Small Molecule Ligands Define a Binding Site on the Immune Regulatory Protein B7.1

The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. These compounds inhibit the binding of B7.1 to both CD28 and CTLA4. Both classes of compounds appear to bind the same site, a relatively small portion of the GFCC′C“ face of the N-terminal V-set domain of human B7.1, not present in the homologous B7.2 or even mouse B7.1. This site may represent a rare hot spot for small molecule antagonist design of inhibitors of cell-cell interactions, whose ligands may yield leads for the development of novel immunomodulatory medicines. The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. These compounds inhibit the binding of B7.1 to both CD28 and CTLA4. Both classes of compounds appear to bind the same site, a relatively small portion of the GFCC′C“ face of the N-terminal V-set domain of human B7.1, not present in the homologous B7.2 or even mouse B7.1. This site may represent a rare hot spot for small molecule antagonist design of inhibitors of cell-cell interactions, whose ligands may yield leads for the development of novel immunomodulatory medicines. Full T cell activation requires both an antigen-specific and a second co-stimulatory signal. Co-stimulation dictates the outcome for T cells through the binding of B7.1 (CD80) and B7.2 (CD86) expressed on antigen-presenting cells to CD28 and CTLA4 on T cells (1Greenfield E.A. Nguyen K.A. Kuchroo V.K. Crit. Rev. Immunol. 1998; 18: 389-418Crossref PubMed Google Scholar,2Lenschow D.J. Walunas T.L. Bluestone J.A. Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2344) Google Scholar). Signaling through CD28 augments the T cell response, whereas CTLA4 signaling attenuates it (3Harding F.A. McArthur J.G. Gross J.A. Raulet D.H. Allison J.P. Nature. 1992; 356: 607-609Crossref PubMed Scopus (1488) Google Scholar, 4Walunas T.L. Lenschow D.J. Bakker C.Y. Linsley P.S. Freeman G.J. Green J.M. Thompson C.B. Bluestone J.A. Immunity. 1994; 1: 405-413Abstract Full Text PDF PubMed Scopus (1778) Google Scholar, 5Waterhouse P. Penninger J.M. Timms E. Wakeham A. Shahinian A. Lee K.P. Thompson C.B. Griesser H. Mak T.W. Science. 1995; 270: 985-988Crossref PubMed Scopus (2365) Google Scholar). Animal studies and clinical trials with protein antagonists of these interactions indicate considerable promise for immunotherapy in transplantation and autoimmune disease (1Greenfield E.A. Nguyen K.A. Kuchroo V.K. Crit. Rev. Immunol. 1998; 18: 389-418Crossref PubMed Google Scholar, 2Lenschow D.J. Walunas T.L. Bluestone J.A. Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2344) Google Scholar,6Abrams J.R. Kelley S.L. Hayes E. Kikuchi T. Brown M.J. Kang S. Lebwohl M.G. Guzzo C.A. Jegasothy B.V. Linsley P.S. Krueger J.G. J. Exp. Med. 2000; 192: 681-694Crossref PubMed Scopus (298) Google Scholar, 7Guinan E.C. Boussiotis V.A. Neuberg D. Brennan L.L. Hirano N. Nadler L.M. Gribben J.G. N. Engl. J. Med. 1999; 340: 1704-1714Crossref PubMed Scopus (375) Google Scholar, 8Larsen C.P. Elwood E.T. Alexander D.Z. Ritchie S.C. Hendrix R. Tucker-Burden C. Cho H.R. Aruffo A. Hollenbaugh D. Linsley P.S. Nature. 1996; 381: 434-438Crossref PubMed Scopus (1276) Google Scholar, 9Leach D.R. Krummel M.F. Allison J.P. Science. 1996; 271: 1734-1736Crossref PubMed Scopus (2689) Google Scholar, 10Linsley P.S. Wallace P.M. Johnson J. Gibson M.G. Greene J.L. Ledbetter J.A. Singh C. Tepper M.A. Science. 1992; 257: 792-795Crossref PubMed Scopus (757) Google Scholar). Identification of potent, orally active, small molecule inhibitors of the extracellular binding of these co-stimulatory molecules faces a number of thermodynamic challenges common to protein-protein interactions between opposing cell surfaces. Principally, these present obstacles to identifying inhibitors with sufficient binding kinetics (slow enough off-rates) to compete effectively. Protein-protein interfaces can be large and relatively shallow (11Janin J. Chothia C. J. Biol. Chem. 1990; 265: 16027-16030Abstract Full Text PDF PubMed Google Scholar, 12Lawrence M.C. Colman P.M. J. Mol. Biol. 1993; 234: 946-950Crossref PubMed Scopus (1098) Google Scholar), without preferred sites for small ligands. Additionally, the multiple interactions between the two cell surfaces results in a high avidity. Further still, T cell signaling occurs in a specialized contact area between cells, termed the immunological synapse (13Grakoui A. Bromley S.K. Sumen C. Davis M.M. Shaw A.S. Allen P.M. Dustin M.L. Science. 