhaled antigen) in all dogs. In ragweed-sensitized dogs, there was an inverse correlation between immunologic responsiveness (reflected by the cutaneous response to antigen and histamine released from lung by inhaled antigen) and nonimmunologic responsiveness of airways (histamine ED200: r = 0.73, P less than 0.05 and r = 0.75, P less than 0.01, respectively). Antigen ED200 was also correlated with histamine release from lung after antigen inhalation (r = 0.74; P less than 0.01). We conclude that airway reactions to inhaled antigen in allergic dogs are dependent not only on immunologic factors but also on the degree of nonimmunologic airway responsiveness to histamine and that these factors are correlated inversely.

Purification of Ascaris suum antigen: its allergenic activity in vitro and in vivo.

Crude aqueous extracts of Ascaris suum (CE) have been used widely to study IgE-mediated reactions in various experimental preparations. Because some CE may contain a polypeptide, a mast cell degranulating peptide (MCDP), that degranulates mast cells by nonimmunologic mechanisms, various protocols have been used to ensure that the Ascaris preparation used did not contain MCPD. In general, these protocols have assumed MCDP had been without providing proof. Even protocols designed to isolate the major antigenic determinants from CE have usually been designed to evaluate immunogenic characteristics of the purified Ascaris; thus, few systematic comparisons of CE with purified Ascaris exist concerning mast cell degranulation, and few studies have demonstrated that MCDP has been removed during purification. Since Ascaris has proved to be useful in a variety of studies of IgE-mediated reactions, particularly in large animals (dog and sheep), we have developed a protocol to purify CE and MCDP and characterize their physiochemical and immunologic properties. We compared the allergenic activity of our purified Ascaris to that of CE and MCDP in skin and lung of natively sensitized dogs and in unsensitized rat peritoneal mast cells. Our results indicate that MCDP probably contaminates CE by less than 1.0%. However, the biologic activity of MCDP in dog lung appears insignificant and probably contributes little to CE-induced reactions in doses of CE commonly used (less than or equal to 100 mg injected). If a purified Ascaris preparation is essential, our protocol will yield an Ascaris preparation that has potent IgE-mediated effects in dog preparations with insignificant contamination by MCDP.

Airway responsiveness to inhaled antigen, histamine, and methacholine in inbred, ragweed-sensitized dogs.

We studied the responses to antigen in animals selected from a colony of inbred dogs sensitized to specific allergens to determine if they had characteristics similar to those of human asthmatics. They were immunized with ragweed and grass pollen extracts (10 micrograms in alum) immediately after routine vaccination with attenuated live virus (distemper and hepatitis) and killed bacteria (Leptospira) at 4, 8, and 12 wk of age. Subsequently, ragweed and grass injections were repeated every 2 months. Immunized dogs made specific IgE-antibodies in serum averaging 3 to 4 times that of control animals (no immunization with pollen or vaccine). They showed positive skin responses to the injection of ragweed pollen extract, whereas control dogs did not respond to ragweed pollen by quantitative skin test or inhalation challenge. In immunized dogs under barbiturate anesthesia, air-flow resistance of the total respiratory system increased from 0.60 +/- 0.07 (mean +/- SEM) before to 12.6 +/- 3.4 cm H2O/lps 5 min after the start of antigen aerosol; respiratory resistance remained increased for 20 min and was associated with 0 hypoxemia and increased arterial plasma histamine. In addition, airway responsiveness to both inhaled histamine and methacholine was greater in immunized dogs than in nonimmunized dogs of comparable age. Airway responses to each agonist were highly reproducible on repeated testing. These results indicate that physiologic responses to antigen by inbred, ragweed-sensitized dogs resemble human asthma closely and that these dogs appear suitable for a variety of experimental studies of asthma with respect to pathogenesis, diagnosis, prevention, and treatment.

Immunologic release of histamine from dog lung: comparison of in vivo and in vitro responses in the same animal.

The purpose of this study was to compare, for the first time, antigen-induced histamine release from the lung in the same natively allergic dogs both in vitro and in vivo. In six dogs, maximal antigen-induced histamine release from the lung correlated closely in vitro and in vivo (r = 0.94), although it varied widely between dogs (0% to 75.5% of total tissue histamine content); similarly, the antigen concentration to produce 50% of maximal histamine release varied sixfold between dogs (40 micrograms/ml to 250 micrograms/ml). In each of five other dogs, terbutaline sulfate administered intravenously caused a dose-dependent inhibition of antigen-induced histamine release from lung fragments in vitro: the maximal inhibition produced by 1 mg/kg was 60 +/- 4.5% (mean +/- SEM). In these same dogs, 10(-5)M terbutaline incubated with lung fragments in vitro caused inhibition of antigen-induced histamine release comparable to 1 mg/kg terbutaline in vivo. Increasing the dose of terbutaline in vitro produced maximal inhibition at 10(-4)M with no greater effect of the drug at 10(-3)M (71.4 +/- 3.8% inhibition). In both experimental situations propranolol caused a dose-dependent inhibition of beta-adrenergic modulation of Ascaris-induced release of histamine. This result supports the conclusion that terbutaline produced its effects by actions mediated by beta-adrenergic receptors on pulmonary mast cells. This experimental approach provides a suitable preparation in which to estimate the effective dose of agonists that modulate antigen-induced mast cell function in vivo.

Cigarette smoke-induced bronchoconstriction in dogs: vagal and extravagal mechanisms.

We reassessed the severity of cigarette smoke-induced bronchoconstriction and the mechanisms involved in anesthetized dogs. To evaluate the severity of smoke-induced bronchoconstriction, we measured airway pressure and airflow resistance (Rrs, forced oscillation method). We studied the mechanisms in other dogs by measuring airway pressure, central airway smooth muscle tone in tracheal segments in situ, and respiratory center drive by monitoring phrenic motor nerve output, including the role of vagal and extravagal nerves vs. the role of blood-borne materials during inhalation of cigarette smoke. Rrs increased more than fourfold with smoke from one cigarette delivered in two tidal volumes. About half the airway response was due to local effects of smoke in the lungs. The remainder was due to stimulation of the respiratory center, which activated vagal motor efferents to the airway smooth muscle. Of this central stimulation, about half was due to blood-borne materials and the rest to vagal pulmonary afferents from the lungs. We conclude that inhalation of cigarette smoke in dogs causes severe bronchoconstriction which is mediated mainly by extravagal mechanisms.