Primary and Tertiary Structures of the Fab Fragment of a Monoclonal Anti-E-selectin 7A9 Antibody That Inhibits Neutrophil Attachment to Endothelial Cells*

Adela Rodríguez-RomeroDagger §, Orna AlmogDagger , Maria TordovaDagger , Zafar Randhawaparallel , and Gary L. GillilandDagger

From the Dagger  Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, Rockville, Maryland 20850, the § Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán México, Distrito Federal 04510, and parallel  Otsuka Pharmaceutical, Inc., Rockville, Maryland 20850

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

The murine monoclonal IgG1 antibody 7A9 binds specifically to the endothelial leukocyte adhesion molecule-1 (E-selectin), inhibiting the attachment of neutrophils to endothelial cells. The primary and three-dimensional structures of the Fab fragment of 7A9 are reported. The amino acid sequence was determined by automated Edman degradation analysis of proteolytic fragments of both the heavy and light chains of the Fab. The sequences of the two chains are consistent with that of the IgG1 class with an associated kappa  light chain with two intrachain disulfide bridges in each of the heavy and light chains. The tertiary structure of the antibody fragment was determined by x-ray crystallographic methods at 2.8 Å resolution. The F(ab')2 molecule, treated with dithiothreitol, crystallizes in the space group P212121 with unit cell parameters a = 44.5 Å, b = 83.8 Å, and c = 132.5 Å with one Fab molecule in the asymmetric unit. The structure was solved by the molecular replacement method and subsequently refined using simulated annealing followed by conventional least squares optimization of the coordinates. The resulting model has reasonable stereochemistry with an R factor of 0.195. The 7A9 Fab structure has an elbow bend of 162° and is remarkably similar to that of the monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody Fab fragment. The 7A9 antigen combining site presents a groove resembling the structure of the anti-ICAM-1 antibody, and other antibodies raised against surface receptors and peptides. Residues from the six complementary determining regions (CDRs) and framework residues form the floor and walls of the groove that is approximately 22 Å wide and 8 Å deep and that is lined with many aromatic residues. The groove is large enough to accommodate the loop between beta -strands beta 4 and beta 5 of the lectin domain of E-selectin that has been implicated in neutrophil adhesion (1).

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Neutrophils are the major type of cell involved in the early stages of many forms of acute inflammation. The neutrophil accumulation in the lung can cause disease by damaging normal host tissue as in the case of adult respiratory distress syndrome (ARDS)1 (2), in the inflammation response (asthma, and graft rejection), or in insult as a result of trauma or bacterial infection. Cell surface receptors such as E-selectin (also called ELAM-1; endothelial leukocyte adhesion molecule) belong to a subclass of the IgG superfamily, including VCAM (vascular cell adhesion molecule) and ICAM (intercellular adhesion molecules 1, 2, and 3). E-selectin reacts with a fucosylated carbohydrate residue on the neutrophil (3), whereas the leukocyte integrins containing the CD18 antigen react with ICAM-1. The expression of E-selectin and ICAM-1 is induced by cytokines produced at the site of inflammation. This serves to enhance neutrophil adherence to endothelial cells and migration from the circulation into extracellular tissues at these sites. Neutrophils contribute further to the inflammatory process by releasing tissue-damaging mediators.

E-selectin is a member of the selectin gene family. Each of these proteins is composed of an amino-terminal lectin domain followed by an epidermal growth factor-like (EGF) domain, five complement regulatory repeat units, a single membrane-spanning region and a carboxyl-terminal cytoplasmic domain. The lectin and the EGF domains are both necessary and sufficient to mediate neutrophil adhesion (4). The three-dimensional structure of the lectin/EGF domains that includes the ligand-binding region has been reported for this molecule (1). A specific surface region of the lectin domain of E-selectin that contains a loop between beta -strands beta 4 and beta 5 with exposed Tyr-94 and Arg-97 side chains has been implicated in ligand binding based on the structural and related biochemical studies (1).

