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Published ahead of print on October 24, 2003, doi:10.1164/rccm.200304-543OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 97-104, (2004)
© 2004 American Thoracic Society

A Monoclonal Antibody to {alpha}1ß1 Blocks Antigen-induced Airway Responses in Sheep

William M. Abraham, Ashfaq Ahmed, Irakli Serebriakov, Amie N. Carmillo, Janine Ferrant, Antonin R. de Fougerolles, Ellen A. Garber, Philip J. Gotwals, Victor E. Koteliansky, Fred Taylor and Roy R. Lobb

Division of Pulmonary Disease and Critical Care Medicine, University of Miami at Mount Sinai Medical Center, Miami Beach, Florida; and Biogen, Inc., Cambridge, Massachusetts

Correspondence and requests for reprints should be addressed to William M. Abraham, Ph.D., Department of Research, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140. E-mail: abraham{at}msmc.com


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The integrin {alpha}1ß1 (very late antigen-1; CD49a/CD29) is a major adhesion receptor for collagen I, IV, and VI, and its induced expression on activated monocytes and lymphocytes plays a central role in their retention and activation at inflammatory sites in autoimmune pathologies. However, the role of {alpha}1ß1 in allergic settings has not been explored. In this study, we show that a single 45-mg dose of aerosolized monoclonal antibody AQC2 to the {alpha}1 chain of human and sheep very late antigen-1, given 30 minutes before challenge, blocks both the allergen-induced late response and the associated airway hyperresponsiveness, functional indicators of allergen-induced inflammation, in sheep. AQC2 does not affect the early response. Consistent with these effects, AQC2 tended to reduce the cell response associated with local antigen instillation. An isotype-matched control antibody had no protective effects. Two humanized versions of AQC2, a wild-type IgG1 and an aglycosyl form of the same monoclonal antibody, which has reduced Fc receptor–mediated effector functions, are equally effective in blocking the antigen-induced late response and airway hyperresponsiveness in the sheep model. These data suggest that mononuclear leukocyte adhesion–dependent pathologies contribute to allergic lung disease and provide proof-of-concept that antagonists of {alpha}1 integrins may be useful in preventing these events.

Key Words: asthma • inflammation • integrin • animal model • CD49a

The development of late airway responses (LAR) and the associated airway hyperresponsiveness (AHR) that follow airway antigen challenge are believed to be the functional indicators of a heightened and continued inflammatory response that is initiated by a single allergen exposure. Studies of patients with asthma and animal models of allergic asthma indicate that recruitment, activation, and retention of mononuclear leukocytes in the airways contribute to these pathophysiologic events (1–6). These findings have led to the use of a number of antiadhesion molecules, including selectin inhibitors (6), inhibitors of the immunoglobulin superfamily (7, 8), and integrin inhibitors (1, 5), to counteract the mechanisms necessary for inflammatory cell recruitment and retention in an attempt to modulate the abnormalities in airway function. Whereas the primary action of some of these molecules, e.g., selectin inhibitors, is to prevent cells from leaving the vasculature, other molecules can affect leukocyte interactions once in the tissue.

Our initial evaluation of the role of matrix-binding integrins was based, in part, on our earlier discovery that pretreatment with aerosolized monoclonal antibodies (mAbs) to the integrin VLA-4 ({alpha}4ß1) was highly effective in blocking the LAR and AHR in the sheep model of allergic asthma (1). The efficacy of local as opposed to systemic delivery was initially a surprising result, although it was rapidly confirmed and extended by others (2). A careful comparison of systemic and local delivery of mAb PS/2 to murine VLA-4 in a murine model of allergic asthma showed that although both systemic and local treatments inhibited antigen-induced eosinophil emigration into bronchoalveolar lavage (BAL) fluid, only local treatment inhibited allergen-induced mucus production, Th2 cytokine production, and AHR (2). This differential treatment effect was likely explained by the observation that immunocytochemical staining of lung tissue after antigen challenge showed only local delivery of mAb PS/2-labeled VLA-4–expressing leukocytes, including CD11c+ cells, within the lung parenchyma, which suggests that local treatment with VLA-4 inhibitors act on cells that have already exited the vasculature. Such a mechanism would be consistent with the finding that aerosol treatment with a small molecule inhibitor of VLA-4, BIO 1211, reversed postantigen-induced AHR in sheep with allergy (5). Collectively, these findings strongly suggest that modulation of cell retention/activation in tissues can be an important site of therapeutic intervention.

Given the importance of interstitial adhesive interactions in inflammation, we have begun to focus on one of the major collagen-binding integrins, {alpha}1ß1. Whereas basal expression of {alpha}1ß1 in the adult is largely confined to mesenchymal cells (9, 10), this integrin is rapidly expressed on activated monocytes and on long-term activated T cells in vitro (11, 12). In addition, infiltrating T cells express {alpha}1ß1 in a variety of chronic inflammatory settings, including rheumatoid synovium (13, 14) and atherosclerotic plaques (15). We have shown that a function-blocking mAb to {alpha}1ß1 significantly inhibited inflammatory responses in murine models of delayed-type hypersensitivity, contact hypersensitivity, and arthritis (16). That {alpha}1ß1 is important in these inflammatory models is supported further by the reduced inflammatory response seen in {alpha}1-deficient mice (16). More recently we have extended these studies, showing that {alpha}1ß1 integrin plays a central role in two rodent models of inflammatory bowel disease (17, 18) in which {alpha}1ß1-expressing monocytes are critical. These results emphasize the central role of extracellular matrix-binding integrins in inflammation.