1999; 285: 221-227Crossref PubMed Scopus (2529) Google Scholar, 14van der Merwe P.A. Davis S.J. Shaw A.S. Dustin M.L. Semin. Immunol. 2000; 12: 5-21Crossref PubMed Scopus (246) Google Scholar) that forms to concentrate receptors and their signaling complexes (15Lee K-M. Chuang E. Griffin M. Khattri R. hong D.K. Zhang W. Straus D. Samelson L.E. Thompson C.B. Bluestone J.A. Science. 1998; 282: 2263-2266Crossref PubMed Scopus (537) Google Scholar). Although some successes have been reported for small molecule antagonism of analogous interactions mediated by integrins (16Chen L.L. Whitty A. Lobb R.R. Adams S.P. Pepinsky R.B. J. Biol. Chem. 1999; 274: 13167-13175Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 17Kelly T.A. Jeanfavre D.D. McNeil D.W. Woska J.R., Jr. Reilly P.L. Mainolfi E.A. Kishimoto K.M. Nabozny G.H. Zinter R. Bormann B.-J. Rothlein R. J. Immunol. 1999; 163: 5173-5177PubMed Google Scholar, 18Lin K. Ateeq H.S. Hsiung S.H. Chong L.T. Zimmerman C.N. Castro A. Lee W. Hammond C.E. Kalkunte S. Chen L.-L. Pepinsky R.B. Leone D.R. Sprague A.G. Abraham W.M. Gill A. Lobb R.R. Adams S.P. J. Med. Chem. 1999; 42: 920-934Crossref PubMed Scopus (206) Google Scholar, 19Miller W.H. Keenan R.M. Willette R.N. Lark M.W. Drug Disc. Today. 2000; 5: 397-408Crossref PubMed Scopus (117) Google Scholar) and selectins (20Kaila N., Xu, G.Y. Camphausen R.T. Xiang Y. Bioorg. Med. Chem. 2001; 9: 801-806Crossref PubMed Scopus (6) Google Scholar, 21Slee D.H. Romano S.J., Yu, J. Nguyen T.N. John J.K. Raheja N.K. Axe F.U. Jones T.K. Ripka W.C. J. Med. Chem. 2001; 44: 2094-2107Crossref PubMed Scopus (71) Google Scholar), co-stimulatory molecule interactions may present a difficult target. This is especially true for interactions involving CTLA4, whose submicromolar affinity for B7.1 is unusually high for interactions between cell surface molecules (22van der Merwe P.A. Bodian D.L. Daenke S. Linsley P. Davis S.J. J. Exp. Med. 1997; 185: 393-403Crossref PubMed Scopus (416) Google Scholar). In the recently solved crystal structure of the human CTLA4/B7.1 complex, CTLA4 and B7.1 pack in a strikingly periodic zipper-like arrangement in which bivalent CTLA4 homodimers bridge bivalent B7.1 homodimers. This reveals the structural basis for formation of unusually stable signaling complexes (23Stamper C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar). The combination of avidity-driven binding and submicromolar affinity is thought to be unique to CTLA4 and B7.1 (27Ikemizu S. Gilbert R.J.C. Fennelly J.A. Collins A.V. Harlos K. Jones E.Y. Stuart D.I. Davis S.J. Immunity. 2000; 12: 51-60Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Although CD28 binds B7 molecules with a lower affinity than CTLA4 (22van der Merwe P.A. Bodian D.L. Daenke S. Linsley P. Davis S.J. J. Exp. Med. 1997; 185: 393-403Crossref PubMed Scopus (416) Google Scholar), each of the other challenges mentioned above for inhibiting protein-protein interactions applies to small molecule antagonism of CD28 binding to the B7s as well. Consequently, the small molecule binding site on human B7.1 identified here may represent an exceptional, albeit challenging opportunity. In a high throughput screen we identified a number of inhibitors of B7.1/CD28 binding, two classes of which were studied further. Equilibrium dialysis demonstrated that these compounds bound specifically to human B7.1 at a common site. Occupancy of this site by the inhibitors blocked B7.1 binding not only to CD28, but also to CTLA4, although at much higher concentrations of inhibitors. Mapping of the binding of these small molecules then located this unique site to the GFCC′C“ face of the N-terminal V-set domain of human B7.1, very near to the site of its interactions with counter receptors. Production and characterization of antibodies to human B7.1 (EW3.1F1 and EW3.3B5), B7.1-Fc chimeras (including domain swaps and point mutants), and CTLA4- Fc were as described previously (23Stamper C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar). 