TLR7 gain-of-function genetic variation causes human lupusAbstract Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease 1–7 , evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA 8 , 9 and binds to guanosine 10 – 12 . We identified a de novo, previously undescribed missense TLR7 Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7 Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP 10–12 , and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c + age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7 Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.
Peripheral CD4+ T cell subsets and antibody response in COVID-19 convalescent individualsFang Gong, Yaping Dai, Ting Zheng et al.|Journal of Clinical Investigation|2020 BACKGROUNDMarked progress is achieved in understanding the physiopathology of coronavirus disease 2019 (COVID-19), which caused a global pandemic. However, the CD4+ T cell population critical for antibody response in COVID-19 is poorly understood.METHODSIn this study, we provided a comprehensive analysis of peripheral CD4+ T cells from 13 COVID-19 convalescent patients, defined as confirmed free of SARS-CoV-2 for 2 to 4 weeks, using flow cytometry and magnetic chemiluminescence enzyme antibody immunoassay. The data were correlated with clinical characteristics.RESULTSWe observed that, relative to healthy individuals, convalescent patients displayed an altered peripheral CD4+ T cell spectrum. Specifically, consistent with other viral infections, cTfh1 cells associated with SARS-CoV-2-targeting antibodies were found in COVID-19 covalescent patients. Individuals with severe disease showed higher frequencies of Tem and Tfh-em cells but lower frequencies of Tcm, Tfh-cm, Tfr, and Tnaive cells, compared with healthy individuals and patients with mild and moderate disease. Interestingly, a higher frequency of cTfh-em cells correlated with a lower blood oxygen level, recorded at the time of admission, in convalescent patients. These observations might constitute residual effects by which COVID-19 can impact the homeostasis of CD4+ T cells in the long-term and explain the highest ratio of class-switched virus-specific antibody producing individuals found in our severe COVID-19 cohort.CONCLUSIONOur study demonstrated a close connection between CD4+ T cells and antibody production in COVID-19 convalescent patients.FUNDINGSix Talent Peaks Project in Jiangsu Province and the National Natural Science Foundation of China (NSFC).
Structural Basis for Recognition of CD20 by Therapeutic Antibody RituximabJiamu Du, Hao Wang, Chen Zhong et al.|Journal of Biological Chemistry|2007 Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. To understand the molecular mechanism of recognition of human CD20 by Rituximab, we determined the crystal structure of the Rituximab Fab in complex with a synthesized peptide comprising the CD20 epitope (residues 163-187) at 2.6-Aå resolution. The combining site of the Fab consists of four complementarity determining regions that form a large, deep pocket to accommodate the epitope peptide. The bound peptide assumes a unique cyclic conformation that is constrained by a disulfide bond and a rigid proline residue (Pro172). The 170ANPS173 motif of CD20 is deeply embedded into the pocket on the antibody surface and plays an essential role in the recognition and binding of Rituximab. The antigen-antibody interactions involve both hydrogen bonds and van der Waals contacts and display a high degree of structural and chemical complementarity. These results provide a molecular basis for the specific recognition of CD20 by Rituximab as well as valuable information for development of improved antibody drugs with better specificity and higher affinity. Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. To understand the molecular mechanism of recognition of human CD20 by Rituximab, we determined the crystal structure of the Rituximab Fab in complex with a synthesized peptide comprising the CD20 epitope (residues 163-187) at 2.6-Aå resolution. The combining site of the Fab consists of four complementarity determining regions that form a large, deep pocket to accommodate the epitope peptide. The bound peptide assumes a unique cyclic conformation that is constrained by a disulfide bond and a rigid proline residue (Pro172). The 170ANPS173 motif of CD20 is deeply embedded into the pocket on the antibody surface and plays an essential role in the recognition and binding of Rituximab. The antigen-antibody interactions involve both hydrogen bonds and van der Waals contacts and display a high degree of structural and chemical complementarity. These results provide a molecular basis for the specific recognition of CD20 by Rituximab as well as valuable information for development of improved antibody drugs with better specificity and higher affinity. CD20 is a pan-B cell marker expressed from pre-B cells until B cells are differentiated into plasma cells (1Stashenko P. Nadler L.M. Hardy R. Schlossman S.F. J. Immunol. 1980; 125: 1678-1685PubMed Google Scholar). It is a tetraspan membrane protein that is predicted to contain a large extracellular loop (about residues 142 to 182) and to form oligomers on the cell surface (2Teeling J.L. Mackus W.J. Wiegman L.J. van den Brakel J.H. Beers S.A. French R.R. van Meerten T. Ebeling S. Vink T. Slootstra J.W. Parren P.W. Glennie M.J. van de Winkel J.G. J. Immunol. 2006; 177: 362-371Crossref PubMed Scopus (515) Google Scholar, 3Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (286) Google Scholar, 4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar). Although the precise function of CD20 remains unclear, biochemical and cell biological data have shown that it seems to form or regulate a voltage-independent calcium channel (3Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (286) Google Scholar, 5Li H. Ayer L.M. Lytton J. Deans J.P. J. Biol. Chem. 2003; 278: 42427-42434Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Despite the limited knowledge about its function, several lines of evidence have clearly demonstrated that CD20 is an ideal target for passive immunotherapy of B-cell lymphoma: it is highly expressed in more than 80% of the B-cell lymphomas but not in stem cells, pro-B cells, normal plasma cells, or other normal tissues; it remains on the cell surface without substantial internalization after cross-linking with antibodies; and it is not shed to the circulation to inhibit the antibody therapy (6Anderson K.C. Bates M.P. Slaughenhoupt B.L. Pinkus G.S. Schlossman S.F. Nadler L.M. Blood. 1984; 63: 1424-1433Crossref PubMed Google Scholar, 7Press O.W. Howell-Clark J. Anderson S. Bernstein I. Blood. 1994; 83: 1390-1397Crossref PubMed Google Scholar, 8Cragg M.S. Walshe C.A. Ivanov A.O. Glennie M.J. Curr. Dir. Autoimmun. 2005; 8: 140-174Crossref PubMed Google Scholar). The CD20-targeted chimeric monoclonal antibody (mAb) 3The abbreviations used are: mAb, monoclonal antibody; CDRs, complementarity determining regions; H, heavy chain; L, light chain. Rituximab (Rituxan,® IDEC-C2B8) was the first Food and Drug Administration approved mAb drug for the treatment of malignancy. Although it was originally used for treating low-grade non-Hodgkin lymphoma, Rituximab has been proven to be also effective against other types of lymphomas (9Leget G.A. Czuczman M.S. Curr. Opin. Oncol. 1998; 10: 548-551Crossref PubMed Scopus (220) Google Scholar, 10Boye J. Elter T. Engert A. Ann. Oncol. 2003; 14: 520-535Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) and some autoimmune diseases (11Check E. Nature. 2004; 428: 786Crossref PubMed Scopus (6) Google Scholar, 12Edwards J.C. Szczepanski L. Szechinski J. Filipowicz-Sosnowska A. Emery P. Close D.R. Stevens R.M. Shaw T. N. Engl. J. Med. 2004; 350: 2572-2581Crossref PubMed Scopus (2202) Google Scholar, 13Cohen Y. Nagler A. Autoimmun. Rev. 2004; 3: 21-29Crossref PubMed Scopus (17) Google Scholar). Multiple mechanisms have been proposed for the action of Rituximab in the depletion of B cells including its ability to mediate complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, and to induce cell apoptosis (reviewed in Refs. 14Smith M.R. Oncogene. 2003; 22: 7359-7368Crossref PubMed Scopus (645) Google Scholar and 15Jazirehi A.R. Bonavida B. Oncogene. 2005; 24: 2121-2143Crossref PubMed Scopus (250) Google Scholar). With the expansion of the clinical application of Rituximab in the treatment of lymphoproliferative diseases, it has been noticed that intensity of CD20 expression on B cells varies in patients that may affect the binding and efficacy of Rituximab therapy (16Keating M. O'Brien S. Semin. Oncol. 