Antibodies and antibody fragments specific for the cell-surface receptors have the potential for modulating the interaction of neutrophils with endothelial cells, and hence, the inflammatory response. A monoclonal anti-ICAM-1 antibody R6.5, that inhibits the attachment of neutrophils to endothelium and also prevents the attachment of major group human rhinovirus (HRV) to ICAM-1, has been reported (5). Recently, the structure of the Fab fragment of this antibody was reported at 2.8 Å resolution (6). The surface contour of the antigen-combining site of this molecule has a groove that resembles more the structure of an antipolypeptide antibody than the structure of an antiprotein antibody.

Another potent blocking antibody directed at a cell surface receptor, E-selectin in this case, has been raised. This antibody binds to the lectin/EGF domain of E-selectin.2 We report here the primary and three-dimensional structures of the Fab fragment determined by automated Edman degradation analysis of proteolytic fragments of the heavy and light chains of the antibody and by x-ray crystallographic techniques at 2.8 Å resolution, respectively. The analysis of the structural studies of the anti-E-selectin 7A9 Fab presented here focuses on the complementary determining regions and the comparison with the three-dimensional structures of an anti-ICAM Fab and the anti-human rhinovirus Fab.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Antibody Production-- The perfusion system used was a 50-liter stirred tank bioreactor utilizing an external rotating filter. SP-20 fusion mouse-mouse hybridoma cells were cultured in a medium with a protein-free formulation, and 0.1% Pluronic F-63 (Sigma) was added for shear protection. The cell densities were maintained at 1 × 107 viable cell counts/ml and perfusion-adjusted to keep nutrients (glucose, etc.) and metabolites at the appropriate levels. Ion exchange and affinity chromatography were used to purify the antibody.

Generation and Purification of F(ab')2-- The conversion of 7A9 antibody to F(ab')2 was accomplished by incubating the IgG1 with pepsin (E:S of 1:100) for 2 h at 37 °C, after adjusting pH to 3.5 using 1.0 M citrate buffer. The digestion products were subjected to cation-exchange chromatography in an S-Sephacryl fast flow column, using a linear sodium chloride gradient (0.1-0.5 M) in 20 mM acetate buffer, pH 5.0, as eluant. F(ab')2 eluted as a single peak with purity greater than 97%, as judged from SDS-polyacrylamide gel electrophoresis analysis (results not shown).

Flow Cytometry Analysis-- Titration analysis of the intact antibody and of the F(ab')2 was performed using a Coulter FACS Elite Analyzer. Samples for the flow cytometry analysis of endothelial cells (human umbilical vein endothelial cell, HUVEC) included stimulation with and without interleukin-1beta (IL-1beta ). This assay measures the number of cells that shift to greater fluorescence intensity after specific binding (positive response). In this analysis, the intact 7A9 antibody and the F(ab')2 fragment showed comparable binding efficiency to the surface of HUVEC cells on a molar basis, indicating that the digestion with pepsin did not affect its binding ability (Fig. 1A).


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Fig. 1.   A, FACS analysis of the intact 7A9 antibody and the F(ab')2 fragment was performed in the presence (+) and absence (-) of IL-1beta (1 mg/ml). The analysis was performed on a Coulter (Epex) FACS analyzer. B, effect of 7A9 F(ab')2 on granulocyte adhesion of HL60. The confluent HL60 in 16-mm wells were left untreated (-) or treated (+) with 1 mg/ml IL-1beta for 4 h. After rinsing to remove IL-1beta , cells were incubated with F(ab')2 at the indicated concentration for 30 min at 37 °C. Results are expressed as cells bound/well.

Biological Activity-- Biological activity was based on inhibition of HL60 (a myeloid leukemia line from ATCC) binding to soluble E-selectin assay (7). Ten µg/ml of the 7A9 antibody F(ab')2 fragment were added in wells, followed by 51Cr-labeled HL60 cells (105/well) and incubated for 30 min at 22 °C in a static adhesion assay. Nonadherent cells were removed by rinsing with 1% bovine serum albumin in 0.1 M phosphate-buffered saline, and the residual activity in the wells was measured in a gamma  counter. The details of the assay have been previously described (8). Fig. 1B shows that the anti-E-selectin antibody F(ab')2 fragment is inhibitory to granulocyte binding to HL60 cells in a dose-dependent manner.

