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J. Biol. Chem., Vol. 279, Issue 17, 17875-17887, April 23, 2004

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Function-blocking Integrin _v6 Monoclonal Antibodies

DISTINCT LIGAND-MIMETIC AND NONLIGAND-MIMETIC CLASSES^*

Paul H. Weinreb, Kenneth J. Simon, Paul Rayhorn, William J. Yang, Diane R. Leone, Brian M. Dolinski, Bradley R. Pearse, Yukako Yokota, Hisaaki Kawakatsu, Amha Atakilit, Dean Sheppard, and Shelia M. Violette

From the Biogen Idec, Inc., Cambridge, Massachusetts 02142 and Lung Biology Center, Cardiovascular Research Institute, and Department of Medicine, University of California, San Francisco, California 94143

Received for publication, November 4, 2003 , and in revised form, January 13, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse _v6 integrin with high affinity (up to 15 pM). A subset of these antibodies blocked the binding of _v6 to the transforming growth factor-1 latency-associated peptide with IC₅₀ values as low as 18 pM, and prevented the subsequent _v6-mediated activation of transforming growth factor-1. The antibodies also inhibited _v6 binding to fibronectin. The blocking antibodies form two biochemical classes. One class, exemplified by the ligand-mimetic antibody 6.8G6, bound to the integrin in a divalent cation-dependent manner, contained an RGD motif or a related sequence in CDR3 of the heavy chain, was blocked by RGD-containing peptides, and was internalized by _v6-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for _v6 and showed no cross-reactivity with the RGD-binding integrins _v3, _v8,or _IIb3. The nonligand-mimetic blocking antibodies, exemplified by 6.3G9, were cation-independent, were not blocked by RGD-containing peptides, were not internalized, and did not contain RGD or related sequences. These two classes of antibody were unable to bind simultaneously to _v6, suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for _v6 and resemble those described for _IIb3. We also report for the first time the relative abilities of divalent cations to promote _v6 binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of _v6 as well as the role of _v6 in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Integrins are heterodimeric cell-surface receptors that have been implicated in regulating a variety of processes by mediating cell adhesion, migration, and signaling (1). At least 24 different integrin heterodimers can be formed from the 18 and 8 chains that have been identified. The pairing of and subunits determines the ligand binding specificity of integrins. For example, some integrin heterodimers specifically bind to ligands containing the tripeptide sequence RGD. This group of RGD-binding integrins includes the platelet integrin _IIb3, some of the ₁ integrins, and all of the _v integrins. Although the _v subunit can pair with multiple subunits (₁, ₃, ₅, ₆, and ₈), the ₆ subunit can only pair with _v and not with any other integrin chains. _v6 is unique in that its expression is restricted to epithelial cells (2). Although it is present at low or undetectable levels in normal adult tissue, _v6 is rapidly up-regulated during development, injury, wound healing, and neoplasia (2-7). Ligands for _v6 that have been identified through in vitro binding experiments include fibronectin, tenascin, and the transforming growth factor- (TGF-)¹ latency-associated peptide (LAP) (8-11). As a result of binding to these ligands, _v6 can mediate cell adhesion, spreading, migration, proliferation, and activation of latent TGF-.

Although the importance of interactions between RGD-binding integrins and extracellular matrix proteins such as fibronectin is well established, the role of LAP as an integrin ligand is just beginning to emerge. There are three different human LAP gene products (LAP1, LAP2, and LAP3) that are produced as N-terminal prodomains of the three closely related TGF- isoforms (TGF-1, TGF-2, and TGF-3). The TGF-s are multifunctional cytokines that influence numerous cellular processes including regulation of inflammation, production of extracellular matrix, and regulation of cell growth and differentiation. Each TGF- isoform is expressed as a propeptide with the corresponding LAP at its N terminus. Following translation, the propeptide undergoes intracellular proteolysis, and the resultant LAP and TGF- fragments associate to form a complex comprising a dimer of TGF- noncovalently associated with a dimer of LAP (termed the "small latent complex"). This complex associates with the latent TGF--binding protein (LTBP-1) prior to secretion, in what is referred to as the "large latent complex" (12). As the name implies, the latent TGF- complex is inactive and requires activation in order to produce active cytokine (13-15). Although a number of in vitro activation methods have been described (e.g. low pH, proteolysis, radiation, and interactions with proteins such as thrombospondin and _v6 integrin), the mechanism(s) of physiological TGF- activation have not been fully elucidated (for reviews see Refs. 13 and 14). All five of the _v integrins (16-18), as well as ₈₁ (19), have been shown to bind to RGD sequences contained in LAP1 and LAP3 in vitro, whereas LAP2, in which the RGD sequence is replaced by SGD, does not interact with any of these integrins. These interactions, as with other integrin-ligand interactions, are dependent on the presence of divalent cations. Integrins _v6 and _v8, but not _v1, _v5, or ₈₁, are able to activate latent TGF-1 upon binding, and _v6, as shown recently, can activate latent TGF-3 (20). The process by which _v6 activates latent TGF- is not fully understood, although one model proposes that integrin binding induces a conformational change in the latent TGF- complex, which allows binding of TGF- to its type II receptor (11).

The ability of _v6 to activate TGF-1 and TGF-3 offers a novel mechanism for local activation of these multifunctional cytokines and suggests that this integrin may contribute to the onset of TGF--mediated disease processes. The in vitro data are supported by studies with ₆-null mice, which have low levels of inflammation in the lung and skin, consistent with inactivation of TGF- (21). These mice are also protected in a bleomycin-induced pulmonary fibrosis model, suggesting that inhibition of _v6 might provide a therapeutic benefit by blocking TGF- activation locally in affected tissues. In addition, blocking the binding of _v6 to other ligands, such as fibronectin, might be useful for treating TGF--independent disease processes by inhibiting the adhesive or migratory functions of _v6.