2000; 27: 86-90PubMed Google Scholar, 17McLaughlin P. Hagemeister F.B. Rodriguez M.A. Sarris A.H. Pate O. Younes A. Lee M.S. Dang N.H. Romaguera J.E. Preti A.H. McAda N. Cabanillas F. Semin. Oncol. 2000; 27: 37-41PubMed Google Scholar). Therefore, it is rational to expect that new antibodies with higher affinity and better specificity developed based on Rituximab might be beneficial in clinical use, especially for patients who have low expression levels of CD20. Besides Rituximab and Zevalin® (the prototype of Rituximab 2B8 attached by a radioactive substance 90Y), another mAb specific to human CD20, namely B1, in both native and radioderivative forms (Bexxar,® Tositumomab and 131I-Tositumomab), was approved by the Food and Drug Administration for the treatment of non-Hodgkin lymphoma in 2003 (18Garber K. J. Natl. Cancer Inst. 2003; 95: 189Crossref PubMed Scopus (5) Google Scholar). These antibody drugs along with some other mAbs against CD20 such as 1F5, AT80, and 2H7 were suggested to most likely recognize the same region (Tyr165 to Tyr182) of the large extracellular loop of human CD20 with fine specificities (2Teeling J.L. Mackus W.J. Wiegman L.J. van den Brakel J.H. Beers S.A. French R.R. van Meerten T. Ebeling S. Vink T. Slootstra J.W. Parren P.W. Glennie M.J. van de Winkel J.G. J. Immunol. 2006; 177: 362-371Crossref PubMed Scopus (515) Google Scholar, 19Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (137) Google Scholar). However, these antibodies vary considerably in their functional activities. For example, treatment of B cells with most mAbs promotes segregation of CD20 into detergent-insoluble lipid raft, whereas B1 is the exception. This property seems to be correlated with the ability of the antibodies to mediate complement-dependent cytotoxicity but irrespective of activation of cell apoptosis pathways (20Deans J.P. Robbins S.M. Polyak M.J. Savage J.A. J. Biol. Chem. 1998; 273: 344-348Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 21Cragg M.S. Morgan S.M. Chan H.T. Morgan B.P. Filatov A.V. Johnson P.W. French R.R. Glennie M.J. Blood. 2003; 101: 1045-1052Crossref PubMed Scopus (332) Google Scholar, 22Chan H.T. Hughes D. French R.R. Tutt A.L. Walshe C.A. Teeling J.L. Glennie M.J. Cragg M.S. Cancer Res. 2003; 63: 5480-5489PubMed Google Scholar). To understand the functional diversity of CD20 mAbs and the underlying mechanisms, identification of the epitope of CD20 recognized by Rituximab and other CD20-targeted mAbs has raised great interest in recent years. Sequence comparison of human and murine CD20 reveals that although the two species share 73% sequence identity, the large extracellular loop is less conserved as 16 of the approximate 43 amino acids are different (19Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (137) Google Scholar). Mutagenesis studies by exchanging variant residues of the large extracellular loop between human CD20 and mouse CD20 at the equivalent position indicate that residues Ala170 and Pro172 of human CD20 are critical determinants for the CD20 epitope (19Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (137) Google Scholar). Using a process of biopanning of a phage display peptide library consisting of randomized 7-mer cyclic peptides, it has been shown that an NPS motif corresponding to 171NPS173 of human CD20 is essential for Rituximab binding and Ala170 can be substituted by Ser (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar). Similar studies have also defined a discontinuous epitope that comprises 170ANPS173 and 182YCYSI185 of CD20 joined together spatially by a disulfide bond between Cys167 and Cys183 (23Binder M. Otto F. Mertelsmann R. Veelken H. Trepel M. Blood. 2006; 108: 1975-1978Crossref PubMed Scopus (110) Google Scholar). However, the underlying mechanism of the recognition and binding of Rituximab with the CD20 epitope remains unclear. We report here the crystal structure of the Rituximab Fab fragment in complex with an epitope peptide of the large extracellular loop (residues 163-187) of CD20 that provides the molecular basis for the antigen-antibody recognition and binding. Analysis of the complex structure explains very well biochemical data from the epitope-mapping studies and provides useful hints for the design and development of improved antibody drugs. Preparation of Antibody and Peptide—Rituximab was purchased from Roche. The Fab fragment was obtained by papain digestion of Rituximab