Primary Structure Determination-- The amino acid sequences of the F(ab')2 heavy and light chains were determined after separating both chains of the reduced, alkylated intact 7A9. The samples were reduced with dithiotreitol, alkylated with vinylpyridine or iodoacetamide, and purified by reverse-phase high performance liquid chromatography. Each purified chain was subjected to automated Edman degradation analysis. Additionally, a reduced alkylated sample from purified F(ab')2 was digested with Lys-C protease, and the resulting fragments were separated by reverse-phase high performance liquid chromatography using a C-18 column. The amino acid sequences of the purified peptides were determined by using a protein microsequencer (ABI, Model 477-A) coupled to an on-line PTH-Analyzer (Model 120-A) and a 900-A data reduction module. Digestion with Lys-C protease resulted in 26 peaks for the heavy and light chains of an Fab fragment (chromatogram not shown). Fig. 2 shows the complete amino acid sequence for each of the peaks in the heavy and light chains of the fragment. The sequences of the two chains are consistent with that of the IgG1 class with an associated kappa  light chain. Both the heavy and light chains have two intra-chain disulfide bridges. The sequences of the six CDRs are indicated in Fig. 2.


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Fig. 2.   Amino acid sequences of the light and heavy chains of antibody 7A9 (variable and constant domains). Residues in the complementary determinant regions (29) are underlined. Numbers are sequential. Peptides obtained after digestion with Lys-C protease are labeled with K.

Crystallization-- The F(ab')2 was incubated before the crystallization trials with dithiotreitol for 19 h at a ratio of 2:1 (w/w). The initial crystallization trials were performed using a "fast screen" approach (9) employing the Hampton Research Crystal Screen kit (10). Experiments were set up using the hanging drop vapor diffusion method (11). For the screen, the crystallization drops were prepared by mixing 3 µl of a solution containing 9.6 mg/ml protein in 10 mM HEPES/HCl at pH 7.0 with an equal volume of the reservoir solution. Initially, small crystals appeared in droplets equilibrated against well solutions containing 0.2 M (NH4)2SO4, 0.1 M sodium cacodylate, 30% (w/v) PEG8k at pH 6.5. Refinement of the crystallization conditions was performed using sitting drop vapor diffusion experiments (12). The final reservoir solution consisted of 0.2 M (NH4)2SO4, 0.1 M sodium cacodylate, and 20% (w/v) PEG8k at pH 6.5. Crystals appeared within 3 days at room temperature and grew to full size within 2 weeks (typical dimensions of 0.2 × 0.3 × 0.7 mm3).

X-ray Data Collection and Processing-- The diffraction data were collected at room temperature using a Siemens electronic area detector. This detector was mounted on a Rigaku RU-200 generator operated at 40 kV, 60 mA with a 0.3-mm focal spot. A graphite monochromator followed by a 0.5-mm collimator was used. During data collection, the area detector was mounted 16 cm from the crystal and 2theta was 16°. Diffraction data collected with the area detector were electronic images, each comprising a 0.2° oscillation counted for 3 min. The determination of unit cell parameters, crystal orientation, and the integration of the reflection intensities were carried out with the XENGEN program system (13). The crystals are orthorhombic, belonging to space group P212121 with unit cell constants a = 44.5 Å, b = 83.8 Å, and c = 132.5 Å. The volume of this cell indicates one Fab molecule in the asymmetric unit with a Vm (14) of 2.66 Å3/dalton, corresponding to a solvent content of 53%. From a total of 42,928 observations extending to 2.8 Å resolution, a unique data set of 10,993 reflections (86.5% complete; I > 2.1 sigma (I)) was obtained with a merging R-factor of 0.068.

X-ray Structure Determination and Refinement-- The structure of the 7A9 Fab fragment was determined by molecular replacement (15) followed by iterations of expanding and adjusting the model to fit the electron density map and refining the structure. The molecular replacement and refinement calculations employed the X-PLOR program package (16). All the model building and correction procedures were carried out using the program QUANTA Version 4.1.1 (Molecular Simulations Inc., Burlington, MA).

The search model used in the molecular replacement calculations was based on the crystal structure of the Fab 8F5 that neutralizes human rhinovirus serotype 2 (Protein Data Bank code 1BBD) (17). This Fab has a high percentage of sequence identity with the 7A9 Fab, 96% for the light chain and 83% for the heavy chain, although its elbow bend angle is the smallest reported to date (127°). The search probe was constructed by truncating the side chains at the CB or CA atoms for all corresponding residues that were different between the 8F5 and the 7A9 Fabs.