Here, we have extended our studies on the role of leukocyte {alpha}1ß1 to the sheep model of asthma to determine the role of {alpha}1ß1 in allergic airway disease. To do this, we have aerosolized mAbs to {alpha}1ß1 before allergen provocation and monitored the effects of the drug on allergen-induced airway responses. In addition, we used local challenge with antigen in the presence and absence of mAb to determine if blocking {alpha}1ß1 could affect the cell response that accompanies the allergen-induced functional abnormalities in these animals. Our results show that aerosolized mAbs to {alpha}1ß1 are highly effective at blocking both allergen-induced LAR and AHR. Cell responses after local allergen challenge were also reduced in the treated airways, though this reduction was not statistically significant. These results extend the role of {alpha}1ß1 to allergic diseases.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
mAbs
Murine mAb AQC2 is one of a previously described panel of mAbs to the {alpha}1 I domain (19). All function-blocking mAbs obtained from this screen, including mAb AQC2, were clonally related. Immunoblotting and fluorescence-activated cell sorting (FACS) analysis of one such mAb, AHJ10, demonstrated that it reacts with human, rabbit, and sheep {alpha}1ß1 integrin, suggesting that all blocking mAbs bind to an evolutionarily highly conserved linear epitope within the {alpha}1 I domain (19). MAb AQC2 was also found to bind sheep {alpha}1 by Western blot analysis using the same methods (data not shown). A humanized IgG1 form of mAb AQC2 is described elsewhere (19). Briefly, the variable domains of the heavy and light chains of murine AQC2 antibody were cloned by reverse transcriptase—polymerase chain reaction. Humanized antibody was designed using consensus human frameworks chosen after homology matching, and the version with optimal {alpha}1ß1 integrin–binding properties was designated as huAQC2. The huAQC2 was expressed in Chinese hamster ovary cells, purified by conventional techniques and shown to be equipotent with murine AQC2. An aglycosyl humanized IgG1 form of AQC2 was created by introducing a N297Q mutation (Kabat EU numbering) to eliminate the N-linked glycosylation site in the CH2 domain of the Fc fragment. This form was expressed and purified identically to the wild-type form. It was shown to bind normally to the {alpha}1 I domain but not to bind to human Fc{gamma}RI (data not shown). Murine mAb 1E6, to human LFA-3, an IgG1, was used as a negative control, and is described elsewhere (1).

Animal Preparation
A total of 26 sheep (24–43 kg) with airway hypersensitivity to Ascaris suum antigen were used. All sheep had previously been shown to develop both early airway responses (EAR) and LAR and postantigen-induced AHR to inhaled A. suum antigen (1). The sheep were conscious and were restrained in a modified shopping cart in the prone position with their heads immobilized. For the sheep used in the airway function studies, anesthesia of the nasal passages with topical 2% lidocaine was achieved and then a balloon catheter was advanced through one nostril into the lower esophagus. The animals were intubated with a cuffed endotracheal tube through the other nostril as described previously. The Mount Sinai Medical Center Animal Research Committee is responsible for assuring the humane care and use of laboratory animals approved the procedures used in this study.

Airway Mechanics
Breath-by-breath determination of mean pulmonary airflow resistance (RL) was measured with the esophageal balloon technique described extensively by us (1, 5, 6). The mean of at least 5 breaths, free of swallowing artifact, were used to obtain RL in cm H2O x L-1 x second.

Aerosol Delivery Systems
All aerosols were generated using a disposable medical nebulizer (Raindrop; Nelcor-Puritan Bennett, Carlsbad, CA). The nebulizer was connected to a dosimeter system, consisting of a solenoid valve and a source of compressed air (20 psi). The output of the nebulizer was directed into a plastic T-piece, one end of which was connected to the inspiratory port of a piston respirator (Harvard Apparatus, Mills, MA). The solenoid valve was activated for 1 second at the beginning of the inspiratory cycle of the respirator. Aerosols were delivered at a VT of 500 ml and a rate of 20 breaths/minute as described previously (1, 5, 6).

Concentration Response Curves to Carbachol Aerosol
Airway responsiveness was determined from cumulative concentration response curves to inhaled carbachol as described previously. RL was measured immediately after inhalation of phosphate-buffered saline (PBS) and after each consecutive administration of 10 breaths of increasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0, and 4.0% wt/vol PBS). The provocation test was discontinued when RL increased over 400% from the post-PBS value or after the highest carbachol concentration had been administered. The cumulative carbachol concentration (in breath units [BU]) that increased RL by 400% over the post-PBS value (PC400) was calculated by interpolation from the dose response curve. One BU was defined as one breath of a 1% wt/vol carbachol aerosol solution (1, 5, 6).

BAL
BAL was performed using a fiberoptic bronchoscope as described previously by us (1, 5, 6). After local anesthesia of the airways, the bronchoscope was wedged into randomly selected subsegmental bronchi. Lung lavage was performed by slow infusion and aspiration of 3 x 30 ml aliquots of PBS. Cell counts were made from unconcentrated samples and cellular differentials from cytospin preparations (1, 5, 6).