The development of monoclonal antibodies that bind to specific integrin heterodimers and block ligand binding and functional activity has provided an important tool for understanding the structure and function of integrins. For this reason, we were interested in generating reagents that would specifically bind to the _v6 heterodimer and block its ability to activate latent TGF-. We describe here the generation and characterization of selective, high affinity monoclonal antibodies that block the binding of _v6 to LAP. These antibodies were generated by immunizing ₆-deficient mice with the _v6 integrin, allowing for the production of antibodies that recognize both the human and murine _v6 integrins. Among these antibodies are the first ligand-mimetic antibodies to be described for _v6. These antibodies provide a means to study the mechanism of _v6-mediated TGF- activation in vitro, and to test whether _v6 inhibition can effectively block TGF--mediated pathologies in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The ₆ -/- mice were prepared as described (21). Recombinant human TGF-1 LAP was purchased from R & D Systems (Minneapolis, MN). Antibody 10D5 (9) was purchased from Chemicon (Temecula, CA). The L230 (anti-_v) and AP-3 (anti-₃) hybridomas were purchased from ATCC, and the antibodies were purified from the supernatant of saturated cultures by affinity chromatography on immobilized protein A. The anti-₈ antibody 14E7 was a generous gift of Steve Nishimura (University of California, San Francisco). Isotyping of antibodies was carried out using the Isostrip kit (Roche Applied Science) according to the manufacturer's instructions. The human ₆-transfected SW480 (human colorectal adenocarcinoma) cell line (SW4806) was prepared as described (8). The synthetic peptide acetyl-GGLRRGDRPSLRYAMDS-CONH₂, derived from the 6.8G6 CDR sequence, was kindly provided by Dr. J. H. Cuervo (Biogen) and was prepared using standard solid-phase synthetic methods. The SCC-14 cell line was a gift from Dr. H. Larjava (University of British Columbia) and Dr. R. Grenman (University of Turku, Finland). HT-29 cells were purchased from the ATCC.

Purification of hs_v6—The recombinant human secreted _v6 protein (hs_v6) was purified from the supernatant of transfected CHO cells, essentially as described (8), by affinity chromatography using anti-_v antibody L230. Purified L230 was cross-linked to cyanogen bromide-activated Sepharose 4B (Sigma) at a ratio of 4.8 mg of anti-body/ml resin. The _v6 supernatant was loaded (0.5 mg of antibody/ml resin) onto the L230 affinity column, and the column was washed with 10 column volumes each of the following: 1) 50 mM Tris-Cl, pH 7.5, 1 M NaCl, 1 mM MgCl₂; 2) 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl₂; and 3) 10 mM sodium phosphate, pH 7.0. The hs_v6 was eluted with 100 mM glycine, pH 2.5, into 1 M sodium phosphate, pH 8.0 (1:10 by volume). Protein was dialyzed with several changes against PBS and stored at -20 °C.

Biotinylation of Proteins—Purified hs_v6 protein or antibody (2 mg) was incubated with 10 M equivalents of sulfo-NHS-biotin (Pierce) in 1 ml of 50 mM NaHCO₃, pH 8.3, at 25 °C for 45 min. Unreacted label was removed by desalting on 20 ml of Sephadex G-25M (Amersham Biosciences).

Generation of Stably ₆-Transfected FDC-P1 and NIH3T3 Cells—Murine ₆-transfected NIH3T3 and FDC-P1 cells were generated by electroporating parent cell lines with a DNA construct containing full-length murine ₆ cDNA cloned from murine lung cDNA and a neomycin selectable marker. Stable transfected cells were selected by passaging cells in culture medium containing 1 mg/ml G418 (Invitrogen) for 14 days followed by flow cytometry to isolate cells expressing the highest level of surface-expressed murine ₆. Transfected FDC-P1 cells were cultured in DMEM supplemented with 4 mM L-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 1.0 mM sodium pyruvate, 10% FBS, 2.5% mouse IL-3 culture supplement, and +1.5 mg/ml active G418. Transfected NIH3T3 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, penicillin/streptomycin, and 1 mg/ml active G418.

Generation of Human LAP-Fc—The 833-bp human LAP cDNA sequence was cloned from human kidney cDNA using reverse-transcriptase PCR. A mutation of cysteine to serine was incorporated at amino acid 33 to eliminate aggregation during production of protein (22). Mutant human LAP cDNA was then ligated with the human IgG1 Fc cDNA (23) into the PV90 expression vector (24). CHO cells were transfected with the human LAP-IgG1 expression construct, and stable transfected cells producing fusion protein were selected by expanding in minus minimum essential medium supplemented with 10% dialyzed fetal bovine serum (Hyclone Laboratories, Logan, UT) and 2 mM glutamine (Invitrogen). The protein was purified from the supernatant of CHO cell cultures on protein A-Sepharose 4 FF, as described for the purification of monoclonal antibodies.

Production of Monoclonal Antibodies—₆ -/- mice were immunized by intraperitoneal injection with hs_v6 in complete Freund's adjuvant (CFA) (fusion 6, designated with the prefix 6). Alternatively, ₆ -/- mice were immunized with ₆-transfected NIH3T3 cells, and the same mice were immunized intraperitoneally at an adjacent site with 100 µl of CFA (fusion 7, designated with prefix 7). Two weeks and 4 weeks after the initial immunization mice were boosted similarly with the same reagents with the exception that incomplete Freund's adjuvant was used in place of CFA. Three days prior to isolating spleens for fusions mice were immunized with 12.5 µg of purified recombinant human _v6 protein by both intraperitoneal and intravenous injection. Mice were sacrificed and spleens were removed, and B-cells were teased into single cell suspensions and immortalized by fusion to a drug-selectable cell fusion partner (FL653). Screening for anti-_v6 antibodies was carried out as described under "Results," and select clones were subcloned using flow cytometry and stored frozen.

Purification of Antibodies—Antibodies were purified from hybridoma supernatants using protein A affinity chromatography. For the IgG2a isotype antibodies, the supernatant was directly loaded onto protein A-Sepharose 4 Fast Flow (Amersham Biosciences). The column was washed with PBS, and the IgG fraction was eluted using 25 mM phosphoric acid, 100 mM NaCl, pH 2.8, into 1:20 volume of 0.5 M sodium phosphate, pH 8.6. For the murine IgG1 isotype antibodies, the super-natant was adjusted to 1.5 M glycine, 3 M NaCl, pH 8.9, prior to loading, and the column was washed with 25 mM sodium phosphate, 3 M NaCl, pH 8.6, prior to elution. The eluate from the protein A chromatographic step was adjusted to pH 8.6 using 2 M Tris base, diluted 10-fold with water, and loaded onto a Q-Sepharose column (20 mg of protein/ml resin) that had been equilibrated in 10 mM sodium phosphate, 25 mM NaCl, pH 8.6. The column was washed with 5 column volumes of equilibration buffer, and protein was eluted using 25 mM sodium phosphate, 150 mM NaCl, pH 7.2. Solutions of purified protein were sterile-filtered (0.22 µm) and stored at -70 °C until use.