The rotation search was done using the intact probe with variable "elbow bend" angles between variable (V) and constant (C) domains ranging from 127° to 167° (16). The largest peak in the rotational function search was 6 sigma  above background at the Eulerian angles alpha  = 94.1°, beta  = 15.0°, and gamma  = 314.1°. The Patterson correlation refinement of the rotation function peak gave final angles of alpha  = 93.5°, beta  = 16.2°, and gamma =315.7°, with a correlation coefficient of 0.018. The solution was obtained at an elbow angle of 162°. Using this rotation function solution, the translation function search produced a solution at x = 8.9, y = 25.1, and z = 57.9. It was 8.5 sigma  above the background and 2.2 sigma  above the next highest peak. The application of the rotation and translation solutions to the probe molecule gave a crystallographic R (R = Sigma |Fo - Fc|/Sigma Fo, summed over all h, k, l indexes) value of 0.49.

The molecular replacement solution was subjected to rigid body refinement using 8-4 Å data with an overall temperature factor of 15 Å2. Each of the four domains, VH, CH, VL, and CL (variable and constant domains of heavy and light chains), was allowed to move independently to correct the position of the molecule in the unit cell. After this, R was reduced to 0.38. Next, a positional refinement followed by simulated annealing with a slow-cooling protocol (18) was carried out using data between 8.0 and 2.8 Å. The Engh and Huber (19) geometric parameters for amino acid residues were used as the basis of the protein force field. At this point, map fitting, using 2Fo-Fc and Fo-Fc maps, was used to expand the model to include omitted residues and side chains. Two additional cycles of simulated annealing refinement and map fitting were then carried out. At this stage, residues His136-His140 and 45% of the residue side chains were included, reducing the R factor to 0.206. Then an overall temperature factor was refined followed by group temperature factor refinement for each amino acid residue. A number of cycles of map fitting followed by stereochemical restrained least squares refinement were carried out to complete the structure determination producing a final crystallographic R factor of 0.195. A summary of the data collection and refinement statistics is presented in Table I. Ten water molecules were included in the model.

                              
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Table I
Data collection and refinement statistics of the Fab fragment of the anti-E-selectin 7A9 antibody

Structure Analysis and Comparison-- Analysis of the stereochemistry of the intermediate and final models was done using the PROCHECK program package (20). The molecular comparisons were carried out with the ALIGN program (21) using only the CA atom positions of the polypeptide backbone. Surface curvature and electrostatic calculations were performed using the program GRASP (22).

    RESULTS AND DISCUSSION
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Introduction
Procedures
Results & Discussion
References

Molecular Structure-- The tertiary and quaternary structure of this molecule is consistent with all the crystallographically determined Fab structures. It presents an elbow angle (between the pseudodyad axes relating the V and C domains of the two chains (23)) of 162°, 35° larger than the search model (Fig. 3). The four domains of the Fab are characterized by two beta -sheets packed closely against each other with a disulfide bridge connecting them (VL, Cys23-Cys94; VH, Cys22-Cys96; CL, Cys140-Cys200; and CH, Cys147-Cys199).


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Fig. 3.   Schematic diagram presenting the 7A9 Fab fragment. The constant (C) and variable (V) domains of the light and heavy chains are labeled. The antigen binding site is formed from CDR loops L1, L2, and L3 from the light chain and H1, H2, and H3 from the heavy chain. The figure was prepared using TKRASTER 3D (developed by Hillary Gilson at the National Institute of Standards and Technology, from RASTER 3D (37)).

The final coordinates of the Fab fragment consist of 437 amino acid residues, 220 residues for the light chain fragment and 217 residues for the heavy chain. cis-Proline residues occur at positions 8, 101, and 147 of the light chain and positions 154 and 156 of the heavy chain (residue numbering refers to Fig. 2). The final 2Fo-Fc electron density map, contoured at 1 sigma , shows good overall agreement with the structure. The electron density map corresponding to the L3 CDR is shown in Fig. 4. The hydrophobic core of the subunits as well as the interfaces between heavy and light chains are in strong, continuous electron density. The regions of the map corresponding to the CDR loop regions are also well defined except for residues 100-104 at the tip of the H3 CDR loop, where weak density is observed (these residues have been omitted from the final model). Also, the electron density for the distal extremity of the constant region, in particular residues 138-140, is ill defined, and their conformation must be considered as tentative. Side chains of residues 17 and 114 of the light chain and 13, 105, 139, 140, and 194 of the heavy chain show poorly defined electron density and were therefore not modeled beyond CB.