Agents
A. suum extract (Greer Diagnostics, Lenoir, NC) was diluted with PBS to a concentration of 82,000 protein nitrogen units/ml and delivered as an aerosol (20 breaths/minute). This same concentration of A. suum was used for the local challenge studies. Carbamylcholine (Carbachol; Sigma Chemical Co., St. Louis, MO) was dissolved in PBS at concentrations of 0.25, 0.50, 1.0, 2.0, and 4.0% wt/vol and delivered as an aerosol.

Protocols
Airway function.
To examine the effects of very late antigen-1 and control antibodies on antigen-induced airway responses, we measured baseline airway responsiveness (i.e., PC400) to carbachol 1 to 3 days before antigen challenge. On the antigen challenge day, RL was measured and then the sheep were treated with one of the experimental antibodies or PBS (control). RL was remeasured 30 minutes later, and then animals were challenged with the A. suum antigen. RL was remeasured immediately after, hourly from 1 to 6 hours after and half-hourly from 6.5 to 8 hours after antigen challenge as described previously (1, 5). Postchallenge determinations of airway responsiveness (PC400) were made 1 day after antigen challenge to assess the development of AHR. For these studies separate groups of sheep (n = 4) were treated with 45 mg of murine AQC2, 1E6 (isotyped control antibody), human AQC2 (wild-type), and human AQC2 (aglycosylated) antibodies. The responses to treatment with the active and control mAbs were compared with responses after allergen challenge when the sheep were given PBS.

Airway Inflammation
To determine if AQC2 had antiinflammatory effects, we used local instillation of antigen into lung segments in the presence and absence of locally instilled AQC2 and evaluated the effects on BAL cell returns. For these studies a baseline BAL was performed in each lung (right and left). Then 45 mg of AQC2 was instilled in a segment of one lung, and in the other lung an equivalent amount of PBS (5.2 ml) was instilled. The lungs receiving active and control treatments were alternated. Thirty minutes later, both airways were challenged with 5 ml of A. suum extract. Postchallenge lavages of these segments were obtained at 8 and 24 hours after challenge.

Statistical Analysis
All values in the text, tables, and figures are reported as mean ± SE. Airway functional responses in each group were compared using a two-tailed paired t test. Cell responses were not normally distributed and therefore were first log-transformed and then analyzed using a two-way repeated analysis of variance to assess overall effects of the treatment over time. Post hoc comparisons were made with a paired t test with Bonferroni correction for multiple comparisons. Significance was accepted when p values were less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Airway Responses
In our previous studies using aerosol delivery of an anti–VLA-4 mAb (HP1/2), we found that 15 mg of mAb delivered 30 minutes before allergen challenge gave complete inhibition of both LAR and AHR. Preliminary studies with AQC2 at this dose were ineffective (data not shown). For the studies presented below a dose of 45 mg AQC2 in all forms was used, this being the highest usable dose based on material constraints.

As illustrated in Figure 1 , treatment with 45 mg of murine AQC2 aerosol had no effect on the EAR to allergen but blocked the LAR (Figure 1, top) and the postantigen-induced AHR (Figure 1, bottom). In the control trial, RL increased 370 ± 97% immediately after challenge. RL returned to near baseline values by 4 hours postchallenge but began to increase again by 5 hours postchallenge. The peak increase in the LAR (defined in Table 1) from 5 to 8 hours was 145 ± 4% over baseline. When these same animals were treated with AQC2, there was no effect on baseline bronchial tone (Table 1) and there was no effect on the EAR to inhaled antigen: RL increased 341 ± 59%. By 4 hours postchallenge, however, RL had returned to baseline and increased only slightly throughout the remainder of the 8 hours challenge period (peak increase in RL 55 ± 12% [p < 0.05 vs. control], Table 1). Thus, aerosolized murine AQC2 at a dose of 45 mg provided a mean 62% protection against the LAR in these animals. AQC2 not only blocked the LAR in these animals but also blocked the postantigen-induced AHR (Figure 1, bottom). In the control trial, PC400 at 24 hours fell to 10.8 ± 2.8 BU from a prechallenge value of 22.4 ± 6.3 BU (p < 0.05), resulting in a decreased post/prechallenge PC400, indicative of AHR. When the animals were treated with AQC2, the post/prechallenge PC400 was unchanged as the postchallenge PC400 (18.6 ± 3.2 BU) was not different from the prechallenge value of 18.6 ± 2.4 BU. Values of RL at 24 hours before the determination of the PC400 for both control (0.95 ± 0.01 cm H2O x L-1 x second) and treatment (0.95 ± 0.02 cm H2O x L-1 x second) trials were not different from their respective prechallenge values (Table 1).



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  		Figure 1. Effect of treatment with aerosol murine AQC2 on the time course of antigen-induced changes in pulmonary airflow resistance (RL, cm H2O x L-1 x second) in sheep with allergy (top). AQC2 (45 mg) was administered 0.5 hours before challenge. AQC2 had no effect on the early airway response but significantly blocked the late airway response. The bottom figure represents the change in airway responsiveness to inhaled carbachol expressed as the postantigen PC400/prechallenge PC400 ratio for the control and treated groups. A ratio of 1 indicates no change in responsiveness, whereas a ratio less than 1 indicates the development of airway hyperresponsiveness. AQC2 inhibited the development of postantigen-induced airway hyperresponsiveness. Values are mean ± SE for four sheep (see Table 1 for statistical analysis).