1S. Wang, M. Stahl, Y. Xing, J. F. Tobin, and D. V. Erbe (2001), submitted for publication. CD28-Fc was produced similarly and kindly provided by Drs. James Wilhelm and Pranab Chanda. Fab fragments of the 1F1 antibody were made by digestion with papain and purified by protein-A affinity chromatography. The affinity of the Fab fragments for human B7.1 were determined with a BIAcore 2000 by surface plasmon resonance measurements using immobilized human B7.1. A calculated K d was obtained by first determining the dissociation rate constant, k off, and then the association rate constant, k on.k off was determined by fitting the data contained in the dissociation phase of the sensorgram to a monoexponential decay function. k on was determined by fitting the data contained in the associations phase of the sensorgram to the integrated rate equation using the calculatedk off. ELISA 2ELISAenzyme-linked immunosorbent assayTBSTris-buffered salineMe2SOdimethyl sulfoxidepNPPp-nitrophenyl phosphateCHOChinese hamster ovary wells were coated with 300 ng of CD28-Fc in carbonate buffer (pH 9.4) overnight at 4 °C, blocked with 1% bovine serum albumin in TBS for 1 h at 22 °C and then washed three times in TBS prior to assay. The detection complex was formed as follows: B7.1-Fc-biotin, prepared using NHS-LC-biotin (Pierce 21335) according to the manufacturer's instructions (4.1 mol of biotin/mol of Fc) was added at 0.8 μg/ml to streptavidin-alkaline phosphatase (Caltag SA1008) at 1:1000 in TBS. Inhibitors or Me2SO (1% final) were added to this complex and incubated 30 min at 22 °C. Detection complex (± inhibitors) was then added to the CD28-coated wells for 25 min at 22 °C, washed five times with TBS, developed with the colorimetric substrate pNPP (Pierce 34045) in diethanolamine/MgCl2 buffer (pH 9.5) and read at 405 nm. The high throughput screen for antagonists used this CD28/B7.1 ELISA in an automated format to test the Wyeth Research of a both as well as compounds and immunosorbent hamster ovary were coated with 300 ng of in overnight at 4 °C, blocked with 1% bovine serum albumin in TBS for 1 h at 22 °C, and then washed three times in TBS prior to assay. was added at μg/ml with or without inhibitors for five times in TBS. phosphatase at 1:1000 in TBS was then added for 1 h for by and development with pNPP as the antibody B7.1-Fc-biotin, antibody 1F1 was added at μg/ml for times prior to and detection with streptavidin-alkaline were in by compounds and in and each on of a and to To an was each an of was by and at to were a with at a rate of 0.8 and with a to was at and for of the integrated were used at compounds at prior to binding contained 1% antibody each was added at a to each of the dialysis the small molecule dialysis with B7.1-Fc and 1 were as above with a of each were then in to 1 in the The was then and the 1 was in Me2SO and the for as cell were human B7.1 or cells were to and washed with prior to assay. cells were by with and in buffer cells were then with for min at °C, washed in and at cells were incubated with inhibitors for 30 min at °C, added to of cells, and incubated 30 min at with were then washed with buffer by and cells were by at and the at nm. In a screen for antagonists of the binding of CD28 to B7.1, a number of compounds were which this interaction with in the of 1 and 30 whose in were for Equilibrium dialysis chimeras was used to the binding and for each in 1 is only in the of the human B7.1-Fc binding to or a Fc was this also not bind to mouse binding with these was for the second these two compounds appear to bind specifically to human B7.1 and not to the very mouse B7.1 These results were by in which interaction with B7.2 or mouse B7.1 by ELISA at concentrations as high as 1 binds only to human dialysis measurements of 1 binding to Fc is as with the protein of each with the these small molecules not bind the mouse B7.1 their binding can be between the two as was recently reported for a small molecule that binds to the 1 E.C. Chen C. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Google Scholar). Additionally, can the binding of antibodies to human B7.1 to small molecule binding as was for an antagonist T.A. Jeanfavre D.D. McNeil D.W. Woska J.R., Jr. Reilly P.L. Mainolfi E.A. Kishimoto K.M. Nabozny G.H. Zinter R. Bormann B.-J. Rothlein R. J. Immunol. 