and purified by cation exchange chromatography using SP-Sepharose FF (GE Healthcare) followed by hydrophobic interaction chromatography using phenyl-Sepharose HP (GE Healthcare). The purity and homogeneity of the Fab fragment was characterized by SDS-PAGE and dynamic light scattering analyses. The protein sample was concentrated to 8 mg/ml and then exchanged into a stock buffer (100 mm and mm for The amino sequence of the Fab fragment was determined to D.R. N. J.E. R.A. S. 1998; Scholar). cyclic peptide corresponding to residues of the large extracellular loop of human CD20 was synthesized in an disulfide bond was between Cys167 and Cys183 The of the peptide was determined by chromatography and with a purity of than and of the Rituximab Fab fragment large not and not be used for structure For the purified Rituximab Fab and the epitope peptide were at a of at for was using the by of the and a calcium and to a of at in For data were by and then to data were to 2.6-Aå at of and using PubMed Scopus Google Scholar). for data are in of data and structure of in to the of and of of of B of bond bond position in to the in a new and structure of the Rituximab Fab in complex with human CD20 epitope peptide was using the molecular as in Biol. 2005; PubMed Scopus Google Scholar). The structure of the Fab fragment of human mAb M.J. A. PubMed Scopus Google Scholar) was used as the was by using consisting of molecular B and B P.D. P. J. M. L.M. T. Biol. 1998; PubMed Scopus Google Scholar). was using several of using M. A. PubMed Scopus Google the was improved and for the epitope peptide without The of the structure was with Biol. 1994; PubMed Scopus Google Scholar). of the structure are also in was using P.D. P. J. M. L.M. T. Biol. 1998; PubMed Scopus Google Scholar) and in the Biol. 1994; PubMed Scopus Google Scholar). The of the Fab fragment was with the by A. B. J. Biol. 2006; PubMed Scopus Google Scholar). were using M. PubMed Scopus Google Scholar) and 2002; Scholar). of the Rituximab studies have the epitope of CD20 recognized by Rituximab a sequence motif at the large extracellular loop of CD20 consisting of 170ANPS173 (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar, 19Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (137) Google Scholar, M. Otto F. Mertelsmann R. Veelken H. Trepel M. Blood. 2006; 108: 1975-1978Crossref PubMed Scopus (110) Google Scholar). To understand the structural basis of the recognition of CD20 by Rituximab, we synthesized a peptide of the epitope of CD20 comprising the CD20 sequence from to to the CD20 To the conformation of the CD20 an disulfide bond was between residues Cys167 and Cys183 of the synthesized peptide such has been in human CD20 expressed in and J.A. H. 2005; PubMed Scopus Google Scholar). This disulfide has also been to an role in the recognition and binding of the epitope by Rituximab of the disulfide bond the binding of CD20 to Rituximab and of the disulfide bond can the binding J.A. H. 2005; PubMed Scopus Google Scholar). The peptide was to a protein and shown to have with Rituximab as the cyclic and (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar) by the binding of the was also by peptide with and complement-dependent cytotoxicity using cells B and The CD20 epitope peptide was in complex with the Rituximab Fab The structure of the complex was using molecular and to a of with an of and a of are two and in the The two very conformation of for that the heavy in complex more residues at the than in complex B and two residues can be at the of the peptide in complex B than in complex A. to the better the structure of complex be used for structural and L, H, and are to the light and heavy of the Fab fragment and the epitope the Rituximab Fab has very residues from to of a loop in the region that have high B with the B of for the The Rituximab Fab has a consisting of four The light comprises residues to that into the and and heavy residues to that into the and The by the two that the of to and to is about are four disulfide bonds between and and and and and and disulfide bond between and other Fab residue of the light in the region of the and forms of the B. J. Biol. 273: PubMed Scopus Google Scholar). The complementarity determining regions of the Rituximab Fab have without residues to sequence data PubMed Scopus Google Scholar). The and to