 

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  		TABLE 1. Summary of pulmonary airflow resistance and airway responses before and after treatment with AQC2

 
To ensure that the protective effect seen in the AQC2-treated animals did not result from nonspecific effects of inhaling 45 mg of mAb, animals were treated with 45 mg of an isotype-matched control antibody 1E6. As shown in Figure 2 , the control antibody had no effect on the EAR, the LAR, or the postchallenge AHR (Figure 2, Table 1). Values of RL at 24 hours before the determination of PC400 for both control (0.99 ± 0.02 cm H2O x L-1 x second) and treatment (0.93 ± 0.02 cm H2O x L-1 x second) trials were not different from their respective prechallenge values (see Table 1).



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  		Figure 2. Effect of treatment with aerosol 1E6 on the time course of antigen-induced changes in RL (cm H2O x L-1 x second) in sheep with allergy (top). 1E6 (45 mg) was administered 0.5 hours before challenge and had no effect on the early airway response or the late airway response. The bottom figure represents the change in airway responsiveness to inhaled carbachol expressed as the postantigen PC400/prechallenge PC400 ratio for the control and treated groups. A ratio of 1 indicates no change in responsiveness, whereas a ratio less than 1 indicates the development of airway hyperresponsiveness. 1E6 did not protect the animals from the development of postantigen-induced airway hyperresponsiveness. Values are mean ± SE for four sheep (see Table 1 for statistical analysis).

 
Humanized Forms of mAb AQC2
We have recently generated a humanized version of mAb AQC2 of isotype human IgG1, as well as an aglycosyl form of the same mAb, both of which are equipotent with murine AQC2 in their ability to bind {alpha}1ß1 in vitro and to block its adhesion to collagen I and IV (19) (data not shown). Removal of the CH2 glycans, resulting in the aglycosyl form of the mAb, is known to greatly reduce binding to Fc{gamma} receptors and to C1q of complement (20–22). Thus, the aglycosyl form of a mAb provides a useful reagent for testing the contribution of Fc receptor–mediated effector functions in the mechanism of action.

The effects of the humanized forms of AQC2 on the antigen-induced responses are shown in Figures 3 and 4 . When animals were treated with the humanized AQC2 IgG1 isotype (wild type) there was again protection against the LAR (AQC2: 32 ± 3% vs. control: 163 ± 8%, p < 0.05) and the postchallenge AHR (AQC2: prechallenge 21.3 ± 3.3 BU, postchallenge 20.5 ± 4.9 BU vs. control: prechallenge 21.5 ± 3.1 BU, postchallenge 10.3 ± 1.8 BU, p < 0.05)(Figure 3). Values of RL at 24 hours before the determination of PC400 for both control (0.97 ± 0.01 cm H2O x L-1 x second) and treatment (1.00 ± 0.02 cm H2O x L-1 x second) trials were not different from their respective prechallenge values (see Table 1). As was seen with the murine antibody, the humanized wild-type form of AQC2 did not protect against the EAR (Table 1). The results obtained with the aglycosyl form of AQC2 were essentially the same: LAR (AQC2: 31 ± 4% vs. control: 145 ± 13%, p < 0.05); AHR: (AQC2: prechallenge 15.4 ± 3.6 BU, postchallenge 15.8 ± 2.3 BU vs. control: prechallenge 16.4 ± 2.5 BU, postchallenge 7.4 ± 1.8 BU, p < 0.05)(Figure 4). Values of RL at 24 hours before the determination of PC400 for both control (0.97 ± 0.01 cm H2O x L-1 x second) and treatment (0.96 ± 0.02 cm H2O x L-1 x second) trials were not different from their respective prechallenge values (Table 1). Neither of the humanized forms of the antibody had an effect on baseline airway tone (Table 1).



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  		Figure 3. Effect of treatment with aerosol AQC2 (human, wild-type) on the time course of antigen-induced changes in RL (cm H2O x L-1 x second) in sheep with allergy (top). AQC2 (45 mg) was administered 0.5 hours before challenge. AQC2 had no effect on the early airway response but significantly blocked the late airway response. The bottom figure represents the change in airway responsiveness to inhaled carbachol expressed as the postantigen PC400/prechallenge PC400 ratio for the control and treated groups. A ratio of 1 indicates no change in responsiveness, whereas a ratio less than 1 indicates the development of airway hyperresponsiveness. AQC2 inhibited the development of postantigen-induced airway hyperresponsiveness. Values are mean ± SE for four sheep (see Table 1 for statistical analysis).

 


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  		Figure 4. Effect of treatment with aerosol AQC2 (human, aglycosylated) on the time course of antigen-induced changes in RL (cm H2O x L-1 x second) in sheep with allergy (top). AQC2 (45 mg) was administered 0.5 hours before challenge. AQC2 had no effect on the early airway response but significantly blocked the late airway response. The bottom figure represents the change in airway responsiveness to inhaled carbachol expressed as the postantigen PC400/prechallenge PC400 ratio for the control and treated groups. A ratio of 1 indicates no change in responsiveness, whereas a ratio less than 1 indicates the development of airway hyperresponsiveness. AQC2 inhibited the development of postantigen-induced airway hyperresponsiveness. Values are mean ± SE for four sheep (see Table 1 for statistical analysis).