1999; 163: 5173-5177PubMed Google Scholar). previously B7.1 chimeras in which mouse were for their human in the human B7.1-Fc 1S. Wang, M. Stahl, Y. Xing, J. F. Tobin, and D. V. Erbe (2001), submitted for were then used to the binding for an of antibodies to the GFCC′C“ face of the N-terminal V-set domain of human B7.1. 1S. Wang, M. Stahl, Y. Xing, J. F. Tobin, and D. V. Erbe (2001), submitted for publication. the results these in dialysis with each of the small 1 to bind to the N-terminal V-set domain of human B7.1. binding with a the human V-set domain to the mouse domain and not with a the mouse V-set domain to the human domain of human B7.1-Fc in the of an antibody specific for the V-set domain binding of whereas with a antibody in also to bind the V-set binding with and not with The human B7.1 V-set domain to be sufficient for binding of a of only this domain to the Fc in the dialysis In these two small molecules appear to a binding site this N-terminal domain of B7.1. A of is to binding of 1 to B7.1 This common binding site is in with the antibody to B7.1 which binds to the two and on the GFCC′C“ face of the B7.1 N-terminal domain as previously 1S. Wang, M. Stahl, Y. Xing, J. F. Tobin, and D. V. Erbe (2001), submitted for publication. Consequently, human B7.1-Fc chimeras with in and near these were used in dialysis we not binding for the two small 1 not bind a B7.1 with the as well as B7.1. also not bind a with the to also binding to these two although not as this with small of to in B7.1 also results in of binding to both of ligands C.A. A. M. M. R. J. Exp. Med. 1995; PubMed Scopus Google Scholar), a in the binding for both small molecules identified here with CD28 and molecule binding to human B7.1. Equilibrium binding of 1 or was using a of B7.1 and with with human B7.1. present in each B7.1 were as follows: for for for and for the binding sites for these small molecules to with the sites for both CD28 and CTLA4, their to binding to was near their for of CD28 binding, of CTLA4 binding to B7.1 in the ELISA by was This is to the that the dissociation rate of for B7.1 is than that of CD28 for B7.1, and even a in CD28 binds to B7.1 as to the binding, which been demonstrated for CTLA4 (22van der Merwe P.A. Bodian D.L. Daenke S. Linsley P. Davis S.J. J. Exp. Med. 1997; 185: 393-403Crossref PubMed Scopus (416) Google Scholar, C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar, S. Gilbert R.J.C. Fennelly J.A. Collins A.V. Harlos K. Jones E.Y. Stuart D.I. Davis S.J. Immunity. 2000; 12: 51-60Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). of small molecule of B7.1 binding to CTLA4 was at higher concentrations and the CTLA4/B7.1 ELISA was used to by 1 at and at a of than times its for of CD28 binding to B7.1. at this which then as the binding is to 1 CTLA4 to compete for B7.1 than the small with CD28-Fc in even 4 h of binding in the of a antibody to B7.1, with a much dissociation rate constant, is to compete with CTLA4 binding at these is at an which the antibody the small molecule These data that the small molecule binding site on B7.1 identified here is near both the binding sites for CD28 and CTLA4. of co-stimulatory through CD28 and CTLA4 be to have the in disease this in in by these small molecule antagonists of B7.1 an a of be used which between CD28 and CTLA4 we the concentrations of 1 to inhibit CD28/B7.1 interactions in In using cells CD28 to cells B7.1, by 1 was at concentrations to in a of on T cell through CD28, specific was with 1 at to with of CTLA4 binding by B7.1, this was to the relatively of the small molecule and its to compete with the interactions between cells. To we a Fab of the antibody This with a K d for B7.1 binding by surface plasmon resonance and a dissociation rate times than the antibody CD28/B7.1 binding with an in this then an of which was than in the CD28 ELISA with an of 4 nm. This whose structure is in a at for CD28/B7.1 which we have reported J. J. L. J. A. J. S. Y. D. V. and J. F. Tobin (2001), submitted for publication. it an in the B7.1/CD28 ELISA to the antibody Fab these two used in a of B7.1/CD28 binding, by is at concentrations to the Fab with an of nm. This in for the Fab in the ELISA to cells the of present in the assay. Although the Fab and may have binding for B7.1 their ELISA the small molecule much and This its to inhibit CD28 interactions in the to this dissociation rate of the small molecule for B7.1, an than the of the may be to an of these inhibitors in cells, as signaling of CD28 by B7.1. J. J. L. J. A. J. S. Y. D. V. and J. F. Tobin (2001), submitted for of CD28/B7.1 interactions by small molecules and antibody cell the of at three with as the of at three with as in a of the may also be the challenging to inhibit with small protein-protein interfaces of relatively shallow surfaces without binding sites for small ligands. Although hot used to protein-protein binding T. J.A. Science. 