Function-blocking Integrin αvβ6 Monoclonal Antibodies
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m312103200
ISSN1083-351X
AutoresPaul H. Weinreb, Kenneth J. Simon, Paul Rayhorn, William J. Yang, Diane R. Leone, Brian Dolinski, Bradley R. Pearse, Yukako Yokota, Hisaaki Kawakatsu, Amha Atakilit, Dean Sheppard, Shelia M. Violette,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoWe have generated a panel of potent, selective monoclonal antibodies that bind human and mouse αvβ6 integrin with high affinity (up to 15 pm). A subset of these antibodies blocked the binding of αvβ6 to the transforming growth factor-β1 latency-associated peptide with IC50 values as low as 18 pm, and prevented the subsequent αvβ6-mediated activation of transforming growth factor-β1. The antibodies also inhibited αvβ6 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 αvβ6-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for αvβ6 and showed no cross-reactivity with the RGD-binding integrins αvβ3, αvβ8,or αIIbβ3. 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 αvβ6, suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for αvβ6 and resemble those described for αIIbβ3. We also report for the first time the relative abilities of divalent cations to promote αvβ6 binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of αvβ6 as well as the role of αvβ6 in vivo. We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse αvβ6 integrin with high affinity (up to 15 pm). A subset of these antibodies blocked the binding of αvβ6 to the transforming growth factor-β1 latency-associated peptide with IC50 values as low as 18 pm, and prevented the subsequent αvβ6-mediated activation of transforming growth factor-β1. The antibodies also inhibited αvβ6 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 αvβ6-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for αvβ6 and showed no cross-reactivity with the RGD-binding integrins αvβ3, αvβ8,or αIIbβ3. 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 αvβ6, suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for αvβ6 and resemble those described for αIIbβ3. We also report for the first time the relative abilities of divalent cations to promote αvβ6 binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of αvβ6 as well as the role of αvβ6 in vivo. Integrins are heterodimeric cell-surface receptors that have been implicated in regulating a variety of processes by mediating cell adhesion, migration, and signaling (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6852) Google Scholar). At least 24 different integrin heterodimers can be formed from the 18 α and 8 β chains that have been identified. The pairing of α and β 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 αIIbβ3, some of the β1 integrins, and all of the αv integrins. Although the αv subunit can pair with multiple β subunits (β1, β3, β5, β6, and β8), the β6 subunit can only pair with αv and not with any other integrin α chains. αvβ6 is unique in that its expression is restricted to epithelial cells (2Breuss J.M. Gillett N. Lu L. Sheppard D. Pytela R. J. Histochem. Cytochem. 1993; 41: 1521-1527Crossref PubMed Scopus (199) Google Scholar). Although it is present at low or undetectable levels in normal adult tissue, αvβ6 is rapidly up-regulated during development, injury, wound healing, and neoplasia (2Breuss J.M. Gillett N. Lu L. Sheppard D. Pytela R. J. Histochem. Cytochem. 1993; 41: 1521-1527Crossref PubMed Scopus (199) Google Scholar, 3Breuss J.M. Gallo J. DeLisser H.M. Klimanskaya I.V. Folkesson H.G. Pittet J.F. Nishimura S.L. Aldape K. Landers D.V. Carpenter W. Gillett N. Sheppard D. Matthay M.A. Albelda S.M. Kramer R.H. Pytela R. J. Cell Sci. 1995; 108: 2241-2251Crossref PubMed Google Scholar, 4Agrez M.V. Bates R.C. Mitchell D. Wilson N. Ferguson N. Anseline P. Sheppard D. Br. J. Cancer. 1996; 73: 887-892Crossref PubMed Scopus (37) Google Scholar, 5Thomas G.J. Jones J. Speight P.M. Oral Oncol. 1997; 33: 381-388Crossref PubMed Scopus (72) Google Scholar, 6Hakkinen L. Hildebrand H.C. Berndt A. Kosmehl H. Larjava H. J. Histochem. Cytochem. 2000; 48: 985-998Crossref PubMed Scopus (56) Google Scholar, 7Zambruno G. Marchisio P.C. Marconi A. Vaschieri C. Melchiori A. Giannetti A. De Luca M. J. Cell Biol. 1995; 129: 853-865Crossref PubMed Scopus (313) Google Scholar). Ligands for αvβ6 that have been identified through in vitro binding experiments include fibronectin, tenascin, and the transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; 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. 1The abbreviations used are: TGF-β, transforming growth factor-β; 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. latency-associated peptide (LAP) (8Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar, 9Huang X. Wu J. Spong S. Sheppard D. J. Cell Sci. 1998; 111: 2189-2195Crossref PubMed Google Scholar, 10Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar, 11Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar). As a result of binding to these ligands, αvβ6 can mediate cell adhesion, spreading, migration, proliferation, and activation of latent TGF-β. 