 
Effect of AQC2 on Airway Inflammation
The effect of AQC2 on antigen-induced airway inflammation is illustrated in Figure 5 . Analysis of variance indicated that there were significant changes in some cell responses over time within the AQC2-treated and control airways. In control airways, total cells increased from a baseline value of 170 ± 24 cells x 103/ml to 495 ± 99 at 8 hours (p < 0.05) and to 742 ± 381 cells x 103/ml (p < 0.05) at 24 hours. Control airways also showed significant increases from baseline in lymphocytes and eosinophils at 24 hours and neutrophils at both 8 hours and 24 hours. Monocytes tended to increase over time, but these changes did not achieve statistical significance. In the mAb-treated airways, total cells increased from a baseline value of 174 ± 30 cells x 103/ml at 8 hours (309 ± 69 cells x 103/ml) and 24 hours (399 ± 106 cells x 103/ml), but these changes were not significant. The only significant change over time in the treated airways was seen in the neutrophil response at both 8 and 24 hours. Macrophages showed no significant changes over time in either the treated or control airways (data not shown). Despite the within-treatment differences, however, analysis of variance failed to show significance between treatment differences.



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  		Figure 5. Effect of AQC2 (murine) on antigen-induced airway inflammation induced by segmental challenge. Segmental allergen challenge was performed 30 minutes after instilling 45 mg AQC2 into a segment of one lung (treated) and an equal volume of PBS into a segment in the opposite lung (control). Within-treatment changes from baseline indicated that there were significant increases in total cells, lymphocytes, eosinophils, and neutrophils in the control segments. In the AQC2-treated segments only the neutrophil response was significantly increased over baseline. Overall there were no significant differences between the AQC2-treated and control segments. *p Value less than 0.05 versus baseline. Values are mean ± SE for six sheep.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study provide the first evidence that acute aerosol treatment with anti-{alpha}1 mAb, AQC2, can significantly reduce antigen-induced LAR and AHR in the sheep model of allergic asthma. The protection seen at these endpoints is not related to an effect on the antigen-induced EAR because neither the murine nor the humanized mAbs affected this parameter. These findings indicate that the antibody is working to modify downstream inflammatory events known to contribute to the antigen-induced LAR and AHR in this model. Such actions would be consistent with the putative inhibition of the cellular targets of the antibody in this study. It is important to note that the abrogated LAR and AHR seen after treatment with AQC2 are specific for this mAb and not due to nonspecific effects of aerosolized proteins. There was no protection afforded against these antigen responses when animals were treated with an equivalent amount of inactive isotype mAb (1E6). The results achieved in the present study provide additional support for the use of aerosolized mAbs and other proteins in the treatment of inflammatory lung diseases (1, 23, 24).

MAb AQC2 derives from a previously described panel of mAbs to the human {alpha}1 I domain (19). We initially identified 19 mAbs that bound to human {alpha}1ß1 integrin and to the {alpha}1 I domain. To examine the clonal origin of this panel of mAbs, we amplified by polymerase chain reaction and sequenced the complementarity determining regions from 12 of the 19 antibodies (25). Sequences from clones producing function-blocking mAbs, including mAb AQC2 and AJH10, were nearly identical across all the complementarity determining regions and the intervening framework regions, suggesting that these hybridomas were clonally related. Immunoblotting and FACS analysis of mAb AJH10 showed that it reacted with human, rabbit, and sheep, but not rat {alpha}1ß1 integrin, suggesting that these function-blocking mAbs bind to an evolutionarily highly conserved linear epitope within the {alpha}1ß1 I domain (19). The epitope recognized by AQC2 was found to lie within a loop that forms part of the metal ion–dependent adhesion site, between helices R3 and R4 (25). MAb AQC2 was also found to bind sheep {alpha}1 by Western blot analysis (data not shown).

The functional protection afforded by the {alpha}1ß1 mAbs in the sheep model is consistent with previous data obtained in several well characterized, but mechanistically distinct, murine models of inflammation (16–18). The effector cells driving these inflammatory responses are believed to be varied and include CD4 + T cells for delayed-type hypersensitivity, CD8 + T cells for contact hypersensitivity, activated monocytes/neutrophils for arthritis, and activated T cells and monocytes for inflammatory bowel disease. Both monocytes and T cells have been shown to express {alpha}1ß1 on activation, and so blocking their movement through, or retention, activation, and survival in the extracellular matrix with the {alpha}1 mAb could explain the observed protection against the late-stage functional changes associated with allergen provocation.

Work in other experimental systems would suggest that an additional action of {alpha}1ß1 antagonism in the sheep model may also be on cells already resident in the lung. Given that the sheep are naturally allergic to A. suum, and that they have repeatedly encountered antigen, it would be expected that many activated monocytes and T cells (including memory T cells) would be resident in the lung. Recent work has shown that memory T cells can be divided into two functional subsets, central memory cells resident in lymphoid organs and effector memory T cells that reside in peripheral tissues throughout the body, including lung (26, 27). In addition, effector memory T cells, but not central memory T cells, are capable of immediately responding to antigenic stimulation, thereby ensuring that reencounters with pathogens are effectively dealt with at the site of infection. Using mouse influenza and lymphocytic choriomeningitis virus infection models, {alpha}1ß1 was found to be specifically expressed on antigen-specific activated and memory T cells, with {alpha}1+ cells being preferentially found within the extracellular matrix–rich peripheral tissues (28). In addition, migration and retention of activated and memory T cells in peripheral tissues, including the lung, were inhibited by anti-{alpha}1 mAb treatment. Because the interaction between {alpha}1ß1 integrin and collagen promotes cell survival and proliferation and regulates the expression of gene products involved in extracellular matrix remodeling (29), we hypothesize that another consequence of {alpha}1 mAb treatment may be modulation of the retention, localization, and activation of memory T cells in the sheep asthma model.