Flow Cytometry—Cells were harvested by trypsinization, washed once in phosphate-buffered saline, and then resuspended in FC buffer (1x PBS, 2% FBS, 0.1% NaN₃,1mM CaCl₂,and1mM MgCl₂). 0.2 x 10⁵ cells were then incubated on ice for 1 h in FC buffer containing hybridoma supernatant or purified antibody in a total volume of 100 µl. After incubation cells were washed two times with ice-cold FACS buffer and resuspended in 100 µl of FC buffer containing 5 µg/ml phycoerythrinconjugated donkey anti-mouse IgG (Jackson ImmunoResearch) and incubated on ice for 30 min. Cells were then washed two times with ice-cold FC buffer and resuspended in 200 µl of FC buffer. Binding of the labeled secondary antibody was monitored by flow cytometry.

Solid-phase _v6 Binding Assay (ELISA)—A 96-well microtiter plate was coated with 50 µl/well of 5 µg/ml hs_v6 at 4 °C, overnight. The plate was washed with 0.1% Tween 20 in PBS in an automated plate washer, and 180 µl/well of 3% BSA in TBS was added for 1 h at 25 °C to block nonspecific binding. The plate was washed as above, and dilutions of either hybridoma supernatant (for screening assays) or purified antibody (for characterization) in buffer A (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mg/ml BSA) and either 1 mM CaCl₂ + 1 mM MgCl₂, 1 mM MnCl₂, or 10 mM EDTA were added (50 µl/well). The plate was incubated for 1 h at 25 °C, washed, and incubated for 1 h with 50 µl/well of peroxide-conjugated goat anti-mouse IgG + A + M antibody (Cappel/ICN, 1:4000 dilution) or goat anti-human Fc antibody (Cappel/ICN, 1:4000 dilution). Bound antibody was detected using 3,3',5,5'-tetramethylbenzidine. In the antibody competition experiments, biotinylated antibody (0.1 µg/ml) and unlabeled antibody were added simultaneously, and the secondary antibody was substituted with 50 µl/well of peroxidase-conjugated neutravidin (Pierce, 1:1000 dilution). For chimeric antibodies, detection was using a goat anti-human Fc antibody (Cappel/ICN, 1:4000 dilution).

Solid-phase LAP Binding Assay—A 96-well microtiter plate was coated with either 0.3 µg/ml of LAP or 2.5 µg/ml of LAP-Fc fusion protein diluted in PBS (50 µl/well, 4 °C, overnight). The coating solution was removed, and plates were blocked with 180 µl/well of 3% BSA/TBS at 25 °C for 1 h. In a separate 96-well round-bottom plate, 60 µl/well of a 2x stock (0.5 µg/ml (1.25 nM) of _v6 which had been labeled with NHS-biotin, 2 mM CaCl₂, and 2 mM MgCl₂ in buffer A) was combined with 60 µl/well of a 2x stock of either hybridoma supernatant (for screening) or purified antibody (also in buffer A) and incubated at 25 °C for 1 h. After washing the LAP-coated plate with 0.1% Tween 20 in PBS in an automated plate washer, 100 µl of the antibody-_v6 mixture was transferred to the LAP-coated plate and incubated for 1 h at 25 °C. The plate was washed as above and incubated with 50 µl/well of a 1:1000 dilution of extravidin-horseradish peroxidase conjugate (Sigma) in buffer A for 1 h at 25 °C. Bound protein was detected using the substrate 3,3',5,5'-tetramethylbenzidine.

Cell Adhesion Assay Using LAP—A 96-well microtiter plate was coated with 50 µl/well of 0.5 µg/ml LAP diluted in 50 mM sodium bicarbonate, pH 9.2, at 4 °C overnight. The plate was washed twice with PBS (100 µl/well), blocked with 1% BSA in PBS (100 µl/well) for 1 h at 25 °C, and washed twice with 100 µl/well of assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂). FDC-P16 cells (5 x 10⁶ cells/ml) were detached from culture flasks with 5 mM EDTA, incubated with 2 µM fluorescent dye (Calcein-AM, Molecular Probes, Eugene, OR) in assay buffer with gentle shaking in a 37 °C water bath for 15 min, collected by centrifugation, and resuspended in assay buffer to 5 x 10⁶ cells/ml. To individual wells of the washed plate were added 25 µl of supernatant (or purified antibody) and 25 µl of FDC-P16 cells labeled with Calcein-AM, and the plate was incubated at 25 °C for 1 h. The plate was washed 4-6 times with assay buffer (100 µl/well), and the fluorescence due to captured cells on the plate was recorded. Percent binding was determined by comparing the fluorescence prior to the final wash step (i.e. total cells added) to that after washing (i.e. bound cells).

For adhesion assays using fibronectin, plates were coated with 50 µl/well of 5 µg/ml recombinant human fibronectin (Chemicon) diluted in 100 mM sodium phosphate, pH 9.0, at room temperature for 1 h, and washes were done using PBS + 0.05% Tween 20 (350 µl/well), and blocking was with 200 µl/well assay buffer (0.1% BSA in PBS + 1 mM CaCl₂, 1 mM MgCl₂) + 0.05% Tween 20 for 1 h at room temperature. HT-29 cells labeled with Calcein-AM as above were incubated at 25 °C for 2 h, and the bound fluorescence was determined as described above. TGF- Bioassay (Mink Lung Epithelial Cell PAI-1 Luciferase Coculture Assay)—TMLC (mink lung epithelial cell line Mv 1 Lu) transfected with PAI-1-luciferase construct (as described in Ref. 25) were grown in DMEM + 10% fetal bovine serum with 2 mM L-glutamine, penicillin/streptomycin, and 200 µg/ml G418. SW4806 cells were grown in DMEM + 10% fetal bovine serum with L-glutamine, penicillin/streptomycin, and 1 mg/ml G418. Cells were lifted from flasks with PBS + 5 mM EDTA, washed in PBS + 0.5% BSA, counted by hemocytometer, and plated in 96-well plates. SW4806 cells were stored on ice for 2 h, whereas TMLC were plated in 96-well plates at 10⁴ cells/well in DMEM + 0.1% FBS and allowed to adhere at 37 °C, after which bound TMLC were washed once with DMEM + 0.1% BSA. Monoclonal antibodies were diluted in DMEM + 0.1% BSA added to SW4806 cells and pre-incubated for 20 min at room temperature. SW4806 were then added to the TMLC at 4 x 10⁴/well in DMEM + 0.1% BSA (100 µl/well). Plates were incubated for 20 h at 37 °C in a humidified, CO₂-enriched incubator. Supernatant was discarded and replaced with 100 µl of PBS + 1 mM Ca²⁺ and 1 mM Mg²⁺. Cells were lysed, and luciferase was detected with a LucLite kit (PerkinElmer Life Sciences) using a micro-plate luminometer.