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 (LAPβ1, LAPβ2, and LAPβ3) that are produced as N-terminal prodomains of the three closely related TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3). The TGF-βs 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-β 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-β fragments associate to form a complex comprising a dimer of TGF-β noncovalently associated with a dimer of LAP (termed the "small latent complex"). This complex associates with the latent TGF-β-binding protein (LTBP-1) prior to secretion, in what is referred to as the "large latent complex" (12Miyazono K. Hellman U. Wernstedt C. Heldin C.H. J. Biol. Chem. 1988; 263: 6407-6415Abstract Full Text PDF PubMed Google Scholar). As the name implies, the latent TGF-β complex is inactive and requires activation in order to produce active cytokine (13Gleizes P.E. Munger J.S. Nunes I. Harpel J.G. Mazzieri R. Noguera I. Rifkin D.B. Stem Cells. 1997; 15: 190-197Crossref PubMed Scopus (222) Google Scholar, 14Lawrence D.A. Mol. Cell. Biochem. 2001; 219: 163-170Crossref PubMed Scopus (102) Google Scholar, 15Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (440) Google Scholar). 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 αvβ6 integrin), the mechanism(s) of physiological TGF-β activation have not been fully elucidated (for reviews see Refs. 13Gleizes P.E. Munger J.S. Nunes I. Harpel J.G. Mazzieri R. Noguera I. Rifkin D.B. Stem Cells. 1997; 15: 190-197Crossref PubMed Scopus (222) Google Scholar and 14Lawrence D.A. Mol. Cell. Biochem. 2001; 219: 163-170Crossref PubMed Scopus (102) Google Scholar). All five of the αv integrins (16Munger J.S. Harpel J.G. Giancotti F.G. Rifkin D.B. Mol. Biol. Cell. 1998; 9: 2627-2638Crossref PubMed Scopus (198) Google Scholar, 17Mu D. Cambier S. Fjellbirkeland L. Baron J.L. Munger J.S. Kawakatsu H. Sheppard D. Broaddus V.C. Nishimura S.L. J. Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (592) Google Scholar, 18Ludbrook S.B. Barry S.T. Delves C.J. Horgan C.M. Biochem. J. 2003; 369: 311-318Crossref PubMed Scopus (96) Google Scholar), as well as α8β1 (19Lu M. Munger J.S. Steadele M. Busald C. Tellier M. Schnapp L.M. J. Cell Sci. 2002; 115: 4641-4648Crossref PubMed Scopus (68) Google Scholar), have been shown to bind to RGD sequences contained in LAPβ1 and LAPβ3 in vitro, whereas LAPβ2, 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 αvβ6 and αvβ8, but not αvβ1, αvβ5, or α8β1, are able to activate latent TGF-β1 upon binding, and αvβ6, as shown recently, can activate latent TGF-β3 (20Annes J.P. Rifkin D.B. Munger J.S. FEBS Lett. 2002; 511: 65-68Crossref PubMed Scopus (125) Google Scholar). The process by which αvβ6 activates latent TGF-β is not fully understood, although one model proposes that integrin binding induces a conformational change in the latent TGF-β complex, which allows binding of TGF-β to its type II receptor (11Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar). The ability of αvβ6 to activate TGF-β1 and TGF-β3 offers a novel mechanism for local activation of these multifunctional cytokines and suggests that this integrin may contribute to the onset of TGF-β-mediated disease processes. The in vitro data are supported by studies with β6-null mice, which have low levels of inflammation in the lung and skin, consistent with inactivation of TGF-β (21Huang X.Z. Wu J.F. Cass D. Erle D.J. Corry D. Young S.G. Farese Jr., R.V. Sheppard D. J. Cell Biol. 1996; 133: 921-928Crossref PubMed Scopus (258) Google Scholar). These mice are also protected in a bleomycin-induced pulmonary fibrosis model, suggesting that inhibition of αvβ6 might provide a therapeutic benefit by blocking TGF-β activation locally in affected tissues. In addition, blocking the binding of αvβ6 to other ligands, such as fibronectin, might be useful for treating TGF-β-independent disease processes by inhibiting the adhesive or migratory functions of αvβ6. 