1995; PubMed Scopus (1778) Google Scholar), the of sufficient for high affinity small molecule binding Chen K. K.A. P.A. S. A. 1999; PubMed Scopus Google Scholar) may be in the protein In although can sites on protein surfaces for very of these sites to D. C. Med. Rev. 1999; PubMed Scopus Google Scholar, A. M. PubMed Scopus Google Scholar, J. Med. Chem. PubMed Scopus Google Scholar, C. D. J. Chem. 1996; Scopus Google Scholar). Consequently, have to of even affinity to ligands for that can then be even for affinity D.R. M. R.M. J.A. S. A. 2000; PubMed Scopus Google Scholar, Science. 1996; 274: PubMed Scopus Google Scholar). The results of the challenges to small molecule at protein the results here indicate that a small molecule binding on B7.1, which is sufficient for the binding of at two classes of small molecules with The that this may be unique the B7 of co-stimulatory This binding site for small molecules on B7.1 near its counter binding site, which been recently by (23Stamper C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar). The crystal structure of B7.1 a protein with an domain with the V-set by a domain with (27Ikemizu S. Gilbert R.J.C. Fennelly J.A. Collins A.V. Harlos K. Jones E.Y. Stuart D.I. Davis S.J. Immunity. 2000; 12: 51-60Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In to other cell surface B7.1 binds CTLA4 with a relatively small with an unusually high of which for their binding kinetics (11Janin J. Chothia C. J. Biol. Chem. 1990; 265: 16027-16030Abstract Full Text PDF PubMed Google Scholar, der Merwe P.A. Bodian D.L. Daenke S. Linsley P. Davis S.J. J. Exp. Med. 1997; 185: 393-403Crossref PubMed Scopus (416) Google Scholar, C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar). In the with CTLA4, it is only the N-terminal V-set domain of B7.1 that CTLA4 (23Stamper C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar). it is that this domain of B7.1 also with CD28 for previously (23Stamper C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar). two surface on the GFCC′C“ face of this binding is by the of which the a surface of B7.1 of and (23Stamper C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google Scholar). These the of the binding at the binding is added through a between on and on B7.1. This of B7.1 is the for CD28 and CTLA4 binding, as by the that binding for an of antibodies also 1S. Wang, M. Stahl, Y. Xing, J. F. Tobin, and D. V. Erbe (2001), submitted for publication. Although the small molecule antagonists appear to bind near this site, of the structure of B7.1 in this is C.C. Zhang Y. Tobin J.F. Erbe D.V. Ikemizu S. Davis S.J. Stahl M.L. Seehra J. Somers W.S. Mosyak L. Nature. 2001; 410: 608-611Crossref PubMed Scopus (359) Google and S. Gilbert R.J.C. Fennelly J.A. Collins A.V. Harlos K. Jones E.Y. Stuart D.I. Davis S.J. Immunity. 2000; 12: 51-60Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). that some surface a or that then small molecule binding to binding of the inhibitors to B7.1 is not and be by a B7.1 or an in the small molecules is also of to that the two classes of antagonists identified here appear to bind to this same site. this portion of B7.1 may represent a of a small molecule binding site, which is not present in mouse B7.1 or in human These results the of this the GFCC′C“ face of the V-set domain as the site for B7.1 antagonist the small molecule B7.1 antagonists reported here not inhibit in the of an at high protein-protein is that through B7.1 with bound compounds off-rates) can be compounds a novel of inhibiting an to in autoimmune disease and in and Stahl for and and for for Zhang for Fc and for for high throughput and the for and of

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