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Fig. 4.   Stereoview of the 2.8 Å |2Fo-Fc| electron density map in the vicinity of L3 CDR contoured at 1 sigma .

The root mean square deviations of coordinates from the stereochemical standards on both bond lengths and angles were 0.015 Å and 2.1°, respectively, and the average temperature factor is 15 Å2. The Ramachandran plot (24) indicates that the main-chain torsion angles (Phi  and Psi ) fall within the energetically favored regions, except for Ala57 of the light chain, that is located in the L2 CDR. This constrained conformation has been reported to occur in other proteins, specifically at the active site (25). The geometry of the side chain dihedral angles was also examined. Nearly all values lie in the expected regions. An analysis of the structure shows that almost 80% of the 437 residues with side chain dihedrals adopt preferred rotamer conformations.

Overall, the heavy chain residues have higher average temperature factors than the light chain residues. The variations of residue-averaged temperature factors follow closely the alternating beta -strands and loops along the polypeptide chain. The loops at the end of the constant region exposed to the solvent and essentially not involved in crystal packing interactions appeared more flexible, with large temperature factors. The highest B factors are in the solvent exposed loop areas indicating their flexibility and/or partial disorder.

At the carboxy ends of both light and heavy chain are regions of unexplained non-continuous density. This could be the result of pepsin digestion and dithiothreitol treatment, indicating different species with different lengths for the carboxy end of the heavy chain.

The Antigen Combining Site-- The most interesting feature of the structure is the surface of the antigen combining site that presents an irregular concave surface (Fig. 5). Currently it is known that five out of the six hypervariable loops that form the antigen-binding site are limited to a few main-chain conformations (canonical) (26). These conformations can be predicted by the size of the CDR and the occurrence of a specific set of residues that produce a known conformation. Recent studies (27) indicate that antibodies exhibit a surprisingly small number of combinations of canonical structures. Moreover, it has been shown (28) that the shapes allowed by these combinations correlate, in some cases, with the type of antigens with which the antibody interacts. Four different groups of antibody-antigen topographies have been distinguished: concave (for small haptens), moderately concave, grooves (for peptides and carbohydrates), and planar (for proteins).


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Fig. 5.   A superposition of the CA chains of the Fv fragments of anti-E-selectin (7A9) (VL in blue and VH in magenta), anti-ICAM-1 (R6.5) (green), and anti-human rhinovirus (8F5) (yellow). The drawing was made using the RIBBONS program (38).

The CDRs of 7A9 are canonical: the L1 CDR belongs to the canonical group 3, the L2 CDR to group 1, the L3 CDR to group 1, the H1 CDR to group 1 and the H2 CDR to group 2. No canonical groups have been identified for the H3 CDR (29). Then it belongs to the class 1-2-3-1-1. Major conclusions drawn from the analysis of the surface of the combining site are that this antibody displays a groove found at the interface between the heavy and light chain variable domains and that L1 CDR points out into the solvent (Fig. 5). Residues from the six CDRs and framework residues form the floor and walls of the groove. This groove is approximately 22 Å wide and 8 Å deep, and it is lined with many aromatic residues. It is worth mentioning that 8F5 is the only antibody of known three-dimensional structure, that belongs to the same canonical class, 1-2-3-1-1, and displays a similar groove in the combining site (17). This type of topography has been observed for peptide binding antibodies

Since the lectin activity of E-selectin is abolished when it combines the 7A9 Fab, it is possible that the loop containing the amino acid residues that are essential for the activity (Tyr94 and Arg97) could partially fit this Fab groove at the combining site. Similar topography has been observed also for other antireceptor antibodies, like the anti-ICAM-1 antibody (6), or those that bind peptides, in particular peptides that adopt a beta -turn conformation (30).