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 αvβ6 heterodimer and block its ability to activate latent TGF-β. We describe here the generation and characterization of selective, high affinity monoclonal antibodies that block the binding of αvβ6 to LAP. These antibodies were generated by immunizing β6-deficient mice with the αvβ6 integrin, allowing for the production of antibodies that recognize both the human and murine αvβ6 integrins. Among these antibodies are the first ligand-mimetic antibodies to be described for αvβ6. These antibodies provide a means to study the mechanism of αvβ6-mediated TGF-β activation in vitro, and to test whether αvβ6 inhibition can effectively block TGF-β-mediated pathologies in vivo. Materials—The β6 -/- mice were prepared as described (21Huang X.Z. Wu J.F. Cass D. Erle D.J. Corry D. Young S.G. Farese Jr., R.V. Sheppard D. J. Cell Biol. 1996; 133: 921-928Crossref PubMed Scopus (258) Google Scholar). Recombinant human TGF-β1 LAP was purchased from R & D Systems (Minneapolis, MN). Antibody 10D5 (9Huang X. Wu J. Spong S. Sheppard D. J. Cell Sci. 1998; 111: 2189-2195Crossref PubMed Google Scholar) was purchased from Chemicon (Temecula, CA). The L230 (anti-αv) and AP-3 (anti-β3) 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-β8 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 β6-transfected SW480 (human colorectal adenocarcinoma) cell line (SW480β6) was prepared as described (8Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar). 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 hsαvβ6—The recombinant human secreted αvβ6 protein (hsαvβ6) was purified from the supernatant of transfected CHO cells, essentially as described (8Weinacker A. Chen A. Agrez M. Cone R.I. Nishimura S. Wayner E. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar), by affinity chromatography using anti-αv 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 αvβ6 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 hsαvβ6 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 hsαvβ6 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 β6-Transfected FDC-P1 and NIH3T3 Cells—Murine β6-transfected NIH3T3 and FDC-P1 cells were generated by electroporating parent cell lines with a DNA construct containing full-length murine β6 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 β6. Transfected FDC-P1 cells were cultured in DMEM supplemented with 4 mml-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 mml-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 (22Gentry L.E. Lioubin M.N. Purchio A.F. Marquardt H. Mol. Cell. Biol. 1988; 8: 4162-4168Crossref PubMed Scopus (209) Google Scholar). Mutant human LAP cDNA was then ligated with the human IgG1 Fc cDNA (23Cunningham B.A. Rutishauser U. Gall W.E. Gottlieb P.D. Waxdal M.J. Edelman G.M. Biochemistry. 1970; 9: 3161-3170Crossref PubMed Scopus (25) Google Scholar) into the PV90 expression vector (24Brezinsky S.C.G. Chiang G.G. Szilvasi A. Mohan S. Sharpiro R.I. MacLean A. Sisk W. Thill G. J. Immunol. Methods. 2003; 277: 141-155Crossref PubMed Scopus (126) Google Scholar). CHO cells were transfected with the human LAP-IgG1 expression construct, and stable transfected cells producing fusion protein were selected by expanding in α 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—β6 -/- mice were immunized by intraperitoneal injection with hsαvβ6 in complete Freund's adjuvant (CFA) (fusion 6, designated with the prefix 6). Alternatively, β6 -/- mice were immunized with β6-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 αvβ6 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-αvβ6 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 (1× PBS, 2% FBS, 0.1% NaN3,1mm CaCl2,and1mm MgCl2). 0.2 × 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 αvβ6 Binding Assay (ELISA)—A 96-well microtiter plate was coated with 50 μl/well of 5 μg/ml hsαvβ6 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 2× stock (0.5 μg/ml (1.25 nm) of αvβ6 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 2× 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-αvβ6 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-P1β6 cells (5 × 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 × 106 cells/ml. To individual wells of the washed plate were added 25 μl of supernatant (or purified antibody) and 25 μl of FDC-P1β6 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-β 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. 25Abe M. Harpel J.G. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. Anal. Biochem. 1994; 216: 276-284Crossref PubMed Scopus (677) Google Scholar) were grown in DMEM + 10% fetal bovine serum with 2 mml-glutamine, penicillin/streptomycin, and 200 μg/ml G418. SW480β6 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. SW480β6 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 SW480β6 cells and pre-incubated for 20 min at room temperature. SW480β6 were then added to the TMLC at 4 × 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 hsαvβ6 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 hsαvβ6 were incubated with 1 × 10-10m (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 hsαvβ6 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 × g. The PRP was removed and platelet-poor plasma (PPP) was prepared by centrifuging the remaining blood specimen at 1500 × g for 15 min. The platelet count in the PRP was adjusted to 2 × 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 × 10-4m 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 genera
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