Kinetic Exclusion Assay—Unactivated PMMA beads (200 mg, Sapi-dyne Instruments, Boise, ID) were coated with 100 µg/ml hs_v6 in PBS by incubation for 1 h at 37 °C and blocked by incubating for 1 h at 37 °C with 10 mg/ml BSA in PBS. Serial dilutions of hs_v6 were incubated with 1 x 10^-10 M (0.015 µg/ml) antibody (6.3G9 or 6.8G6) in a buffer consisting of either PBS (for 6.3G9) or PBS containing 1 mM MgCl₂ and 1 mM CaCl₂ (for 6.8G6). After 3 h at 25 °C, the concentration of free antibody in solution was determined by measuring the binding to hs_v6 beads, using a kinetic exclusion assay instrument (Sapidyne) according to the manufacturer's instructions. Cy5-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used for detection of bead-bound antibodies.

Platelet Aggregation Assay—50 ml of whole human blood was collected into 10-ml vacutainer tubes containing 1 ml of 3.8% sodium citrate. Platelet-rich plasma (PRP) was prepared by centrifuging the citrate-treated blood for 5 min at 200 x g. The PRP was removed and platelet-poor plasma (PPP) was prepared by centrifuging the remaining blood specimen at 1500 x g for 15 min. The platelet count in the PRP was adjusted to 2 x 10⁸ platelets/ml using PPP. The Biodata 4-channel platelet aggregation profiler (PAP-4; Biodata Corp., Hatboro, PA) was blanked using a cuvette containing only PPP. 350 µl of PRP plus 100 µl of antibody were added to a cuvette containing a stir bar. To start aggregation, 50 µl of ADP at 2 x 10^-4 M was added to each stirring sample. A buffer control (24 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, containing 2 mM glucose, 0.1% BSA, and 1 mM MnCl₂, pH 7.4) was run with each set of test samples. A 4-min aggregation tracing was generated for each sample, and % aggregation was calculated. The peptide RGD was run on each day as a positive control and effectively inhibited platelet aggregation.

Determination of Antibody CDR Sequences—To determine the variable region sequences of the described antibodies, messenger RNA from the hybridoma cell lines was prepared on RNeasy columns (Qiagen, Valencia, CA) according to the manufacturer's protocol. The cDNAs for antibody heavy and light chain genes were prepared using the First Strand cDNA Synthesis kit (Amersham Biosciences) and primers 5'-ATTAAGTCGACCKYGGTSYTGCTGGCYGGGTG-3' for the heavy chain and 5'-GCGTCTAGAACTGGATGGTGGGAGATGGA-3' for the light chain. The cDNAs were amplified in a PCR with Pfu polymerase (Stratagene, La Jolla, CA) with the 5' oligonucleotides listed above and the degenerate 3' oligonucleotide pools 5'-GGGGATATCCACCATGGRATGSAGCTGKGTMATSCTCTT-3', 5'-GGGGATATCCACCATGRACTTCGGGYTGAGCTKGGTTTT-3', 5'-GGGGATATCCACCATGGCTGTCTTGGGGCTGCTCTTCT-3', for the heavy chain and 5'-GGGGATATCCACCATGGATTTTCAGGTGCAGATTTTCAG-3', 5'-GGGGATATCCACCATGRAGTCACAKACYCAGGTCTTYRTA-3', 5'-GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG-3', and 5'-GGGGATATCCACCATGAGGKCCCCWGCTCAGYTYCTKGGR-3' for the light chain. The resultant amplified antibody variable region genes were cloned into the pCR4Blunt-Topo vector (Invitrogen), and sequence analysis was performed on multiple isolates to generate consensus sequences.

Internalization Studies—Cells were plated onto 4-chambered glass slides at 25% confluency for overnight incubation at 37 °C, 5% CO₂. Antibodies at 10 µg/ml or medium alone were added to appropriate wells. Internalization was stopped by removing the antibody and by washing the cell layer with buffer. Cytofix/Cytoperm solution (Pharmingen, San Diego, CA) was added for 20 min at 4 °C to fix and permeabilize the cells. The cells were washed again, and the secondary anti-mouse Alexa 594 (red fluorescence) was added for 20 min at 4 °C to label the bound or internalized murine _v6 antibody. Cells were washed and fixed by addition of 2% paraformaldehyde and examined by confocal microscopy.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Generation and Characterization of _v6-specific Antibodies—To generate antibodies specific for the _v6 heterodimer, with minimal cross-reactivity to other _v integrins, we carried out immunizations in ₆-deficient mice (₆ -/-). Although previous attempts to generate _v6 antibodies by immunizing wild-type mice produced antibodies that bound to human, but not murine _v6 (8), the ₆ -/- mice were used successfully to generate two antibodies that recognized both murine and human _v6 (9). The antibodies described here were generated from ₆ -/- mice immunized with either human soluble _v6 or transfected NIH3T3 cells expressing murine _v6. Initial selection of positive clones was based on binding to purified hs_v6 and selective binding to murine or human ₆-transfected cells, but not to untransfected control cells. Further selection for neutralizing antibodies was based on the ability of the antibodies to block both soluble biotinylated hs_v6 and human or murine ₆-transfected cells binding to LAP, as described below. Monoclonal antibodies were further confirmed as functional blockers based on their ability to inhibit _v6-mediated TGF- activation. Based on these criteria, a panel of blocking and nonblocking antibodies was selected for further analysis and characterized for functional binding affinity, potency in blocking the interaction of _v6 with the ligand LAP, and potency in blocking the _v6-mediated activation of TGF-. Data from a selected set of 14 of these antibodies are shown in Table I.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Binding of antibodies to v6 by solid-phase immuno-assay (A) or flow cytometry (B). Representative antibodies 6.3G9 (squares), 6.8G6 (circles), and 6.4B4 (triangles) are shown. A, purified antibodies were incubated with immobilized hsv6 in an ELISA format in the presence of 1 mM Ca2+ + 1 mM Mg2+. B, purified antibodies were incubated with 6-transfected NIH3T3 cells in the presence of 1 mM Ca2+ + 1 mM Mg2+ prior to flow cytometry. Curve fits for this and subsequent figures were generated by nonlinear regression using a four-parameter (sigmoidal) equation. The data shown in A represent an average of duplicate measurements, and error bars (not visible for all data points) represent the S.D. at each data point. The data shown in B are single measurements at each data point and are representative of multiple experiments giving similar results.