B. J. Biol. 273: PubMed Scopus Google Scholar) and The and together form a large, deep pocket to accommodate the epitope whereas and are and The for the bound epitope peptide is well defined without in the of the and and of the peptide form a disulfide bond that the of the peptide together and the peptide to a unique cyclic conformation The of the peptide to forms a that is by the of the disulfide bond and the interactions between residues of the and the of the Rituximab The of the peptide forms a to and a loop to that are by interactions with the other regions and The of the peptide to forms a of hydrophobic and it is at the of the extracellular loop and be to the cell might be the of a of CD20 as predicted by B. J. Res. 2004; PubMed Scopus Google interactions between the of the peptide and the other regions of the in a new between the Rituximab Fab and the Peptide—Rituximab can to CD20 on B cells with a binding affinity of K. J.E. R. R.A. N. Anderson D.R. Blood. 1994; 83: PubMed Google Scholar). the complex the epitope peptide of human CD20 is bound at the large pocket by and of the Rituximab Fab is with the that the heavy more than the light in binding especially Fab with a D.R. S. Rev. PubMed Scopus Google Scholar, Curr. Opin. Biol. 1994; PubMed Scopus Google Scholar). The binding of the epitope peptide with the Fab a surface of about with a of is about of the peptide surface or of the Fab surface Although the surface is the of the Curr. Opin. Biol. PubMed Scopus Google the peptide the regions of the Rituximab Fab well with a high degree of structural and chemical complementarity as by the high complementarity of with with the of for J. Biol. 1993; PubMed Scopus Google Scholar). The and of the Fab in interactions with four of the epitope peptide that have been shown to be a critical motif on the CD20 surface for antibody recognition (2Teeling J.L. Mackus W.J. Wiegman L.J. van den Brakel J.H. Beers S.A. French R.R. van Meerten T. Ebeling S. Vink T. Slootstra J.W. Parren P.W. Glennie M.J. van de Winkel J.G. J. Immunol. 2006; 177: 362-371Crossref PubMed Scopus (515) Google Scholar, 4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar, M. Otto F. Mertelsmann R. Veelken H. Trepel M. Blood. 2006; 108: 1975-1978Crossref PubMed Scopus (110) Google Scholar). of the motif form a of hydrogen bonds with residues of the and and the of forms two hydrogen bonds with the of and of the The of forms a hydrogen bond with the of of loop a and the and of two hydrogen bonds with the of of loop the residues the motif including and also to the interactions of the peptide with the Fab by hydrogen bonds with and and and to these van der Waals contacts are between residues and of the peptide and the Fab the 170ANPS173 motif of the van der Waals contacts between the peptide and the Fab These interactions can the high affinity of Rituximab with human interactions between the Rituximab Fab and the epitope bond by The the is the between the Fab and the and the after the is the between the and the peptide bond by The the is the between the Fab and the and the after the is the between the and the peptide in a new der Waals contacts between the Rituximab Fab and the epitope peptide Fab in to the of van der Waals contacts in to the of van der Waals contacts in a new of the 170ANPS173 motif that has been shown to an essential role in the antigen-antibody recognition (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar, 19Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (137) Google is at the of the pocket by residues and of loop of the Fab and residue of loop and has both and hydrophobic interactions with the residues of the The position of at the of the and the rigid conformation of proline might an role in the unique conformation of the peptide and in the recognition and binding of Rituximab. for the of CD20 is an drug target for the treatment of Rituximab is the first Food and Drug Administration approved mAb drug against CD20 for the treatment of B cell non-Hodgkin lymphoma, and has been used to autoimmune diseases and the in crystal structure of the Rituximab Fab in complex with a peptide the epitope on the large extracellular loop of human CD20 has a molecular basis for the underlying mechanisms of the recognition and binding of CD20 with its antibodies and be valuable in the of Rituximab for development of more effective mAb drugs against non-Hodgkin lymphoma and other diseases. of phage display peptide that can 7-mer cyclic and have shown that the sequence motif corresponding to 170ANPS173 of the large extracellular loop of human CD20 is the most region for Rituximab recognition and binding (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar, M. Otto F. Mertelsmann R. Veelken H. Trepel M. Blood. 