To begin to evaluate possible mechanisms of action, we determined if movement of inflammatory cells into the alveolar space was inhibited by mAb AQC2. We used segmental antigen challenge in the presence and absence of the mAb and measured the resultant inflammatory response. We opted for local challenge because it provides a more consistent and formidable inflammatory response than global lung challenge. The small sample size, due to a limited amount of available antibody, also prompted us to use BAL rather than biopsies as the primary endpoint for these studies. For these experiments we used the same dose of mAb that we used in the aerosol studies. Although we were unable to show significant differences between the control and treated airway cellular responses, AQC2-treated animals tended to show reduced cellular infiltration at 24 hours, despite larger mean changes over baseline in the control airways at 24 hours in total cells (control airway: 4.4-fold vs. treated: 2.3-fold), eosinophils (186-fold vs. 28-fold), lymphocytes (3.9-fold vs. 1.8-fold), and monocytes (25-fold vs. 3.5-fold). The neutrophil response in both the treated and untreated airways was almost identical, which would be expected because neutrophils do not express {alpha}1ß1.

There are a number of reasons that could explain the lack of significance between treatment arms, but most likely the major effect of the mAb in these studies was not on cell movement but on retention or activation of pertinent cell types within the lung parenchyma, as discussed previously. Interestingly, in both animal models and humans, changes in inflammatory indices in allergen challenge studies do not always correlate with the primary endpoint, i.e., changes in airway function (1, 2, 30–32). For example, in studies using the interleukin-5 antibody, it was shown that significant reductions in circulating and sputum eosinophils had no effect on pulmonary function changes after allergen provocation, suggesting that resident tissue cells and not circulating cells may be the most crucial targets (33, 34).

The lack of effect seen on the EAR differs from studies in both this model and other lung allergic challenge models using other adhesion molecule inhibitors. For example, VLA-4 inhibitors and/or selectin inhibitors were seen, under some circumstances, to reduce the EAR (5, 6). In the latter instances, the reduction of the EAR was associated with reduced mediator levels, suggesting that engagement of these cell surface molecules can affect signal transduction important for the release of mediators involved in the EAR. The present data indicate that at the doses used in this study, the {alpha}1 mAbs do not affect early mediator release.

Systemic blockade of {alpha}1ß1 is efficacious in numerous rodent models of inflammation (16–18). In preliminary experiments in the sheep model, a 45 mg dose of murine AQC2 given intravenously to two animals had no effect on the antigen-induced changes in airway function (data not shown). However, in our study in this same model, with a mAb HP1/2 to VLA-4 (1), we found that higher doses were required intravenously than aerosolized to achieve the same protection. This finding presumably reflects the increased efficacy of delivering drugs to the target organ. Studies of higher doses of mAb AQC2 were precluded because of material constraints, therefore the efficacy in allergic models of systemic {alpha}1 mAb treatment remains unresolved.

Both the human wild-type form and the human aglycosyl form of AQC2 appeared to provide equipotent protection against antigen-induced LAR and AHR when delivered as aerosols in the sheep model. Because aglycosyl mAbs have reduced Fc receptor–binding and therefore reduced cytotoxic activity in vitro and in vivo (20–22, 35), the comparable activity of the aglycosyl form suggests that blockade of {alpha}1-dependent adhesive interactions in the tissues, rather than Fc-mediated effects, are sufficient for efficacy. Such results would be consistent with previous data obtained in this model, showing that monovalent antibody binding fragments of HP1/2 provided equivalent protection to HP1/2 IgG against antigen-induced responses (36). Collectively, these findings support a mechanism related to inhibition of adhesion-dependent cell function.

The use of protein therapeutics to target asthma and other allergic diseases is increasing and includes anti-IgE mAbs (37) and interleukin-4 receptor blockade (38). Our experiments provide a clear demonstration of a role for the {alpha}1 pathway in allergic asthma, and as such, a humanized form of AQC2 might be a viable antagonist for intervention in human asthma. In addition, {alpha}1 belongs to a subset of integrin subunits that contain a 200 amino acid I domain within their extracellular region (25). Recent studies show that a related I domain containing integrin, LFA-1, can be antagonized by small molecules that bind to an allosteric pocket in the I domain (39). Whether the {alpha}1 I domain contains a similar allosteric site and whether small molecules selective for this site can be obtained remains to be determined.

In summary we have provided the first evidence that blockade of {alpha}1 binding can prevent the later pathophysiologic responses to allergen inhalation. These findings continue to add support for a central role for the extracellular matrix in inflammatory responses and for the use of antiadhesion therapy in asthma.