To identify antibodies that could block both human and murine _v6 function, each antibody was next evaluated for its ability to inhibit LAP binding to either biotin-conjugated hs_v6 (in a solid-phase assay) or ₆-transfected murine FDC-P1 cells (in a cell adhesion assay) (Table I). Seven antibodies blocked the _v6-LAP interaction (6.3G9, 6.8G6, 6.1A8, 6.2B1, 7.1C5, 7.1G10, and 7.7G5). Two of the higher affinity antibodies, 6.3G9 and 6.8G6, were among the most potent inhibitors in the solid-phase assay, with IC₅₀ values of 2.7 and 6.9 ng/ml, respectively (Fig. 2A). In the cell adhesion assay, most of the antibodies had IC₅₀ values of approximately an order of magnitude higher than in the solid-phase immunoassay, and a good correlation between the relative potencies in the biochemical and cellular binding assays was observed. For example, 6.3G9 had an IC₅₀ of 25 ng/ml in the cell adhesion assay (Fig. 2B). Notable exceptions to this trend, however, included 6.1A8 (>25 µg/ml), 6.8G6 (416 ng/ml), and 7.7G5 (422 ng/ml), which were all relatively less potent inhibitors in the cell adhesion format. This result suggested that these antibodies might be distinguishing certain biochemical or structural features of the soluble integrin that are not present in the cell-expressed integrin. In particular, 6.1A8 was completely unable to block cell adhesion while being a measurable inhibitor in the solid-phase assay (129 ng/ml). In separate experiments, immobilized 6.1A8 bound to both mouse 6FDC-P1 and human 6SW480 cells at roughly equivalent concentrations (data not shown), indicating that the low potency of this antibody was not due to a lack of cross-reactivity to mouse _v6. However, the much lower affinity of this antibody for soluble (Table I) and cell-expressed (data not shown) _v6 translated into a very low blocking potency in the cell adhesion format. For comparison, we included 10D5, the only other monoclonal antibody that has been shown to block _v6-ligand binding (9, 11). In both assay formats, 6.3G9 was found to be 20-fold more potent at blocking the _v6-LAP interaction than 10D5 (Fig. 2, A and B). The remaining antibodies (6.2A1, 6.2E5, 6.2G2, 6.4B4, 7.8B3, and 7.8C9) were classified as nonblockers. One of these (6.4B4) did show some partial inhibition (50%) in the solid-phase format (Fig. 2A) but not in the cell adhesion format. This was not due to a difference in species specificity, since 6.4B4 did bind to murine 6FDC-P1 cells with high affinity (data not shown). The other antibodies were completely unable to block the _v6-LAP interaction in either assay format.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Inhibition of v6 binding to LAP or fibronectin by solid-phase LAP binding assay (A) or cell adhesion assay (B and C). A, biotin-labeled hsv6 was preincubated with varying concentrations of either 6.3G9 (squares), 6.8G6 (circles), 10D5 (triangles), or 6.4B4 (diamonds), and then bound to a plate coated with human LAPFc. B, FDC-P16 cells were preincubated with antibodies and then bound to a plate coated with human LAP. C, HT29 cells were incubated with antibodies L230 (anti-v), 6.8G6, 6.3G9, 7.8C9, and P1F6 (anti-v5) and then bound to a plate coated with human plasma fibronectin. All data represent averages of duplicate measurements, and error bars represent the standard deviation at each data point.

Finally, the antibodies were tested for their abilities to block _v6-mediated activation of latent TGF-. In this experiment, ₆-transfected SW480 cells were cocultured with TGF--responsive mink lung epithelial reporter cells stably expressing a portion of the plasminogen activator inhibitor 1 promoter (TMLC). The ₆-transfected cell line, but not the untransfected parent, converts latent TGF-1 and/or TGF-3 to active, receptor-binding forms (11, 20). The two representative blocking antibodies 6.3G9 and 6.8G6 were potent inhibitors of _v6-mediated TGF- activation, with IC₅₀ values of 0.5 (3.5 pM) and 1.5 ng/ml (10 pM), respectively. The antibody 6.4B4, which did not block LAP binding, was not an inhibitor of TGF- activation (Fig. 3). Although the sensitivity of this assay precluded a more thorough quantitative comparison between antibodies, experiments using the entire panel of antibodies demonstrated a general correlation between the ability to block the interaction of _v6 with LAP and the ability to inhibit activation of TGF- in vitro (data not shown). Once again, these antibodies were significantly more potent than 10D5, which inhibited TGF- activation with an IC₅₀ 100-fold higher than that of 6.3G9 or 6.8G6 (100 ng/ml) (Fig. 3).

View larger version (23K): [in this window] [in a new window]   FIG. 3. Inhibition of TGF- activation by anti-v6 antibodies 6.3G9 (squares), 6.8G6 (circles), 10D5 (triangles), or 6.4B4 (diamonds). Antibodies were incubated with SW4806 cells and TMLC reporter cells overnight, and active TGF- was detected by its ability to induce luciferase production. No inhibition was observed using 6.4B4, and both 6.3G9 and 6.8G6 were significantly more potent than 10D5. All data represent averages of duplicate measurements, and error bars represent the standard deviation at each data point.

View larger version (46K): [in this window] [in a new window]   FIG. 4. Specificity of antibodies for v6 determined by solid-phase immunoassay (A) or flow cytometry (B). A, antibodies 6.8G6, 6.3G9, 6.4B4, or L230 (anti-v) (10 µg/ml) or buffer alone (ctrl) were incubated with either purified human v3 (solid bars), purified human v5 (open bars), or hsv6 (hatched bars) immobilized on an ELISA plate at 1 µg/ml. Binding was carried out in buffer containing 1 mM Ca2+ + 1 mM Mg2+. All data represent averages of duplicate measurements, and error bars represent the standard deviation at each data point. B, binding of v6 or control (AP-3, anti-3; 14E7, anti-8) antibodies to SW480 cells either mock-transfected or transfected with 3, 6, or 8 in a buffer containing 1 mM Ca2+ + 1 mM Mg2+ was measured by flow cytometry. No binding of 6.3G9 or 6.8G6 to the SW480, SW4803, or SW4808 cells was detectable.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Cation dependence of LAP binding to v6. A, binding of biotin-v6 to immobilized LAP in the presence of either 1 mM Ca2+ + 1 mM Mg2+ (filled diamonds), 1 mM Mn2+ (filled squares), 5 mM Ca2+ (filled triangles), 5 mM Mg2+ (open diamonds), or 10 mM EDTA (open circles) was measured using the solid-phase LAP binding assay. B, binding of FDC-P16 cells to immobilized LAP-Fc was measured using the cell adhesion assay as described under "Experimental Procedures" in the presence of either 1 mM Ca2+ + 1 mM Mg2+ (closed diamonds) or 1mM Mn2+ (closed squares). Data represent averages of duplicate (A)or triplicate (B) measurements, and error bars represent the standard deviation at each data point.