2006; 108: 1975-1978Crossref PubMed Scopus (110) Google Scholar). Ala170 and Pro172 are the most critical determined by of phage and peptide (2Teeling J.L. Mackus W.J. Wiegman L.J. van den Brakel J.H. Beers S.A. French R.R. van Meerten T. Ebeling S. Vink T. Slootstra J.W. Parren P.W. Glennie M.J. van de Winkel J.G. J. Immunol. 2006; 177: 362-371Crossref PubMed Scopus (515) Google Scholar, 4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar, 19Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (137) Google Scholar). structure of the Rituximab four residues 170ANPS173 of the motif are deeply in the pocket by four and Ala170 is in a hydrophobic by and of the The of the is to accommodate other residues with a large to an for the that is with Ala170 (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar). Pro172 is at the of the and is bound at the of the pocket that a residue at the same However, the of the rigid conformation of Pro172 might the of the and the specificity and binding explains the that Pro172 was substituted by in the cyclic 7-mer the peptide not to Rituximab (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar). The and of the two other and of the motif are also by crystal structure that residue two of hydrogen bonds as well as van der Waals contacts with the Rituximab the structure residues and of the epitope peptide and and van der Waals contacts with the Rituximab Although 170ANPS173 of CD20 were to be in the Rituximab the other residues might also some in the recognition and binding of CD20 by Rituximab. on phage display it was suggested that fragment at the of the large extracellular loop of CD20 is also in Rituximab binding (23Binder M. Otto F. Mertelsmann R. Veelken H. Trepel M. Blood. 2006; 108: 1975-1978Crossref PubMed Scopus (110) Google Scholar). region forms of the and has interaction with the However, residues and of the epitope peptide form a disulfide bond that the peptide a unique cyclic for the epitope of CD20 with phage display peptide a of cyclic and were the cyclic the sequence of human CD20 (4Perosa F. Favoino E. Caragnano M.A. Dammacco F. Blood. 2006; 107: 1070-1077Crossref PubMed Scopus (79) Google Scholar). data have shown that of the disulfide bond on the large extracellular loop of CD20 the binding of CD20 with Rituximab J.A. H. 2005; PubMed Scopus Google Scholar). It is very likely that the of the fragment of the large extracellular loop in the recognition and binding of Rituximab is the of the disulfide bond and the and of the cyclic conformation of the epitope than interaction with the Analysis of the crystal structure of the Rituximab Fab in complex with its epitope peptide also provides valuable information for of the antibody to the specificity and binding affinity. of residues on the of the Rituximab Fab that more interactions with residues of the epitope of CD20 might its binding affinity and specificity with CD20. For of with a residue a interactions with the of of to more interactions with the of of to might form hydrogen bonds with the of of with a residue such as might form a with the of interaction with the of of with a residue might form new interactions with the of and we report here the crystal structure of the Fab fragment of antibody Rituximab in complex with its epitope peptide of human CD20. reveals the molecular basis of the specific recognition and binding of CD20 by Rituximab. the most epitope region 170ANPS173 on the large extracellular loop of CD20 is bound at a pocket by four of the Rituximab Fab and recognized by residues of the a of interactions and van der Waals The unique cyclic conformation of the epitope is to the of a disulfide bond between and and the of a rigid forms the basis of the specificity of Rituximab. structural results also provide useful hints for the development of new antibodies with higher binding affinity and better specificity for the treatment of non-Hodgkin We are to the at for in data and other of for and with