   Acknowledgments
 
W.M.A. received $10,000 in support of this project and these monies were used to offset expenses associated with the experiments; A.A. received $10,000 in support of this project and these monies were used to offset expenses associated with the experiments; I.S. received $10,000 in support of this project and these monies were used to offset expenses associated with the experiments; A.N.C. works as a full-time employee of Biogen Inc. and has stock and stock options in Biogen; J.F. works as a full-time employee of Biogen Inc. and has stock and stock options in Biogen; A.R.dF. worked as a full-time employee of Biogen Inc. and has stock and stock options in Biogen; E.A.G. works as a full-time employee of Biogen Inc. and has stock and stock options in Biogen; P.J.G. works as a full-time employee of Biogen Inc. and has stock and stock options in Biogen; V.E.K. works as a full-time employee of Biogen Inc. and has stock and stock options in Biogen; F.T. works as a full-time employee of Biogen Inc. and has stock and stock options in Biogen; R.R.L. worked as a full-time employee of Biogen Inc. from 1987 until 10/01, then as a part-time consultant until 10/02.

Received in original form April 17, 2003; accepted in final form October 16, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abraham WM, Sielczak MW, Ahmed A, Cortes A, Lauredo IT, Kim J, Pepinsky B, Benjamin CD, Leone DR, Lobb RR, et al. Alpha4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep. J Clin Invest 1994;93:776–787.
  2. Henderson WR, Chi EY, Albert RK, Chu SJ, Lamm WJ, Rochon Y, Jonas M, Christie PE, Harlan JM. Blockade of CD49d (alpha4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma. J Clin Invest 1997;100:3083–3092.[Medline]
  3. Jarjour NN, Calhoun WJ, Kelly EA, Gleich GJ, Schwartz LB, Busse WW. The immediate and late allergic response to segmental bronchopulmonary provocation in asthma. Am J Respir Crit Care Med 1997;155:1515–1521.[Abstract]
  4. de Monchy JGR, Kauffman HF, Venge P, Koeter GH, Jansen HM, Sluiter HJ, de Vries K. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am Rev Respir Dis 1985;131:373–376.[Medline]
  5. Abraham WM, Gill A, Ahmed A, Sielczak MW, Lauredo IT, Botvinnikova Y, Lin KC, Pepinsky B, Leone DR, Lobb RR, et al. A small-molecule, tight binding inhibitor of the integrin alpha(4)beta(1) blocks antigen-induced airway responses and inflammation in experimental asthma in sheep. Am J Respir Crit Care Med 2000;162:603–611.[Abstract/Free Full Text]
  6. Abraham WM, Ahmed A, Sabater JR, Lauredo IT, Botvinnikova Y, Bjercke RJ, Hu X, Revelle BM, Kogan TP, Scott IL, et al. Selectin blockade prevents antigen-induced late bronchial responses and airway hyperresponsiveness in allergic sheep. Am J Respir Crit Care Med 1999;159:1205–1214.[Abstract/Free Full Text]
  7. Wegner CD, Gundel RH, Reilly P, Haynes N, Letts LG, Rothlein R. Intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of asthma. Science 1990;247:456–459.[Abstract/Free Full Text]
  8. Gundel RH, Wegner CD, Torcellini CA, Clarke CC, Rothlein R, Smith CW, Letts LG. Endothelial-leukocyte adhesion molecule-1 mediates antigen-induced acute airway inflammation and late-phase bronchoconstriction in monkeys. J Clin Invest 1991;88:1407–1411.
  9. Belkin VM, Belkin AM, Koteliansky VE. Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development. J Cell Biol 1990;111:2159–2170.[Abstract/Free Full Text]
  10. Duband JL, Belkin AM, Syfrig J, Thiery JP, Koteliansky VE. Expression of alpha 1 integrin, a laminin-collagen receptor, during myogenesis and neurogenesis in the avian embryo. Development 1992;116:585–600.[Abstract]
  11. Hemler ME. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol 1990;8:365–400.[CrossRef][Medline]
  12. Rubio MA, Sotillos M, Jochems G, Alvarez V, Corbi AL. Monocyte activation: rapid induction of a1/b1 (VLA-1) integrin expression by lipopolysaccharide and interferon-g. Eur J Immunol 1995;25:2701–2705.[Medline]
  13. Bank I, Roth D, Book M, Guterman A, Shnirrer I, Block R, Ehrenfeld M, Langevitz P, Brenner H, Pras M. Expression and functions of very late antigen 1 in inflammatory joint diseases. J Clin Immunol 1991;11:29–38.[CrossRef][Medline]
  14. Takahashi H, Soderstrom K, Nilsson E, Kiessling R, Patarroyo M. Integrins and other adhesion molecules on lymphocytes from synovial fluid and peripheral blood rheumatoid arthritis patients. Eur J Immunol 1992;22:2879–2885.[Medline]
  15. Stemme S, Holm J, Hansson GK. T lymphocytes in human atherosclerotic plaques are memory cells expressing CD45RO and the integrin VLA-1. Arterioscler Thromb 1992;12:206–211.[Abstract/Free Full Text]