To determine whether the _v6 antibodies described here distinguish between the cation-bound and apo forms of the integrin, we evaluated the ability of each antibody to bind hs_v6 in buffer alone, 1 mM Mn²⁺, or in 1 mM Ca²⁺ + 1 mM Mg²⁺. The 1 mM Ca²⁺ + 1 mM Mg²⁺ state approximates physiological cation concentrations and accordingly should provide the best model for the in vivo properties of the antibodies. As a control, binding was measured in the presence of 10 mM EDTA. We also examined antibody binding to human ₆-transfected SW480 cells by flow cytometry in the presence of either 1 mM Mn²⁺ or 1 mM Ca²⁺ + 1 mM Mg²⁺, or in the absence of cations. The majority of the antibodies, including 6.3G9, showed no dependence upon divalent cations for binding to hs_v6 or SW4806 cells (Fig. 6, A and B). However, clone 6.8G6 showed a distinct cation requirement for binding hs_v6 or SW4806 cells (Fig. 6, C and D). This antibody bound equally well in either Mn²⁺ or Ca²⁺ + Mg²⁺, but in the absence of cations did not bind to the integrin in any measurable way. An identical cation dependence was observed for antibodies 6.1A8 and 7.7G5 (data not shown). This pattern closely resembled that observed for the _v6-LAP interaction (Fig. 5), suggesting that these antibodies may bind _v6 in a way that structurally mimics the binding of _v6 to LAP.

View larger version (34K): [in this window] [in a new window]   FIG. 6. Cation dependence of antibody binding to v6. The binding of antibodies 6.3G9 (A and B), 6.8G6 (C and D), and 10D5 (E and F) to v6 was measured using either the solid-phase binding assay on hsv6 (A, C, and E) or flow cytometry with SW4806 cells (B, D, and F). Binding was carried out in the presence of either 1 mM Ca2+ + 1 mM Mg2+ (diamonds), 1 mM Mn2+ (squares), 10 mM EDTA (triangles in A, C, and E), or buffer without divalent cations (triangles in B, D, and F). All data represent averages of duplicate measurements, and error bars represent the standard deviation at each data point.

Cation-dependent Antibodies Contain Functional Ligand-mimetic Sequences in CDR H3—Because the metal ion-dependent binding properties of 6.8G6, 6.1A8, 7.7G5, and 10D5 resembled those of LAP, we examined the variable regions of these antibodies to determine whether any sequence similarities to _v6 ligands could be identified. Ligands for _v6 contain the sequence RGD, a tripeptide recognition motif common to numerous integrin ligands and other cell adhesion molecules. This RGD sequence, or the related sequence RYD, has been identified in the CDRs of antibodies raised against the platelet integrin _IIb3 (31-34). The predicted amino acid sequence of the heavy and light chains of each anti-_v6 antibody was determined by nucleotide sequencing (Table II). Most interesting, the cation-dependent antibodies 6.8G6 and 6.1A8 both contain an RGD tripeptide within CDR3 of the heavy chain (CDRH3), and 10D5 contains an RYD sequence in the same region. The other cation-dependent antibody, 7.7G5, has adjacent Asp and Arg residues but no RGD tripeptide in CDRH3; however, this antibody does contain an Asn-Gly-Asp sequence in CDRH2. The cation-independent blocking and nonblocking antibodies, on the other hand, were not found to contain any RGD or related sequences. Based on this sequence information, in conjunction with the cation dependence of _v6 binding, we classified four of the blocking antibodies (6.8G6, 6.1A8, 7.7G5, and 10D5) as "ligand-mimetic," whereas the other group of blocking antibodies was labeled "nonligand-mimetic" (Table II).

View this table: [in this window] [in a new window]   TABLE II CDR sequences of anti-v6 monoclonal antibodies

View larger version (24K): [in this window] [in a new window]   FIG. 7. Inhibition of antibody binding by an RGD-containing peptide. Antibodies 6.3G9 (open diamonds), 6.8G6 (filled triangles), 7.7G5 (filled squares), 6.1A8 (filled circles), 10D5 (filled diamonds), or 6.4B4 (open squares) were preincubated with an RGD-containing peptide (acetyl-GGLRRGDRPSLRYAMDS-CONH2) derived from the 6.8G6 CDRH3 sequence and then bound to immobilized hsv6 in the solid-phase immunoassay in the presence of either 1 mM Mn2+ (A) or 1 mM Mg2+ + 1mM Ca2+ (B). Binding of the ligand-mimetic antibodies 6.8G6, 7.7G5, 6.1A8, and 10D5 was effectively blocked by the peptide, whereas binding of the nonligand-mimetic antibodies 6.3G9 and 6.4B4 was unaffected by the peptide. The concentration of peptide required to inhibit antibody binding in Mn2+ was 15-20-fold lower than in Ca2+ + Mg2+ (note the difference in scale on the x axis). All data represent averages of duplicate measurements, and error bars represent the standard deviation at each data point.

View larger version (24K): [in this window] [in a new window]   FIG. 8. Epitope mapping by antibody competition. The ability of unlabeled antibodies (50 µg/ml) to compete with biotin-conjugated 6.8G6 (A) or 6.3G9 (B) for binding to immobilized hsv6 was measured. Antibodies are grouped, from left to right, as follows: nonligand-mimetic blocking antibodies, ligand-mimetic blocking antibodies, nonblocking antibodies, antibody L230 (anti-v), and control (ctrl, buffer alone). C, dose-dependent inhibition of biotin-3G9 by unlabeled antibodies. As observed in B, the inhibition by 6.8G6 and 7.7G5 is incomplete at 50 µg/ml. The data shown in A represent an average of duplicate measurements, and error bars represent the standard deviation at each data point. The data shown in B are single measurements at each data point and are representative of multiple experiments giving similar results.