Comparison of Anti-E-selectin, Anti-ICAM, and Anti-human Rhinovirus Fabs-- Fig. 5 shows the CA superposition for the variable fragments (Fv) of the three antibodies, 7A9, and R6.5 (Protein Data Bank code 1RMF) (6) directed against cell receptors E-selectin and ICAM-1, and the Fv of the anti-human rhinovirus 85F (Protein Data Bank code 1BBD) (17), used for the molecular replacement analysis. The combining sites of 7A9 and 8F5 belong to the same canonical class, while R6.5 has an extra residue in the L1 CDR and belongs to class 1-2-4-1-1. Structurally it is clear that these Fv fragments are remarkably similar. For the VL domain, the main differences can be appreciated in the L1 CDR loops, due mainly to differences in length. However, in the three structures, this loop is pointing into the solvent. A greater variation is observed for the VH domain. At this stage of the refinement, we cannot conclude anything concerning H3 CDR because we could not find clear density for the five residues at the tip of the loop. On the other hand, this is the most variable loop among antibodies.

The conformation of H2 CDR is very similar for 7A9 and 8F5; however, R6.5 shows a conformation not observed before for this type of region. It is possible that this loop had been incorrectly built (31). Evidently, the topology of the binding site is approximately the same, despite some slight differences in the conformation of some loops. To stress this fact, the molecular surface of 7A9 and R6.5 Fabs is shown in Fig. 6. Clearly, both antibodies present a groove at the antigen binding site.


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Fig. 6.   A comparison of the surface contour of R6.5 and 7A9 CDRs. Concave features are shown in green; convex features are shown in gray. The drawings were made using the GRASP program (22).

Conclusions-- The feature most frequently observed in antibody-peptide complex structures is a beta -turn in the peptide that is embedded in the antibody-antigen interface. Several examples of this recognition motif have been described to date and include a type I beta -turn, reported for the VP2 peptide in complex with 8F5 (32) and a type II beta -turn for the HIV-1 peptide (gp120) in the 59.1 complex (33). Shoham (30) had suggested that antipeptide antibodies seemed to induce a beta -turn conformation in the bound peptide, irrespective of the peptide sequences. However secondary structure predictions for the sequences have shown that they have a tendency to form the turn types found in the Fab-peptide structures. Therefore, it would be expected for these peptides to mimic their protein counterparts (30, 34, 35). In 1995, Tormo et al. (36) reported a model for 8F5 IgG docked onto the viral surface through the peptide in VP2, which adopted the same conformation. On the other hand, Jedrzejas et al. (6) proposed that R6.5 could bind ICAM-1 through a beta -turn in domains D1 or D2.

The antigen combining site of 7A9 is consistent with those observed for Fab directed against proteins that have epitopes that adopt a loop conformation. It is very possible that 7A9 antibody could bind the loop connecting beta 4 and beta 5 on the lectin domain of E-selectin, which contains the two residues essential for neutrophil adhesion (1). Docking experiments of 7A9 Fab fragment and the lectin/EGF domains of E-selectin are now in progress. Oligopeptide libraries are also being tested for this antibody.

    ACKNOWLEDGEMENTS

We thank Drs. Patricia Hughes, James Wilkins as well as Tom Rohrer, Maurice Guertin, Cathy Kletke, Bob Dunn, and Magdalena Bystricka for performing fermentation, purification, and characterization work, Norma Gabor and Dawson Beall for the contribution in performing bioassay, and Dr. Juan Carlos Almagro for valuable comments. Dr. Adela Rodríguez-Romero thanks the CONACyT and the Dirección General de Asuntos del Personal Academico, UNAM, for support for sabbatical leave at CARB.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1a5f and r1a5fsf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

To whom correspondence should be addressed: The National Institute of Standards and Technology, 9600 Gudelsky Dr., Rockville, MD 20850.

1 The abbreviations used are: ARDS, adult respiratory distress syndrome; E-selectin, endothelial leukocyte adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; EGF, epidermal growth factor; FACS, fluorescence-activated cell sorter; Fv, variable domain fragment; HUVEC, human umbilical vein endothelial cell; IL-1beta , interleukin-1beta ; PEG8k, polyethylene glycol with an average molecular weight of 8,000; CDR, complementary determining regions.

2 W. Newman, unpublished observation.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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