  16. de Fougerolles AR, Sprague AG, Nickerson-Nutter CL, Chi-Rosso G, Rennert PD, Gardner H, Gotwals PJ, Lobb RR, Koteliansky VE. Regulation of inflammation by collagen-binding integrins alpha1beta1 and alpha2beta1 in models of hypersensitivity and arthritis. J Clin Invest 2000;105:721–729.[Medline]
  17. Krieglstein CF, Cerwinka WH, Sprague AG, Laroux FS, Grisham MB, Koteliansky VE, Senninger N, Granger DN, de Fougerolles AR. Collagen-binding integrin {alpha}1ß1 regulates intestinal inflammation in experimental colitis. J Clin Invest 2002;110:1773–1782.[CrossRef][Medline]
  18. Fiorucci S, Mencarelli A, Palazzeti B, Sprague AG, Distrutti A, Morelli A, Novobrantseva G, Chirino G, Kotelianksy VE, de Fougerolles AR. Importance of innate immunity and collagen-binding integrin {alpha}1ß1 in TNBS-induced colitis. Immunity 2002;17:769–780.[CrossRef][Medline]
  19. Karpusas M, Ferrant J, Weinreb PH, Taylor FR, Garber EA. Crystal structure of the alpha1beta1 integrin I domain in complex with a antibody Fab fragment. J Mol Biol 2003;327:1031–1041.[CrossRef][Medline]
  20. Leatherbarrow RJ, Rademacher TW. Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor. Mol Immunol 1985;22:407–415.[CrossRef][Medline]
  21. Nose M, Wigzell H. Biological significance of carbohydrate chains on monoclonal antibodies. Proc Natl Acad Sci USA 1983;80:6632–6636.[Abstract/Free Full Text]
  22. Tao MH, Morrison SL. Studies of aglycosylated chimeric mouse-human IgG: role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol 1989;143:2595–2601.[Abstract]
  23. Forteza R, Botvinnikova Y, Ahmed A, Cortes A, Gundel RH, Wanner A, Abraham WM. The interaction of {alpha}1-proteinase inhibitor and tissue kallikrein in controlling allergic ovine airway hyperresponsiveness. Am J Respir Crit Care Med 1996;154:36–42.[Abstract]
  24. Scuri M, Botvinnikova Y, Lauredo IT, Abraham WM. Recombinant {alpha}1-proteinase inhibitor blocks antigen- and mediator-induced airway responses in sheep. J Appl Physiol 2002;93:1900–1906.[Abstract/Free Full Text]
  25. Gotwals P, Chi-Rosso G, Ryan ST, Sizing I, Zafari M, Benjamin C, Singh J, Venyaminov SY, Pepinsky RB, Koteliansky VE. Divalent cations stabilize the a1b1 integrin I domain. Biochemistry 1999;38:8280–8288.[CrossRef][Medline]
  26. Masopust D, Vezys V, Marzo A, Lefrancois L. Preferential localization of effector memory T cells in nonlymphoid tissue. Science 2001;291:2413–2417.[Abstract/Free Full Text]
  27. Reinhardt RL, Khoruts A, Merica R, Zell T, Jenkins MK. Visualizing the generation of memory CD4 cells in the whole body. Nature 2001;410:101–105.[CrossRef][Medline]
  28. Andreasen SO, Thomsen AR, Koteliansky VE, Novobrantseva TI, Sprague AG, de Fougerolles AR, Christensen JP. Expression and functional importance of collagen-binding integrins, {alpha}1ß1 and {alpha}2ß1, on virus-activated cells. J Immunol 2003;171:2804–2811.[Abstract/Free Full Text]
  29. de Fougerolles AR, Koteliansky VE. Regulation of monocyte gene expression by the extracellular matrix and its functional implications. Immunol Rev 2002;186:208–220.[CrossRef][Medline]
  30. Henderson WRJ, Tang LO, Chu SJ, Tsao SM, Chiang GK, Jones F, Jonas M, Pae C, Wang H, Chi EY. A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am J Respir Crit Care Med 2002;165:108–116.[Abstract/Free Full Text]
  31. Abraham WM. Of mice and men. Am J Respir Cell Mol Biol 2003;28:1–4.[Free Full Text]
  32. Holtzman MJ. Drug development for asthma. Am J Respir Cell Mol Biol 2003;29:163–171.[Abstract/Free Full Text]
  33. Leckie MJ, ten Brinke A, Khan J, Diamant Z, O'Connor BJ, Walls CM, Mathur AK, Cowley HC, Chung KP, Djukanovic R, et al. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyperresponsiveness, and the late asthmatic response. Lancet 2000;356:2144–2148.[CrossRef][Medline]
  34. Flood-Page PT, Menzies-Gow AN, Kay AB, Robinson DS. Eosinophil's role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003;167:199–204.[Abstract/Free Full Text]
  35. Isaacs JD, Clark MR. Therapy with monoclonal antibodies: an in vivo model for the assessment of therapeutic potential. J Immunol 1992;148:3062–3071.[Abstract]
  36. Lobb RR, Hemler ME. The pathophysiologic role of {alpha}4 integrins in vivo. J Clin Invest 1994;94:1722–1728.
  37. Babu KS, Arshad SH, Holgate ST. Omalizumab, a novel anti-IgE therapy in allergic disorders. Expert Opin Biol Ther 2001;1:1049–1058.[CrossRef][Medline]
  38. Borish LC, Nelson HS, Corren J, Bensch G, Busse WW, Whitmore JB, Agosti JM, Asthma Study Group. Efficacy of soluble IL-4 receptor for the treatment of adults with asthma. J Allergy Clin Immunol 2001;107:963–970.[CrossRef][Medline]
  39. Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C, Cottens S, Takada Y, Hommel U. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 2001;7:687–692.[CrossRef][Medline]



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