View larger version (114K): [in this window] [in a new window]   FIG. 9. Internalization of ligand-mimetic v6 antibodies. Confocal imaging and internalization of the integrin v6 receptor-antibody complex was observed in human SCC-14 carcinoma cells that were treated with either LAP-Fc (A) or the antibodies 6.4B4 (B), 6.3G9 (C), 7.1C5 (D), 6.8G6 (E), or 6.1A8 (F). The nonligand-mimetic antibodies (B, C, and D) localize to the exterior of the cell membrane, whereas the ligand-mimetic antibodies (E and F) and the ligand (A) internalize and can be identified in the cytoplasm. No staining was observed in the nucleus. Differential interference contrast images and red fluorescent overlays were taken with a Leitz Plan-Apochromatic 63x (1.32 numerical aperture, oil immersion) objective (Leica) with a x2 digital zoom. Each frame represents a single optical section from the middle section of the cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Biochemical characterization led to the identification of two distinct classes of blocking antibody, which we designated as ligand-mimetic and nonligand-mimetic. Three of the antibodies (6.8G6, 6.1A8, and 7.7G5) required divalent cations to bind to _v6. Because integrin-ligand interactions are cation-dependent, we examined the sequences of the variable regions of these antibodies to determine whether RGD sequences were present. In fact, nucleotide sequencing of the variable region revealed the presence of an RGD sequence in the third CDR of the IgG heavy chain (CDRH3) of two of these three antibodies (6.8G6 and 6.1A8). The third antibody, 7.7G5, did not contain an RGD tripeptide per se, although the sequences DRYG at the start of CDRH3 and Asn-Gly-Asp in CDRH2 might be responsible for the cation-dependent binding properties of this antibody. The discovery of RGD sequences only in the cation-dependent antibodies, and not in any of the cation-independent antibodies or nonblocking antibodies, strongly suggested that these sequences were critical for the antibody-_v6 interaction. These findings prompted us to reexamine the biochemical and molecular properties of 10D5 (9), the only previously reported _v6 blocking antibody and a significantly less potent inhibitor of the _v6-LAP interaction. 10D5 was also found to require divalent cations for binding to _v6 and contained the sequence RYD in CDRH3, allowing us to classify it as a fourth ligand-mimetic antibody. The functional role of the RGD (or related) sequence in the ligandmimetic antibodies was confirmed by showing that an RGD-containing peptide can inhibit the binding of all four antibodies to _v6. The second class of blocking antibodies, which we designated as nonligand-mimetic, did not require cation for binding, was not inhibited by RGD-containing peptides, and did not contain any apparent sequence similarities.

Ligand-mimetic antibodies have been described previously (37) for a number of different integrins. These antibodies compete for binding to the same site as the ligand and by definition are blocking antibodies. Ligand-mimetic antibody binding is supported or enhanced by the same stimuli that promote ligand binding, including agonists, integrin-activating antibodies, and divalent cations. In some examples, the similarity to ligand can be further traced to a specific sequence contained within the antibody, just as we observed for the _v6 ligand-mimetic antibodies. The prototypical ligand-mimetic antibodies are the anti-_IIb3 antibodies PAC-1 (32), OPG2 (34), and LJ-CP3 (33), which contain RYD sequences within the third CDR of the IgG heavy chain (CDRH3), and mutation of the aspartate residue to glutamate eliminates integrin binding (38). Another anti-_IIb3 antibody, 16N7C2, contains an RGD sequence in this same CDR (39). As with the _v6 antibodies, these antibodies bind to _IIb3 in a cation-dependent manner. Another more recent example of a ligand-mimetic antibody is the anti-₁₁ antibody AQC2, which blocks the ₁₁-collagen interaction and binds ₁₁ with 20-fold higher affinity in the presence of cations (40). A crystal structure of the complex between the AQC2 Fab fragment and the ₁ integrin I domain revealed a direct contact between an Asp residue in CDRH3 of the antibody and a divalent cation contained in the ligand-binding site of the integrin, in a manner strikingly similar to the way a Glu residue from collagen interacts with ₁₁ (41). Most interesting, the _IIb3, ₁₁, and _v6 (described here) ligand-mimetic antibodies all contain key aspartate residues in CDRH3, suggesting that this region of anti-integrin blocking antibodies may be particularly important for binding. Because ligand-mimetic antibodies resemble natural ligands and may, in some cases, have higher affinity, they have provided useful tools to study the nature of integrin-ligand interactions.

The antibody 6.8G6, an _v6 ligand-mimetic antibody, shows a striking resemblance to PAC-1. Both antibodies are cation-dependent; both can be blocked by RGD-containing peptides, and both contain an RGD sequence in CDRH3. Despite these similarities, 6.8G6 is highly specific for _v6, whereas PAC-1 is also very specific for _IIb3. It is interesting to consider the basis of specificity of these antibodies. One explanation is that sequences flanking the RGD contribute to specificity by altering the relative affinity for different integrins, as has been demonstrated for some snake venom-derived disintegrins (42), antibodies (43-45), and synthetic RGD-containing peptides (46). The motif RXDLXXL was identified through phage display to bind specifically to _v6 and was proposed to be acting as a ligand mimetic (47). A similar motif (RGDLATI) is contained in LAP itself, although the ligand-mimetic _v6 antibodies do not have this motif conserved (Table II). An alternative explanation for the observed differences in antibody specificity is that specificity is determined by regions other than the RGD-containing sites. This explanation is supported by mapping experiments using human-to-mouse chimeras that identified multiple discontinuous ligand-mimetic antibody-binding sites on the integrin _IIb3 (48). One way to explore this issue will be to exchange the 6.8G6 and PAC-1 CDRH3 sequences and test the effects of the exchange on antibody specificity.

Because potent _v6 blocking antibodies from both the ligand-mimetic and nonligand-mimetic classes were observed, we carried out antibody competition experiments with 6.3G9 and 6.8G6 to determine whether these antibodies could be binding to overlapping epitopes. Antibodies from each class competed effectively with 6.8G6 and with 6.3G9, although the ligand-mimetic group was less effective due to the lower relative affinities of the ligand-mimetic antibodies. In contrast, the nonblocking antibodies did not compete with 6.3G9 or 6.8G6. These results demonstrating mutual cross-competition between and within the two antibody classes, taken together with the inability of RGD peptides to block binding of the nonligandmimetic antibodies, suggest that the two classes bind to non-identical but possibly overlapping epitopes on _v6. The mutually exclusive binding observed may be due either to direct steric interactions between antibodies occupying proximal epitopes or to indirect competition through a long distance conformational change. The recently solved crystal structure of _v3 integrin bound to a cyclic RGD-containing peptide (49) demonstrated that the ligand-binding site is on the - subunit interface. The _v6 blocking antibodies from both classes do not bind to the isolated, denatured ₆ subunit,² indicating that they bind a conformational epitope requiring the intact _v6 heterodimer. The simplest explanation for these results, given the competition between the two classes of antibody and among the different nonligand-mimetic antibodies (which have quite divergent CDR sequences), is that each of the _v6 blocking antibodies binds in such a way as to sterically block a single ligand-binding site at the _v/₆ interface. However, further work will be needed to distinguish this scenario from one involving allosteric binding sites. In either case, the interactions likely involve multiple discontinuous sites on the ₆ and/or the _v subunits, as in the PAC-1/_IIb3 interaction.

All integrins have multiple divalent cation-binding sites that can contribute to either promoting or inhibiting ligand binding (for review see Ref. 50). In the structure of the _v3-RGD peptide complex, a Mn²⁺ ion was an integral component of the integrin-ligand interface (49). In general, Mn²⁺ or Mg²⁺ ions tend to promote ligand binding, whereas Ca²⁺ can serve to either promote or inhibit binding. In particular, Mn²⁺ is often able to activate integrins which, in the absence of such stimulus, would fail to bind ligand. The structural basis for Mn²⁺-dependent activation is unclear, since structurally identical Mg²⁺- and Mn²⁺-bound states of _v3 (49), as well as I domain containing-integrins (51), have been reported. In addition to ligands, some anti-integrin antibodies also exhibit cation-dependent changes in affinity (40), presumably because they either interact directly with the ligand-binding pocket or are binding to a site that is allosterically affected by metal ion binding. Binding of LAP to hs_v6 was supported to similar extents by Mn²⁺, Ca²⁺, Mg²⁺, and Ca²⁺ + Mg²⁺, whereas binding of LAP to _v6-expressing cells was supported by Ca²⁺ + Mg²⁺ but was significantly improved by Mn²⁺. This result differs from several other in vitro integrin-ligand interactions that require activation either by Mn²⁺ or by other stimuli (e.g. Refs. 52 and 53).

In contrast to LAP, the ligand-mimetic antibody 10D5 bound _v6 in Mn²⁺ but in Ca²⁺ + Mg²⁺ binding was barely detectable. The sequence of ligand-mimetic antibody 10D5 contains the tripeptide RYD in place of the RGD sequence found in 6.8G6. RYD is one of the most common functional substitutions for RGD. When substituted for RGD in the snake venom disintegrin dendroaspin, for example, the RYD motif had no effect on function (54). However, subtle differences in ligand binding by RGD- and RYD-containing antibodies have been reported. For example, the RYD-containing antibody OPG2 binds to _IIb3, but not _v3, in buffers containing either Mn²⁺ or Ca²⁺ + Mg²⁺ (43). The corresponding RGD variant of OPG2 retains binding to _IIb3 with no detectable difference in affinity (38), but also binds to _v3 in the presence of Mn²⁺ (43). Studies using RYD and RGD variants of 10D5 and LAP may allow further exploration of the role that the central residue in the RGD/RYD tripeptide plays in affecting the cation specificity of antibody and/or ligand binding.

We extended the comparison between ligand- and nonligandmimetic antibodies by testing if the antibodies induce _v6 internalization. LAP (expressed as a functional Fc fusion protein) and the ligand mimetic antibodies 6.8G6 and 6.1A8 induced internalization, but the nonligand-mimetic antibodies 6.3G9, 6.4B4, and 7.1C5 did not. This result further emphasizes the ligand-mimetic nature of the cation-dependent antibodies and suggests that ligand- and nonligand-mimetic antibodies may initiate different signaling pathways or integrin clustering in _v6-expressing cells. Because preliminary studies with 7.7G5, a non-RGD-containing antibody, indicate this antibody is not internalized,² it is likely that cation-dependent binding to _v6 is not sufficient for promoting internalization. Although _v3 (55) and _v5 (56) can function as endocytic receptors for vitronectin, this is the first demonstration of ligand internalization by _v6. The discovery that LAP itself can be internalized suggests an alternative mechanism by which _v6 could activate TGF- through internalization of the latent TGF- complex and subsequent recycling of active TGF- generated through intracellular proteolysis or nonenzymatic cleavage.

Blocking antibodies have provided useful tools for studying integrin structure and biology. Furthermore, anti-integrin antibodies have been validated as legitimate clinical candidates, and one (the anti-_IIb3 antibody abciximab) has been approved for the treatment of coronary angioplasty (57, 58). The antibodies described here block the interaction of _v6 with the ligand LAP, prevent the subsequent activation of latent TGF-, and block the interaction of _v6 with fibronectin. These antibodies should provide useful reagents to study _v6 in vitro and _v6-mediated pathologies in vivo including fibrosis and epithelial cancers. One could imagine that the two classes of antibody may have different therapeutic properties; a nonligand-mimetic might provide a more inert blocking reagent, whereas a readily internalized ligand-mimetic might be of value in oncology applications (e.g. to permit delivery of toxins or radioisotopes). Characterization of the antibodies in animal models of disease will determine whether the two different biochemical classes of antibodies have different effects on cellular function in vivo.

	FOOTNOTES

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

To whom correspondence should be addressed: Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142. Tel.: 617-679-2351; Fax: 617-679-3148; E-mail: Paul.Weinreb{at}Biogenidec.com.

¹ The abbreviations used are: TGF-, transforming growth factor-; LAP, latency-associated peptide; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; CFA, complete Freund's adjuvant; BSA, bovine serum albumin; CDR, complementarity-determining region; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; CHO, Chinese hamster ovary; PRP, platelet-rich plasma; PPP, platelet-poor plasma; hs, human secreted.

² P. H. Weinreb, B. M. Dolinski, D. R. Leone, and S. M. Violette, unpublished data.

	ACKNOWLEDGMENTS

 
We are grateful to Hannu Larjava and Reidar Grenman for providing the SCC-14 cell line; Cell Essentials Inc. (Medford, MA) for hybridoma production; J. Hernan Cuervo for synthesis of RGD-containing peptides; Konrad Miatkowski and Joseph Amatucci for assistance with antibody production and purification; and Ellen Garber, Roy Lobb, Blake Pepinsky, and Matvey Lukashev for invaluable advice and assistance.

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