Design, Engineering, and Production of Human Recombinant T Cell Receptor Ligands Derived from Human Leukocyte Antigen DR2 (2001)

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84–102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (α1 and β1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., Bächinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng.12, 771–778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366–6371). Single-chain human recombinant T cell receptorligands (RTLs) of ∼200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel β-sheet platform and antiparallel α-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85–99)) showed increased stability to thermal unfolding relative to the empty DR2-derived RTLs. These novel molecules represent a new class of small soluble ligands for modulating the behavior of T cells and provide a platform technology for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease. Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84–102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (α1 and β1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., Bächinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng.12, 771–778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366–6371). Single-chain human recombinant T cell receptorligands (RTLs) of ∼200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel β-sheet platform and antiparallel α-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85–99)) showed increased stability to thermal unfolding relative to the empty DR2-derived RTLs. These novel molecules represent a new class of small soluble ligands for modulating the behavior of T cells and provide a platform technology for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease. major histocompatibility complex T cell receptor antigen-presenting cell recombinant T cell receptor ligands myelin basic protein human polymerase chain reaction circular dichroism phosphate-buffered saline antigen human leukocyte antigen The pathogenesis of a variety of human diseases including multiple sclerosis, rheumatoid arthritis, diabetes, autoimmune uveitis, transplant rejection, chronic beryllium disease, and graft-versus-host disease appear to involve antigen-specific CD4+ T cells (1Swanborg R.H. J. Immunol. 1983; 130: 1503-1505PubMed Google Scholar, 2Steinman L. Sci. Am. 1993; 269: 106-114Crossref PubMed Scopus (78) Google Scholar, 3Cush J.J. Lipsky P.E. Arthritis Rheum. 1988; 31: 1230-1238Crossref PubMed Scopus (233) Google Scholar, 4Quill H. Schwartz R.H. J. Immunol. 1987; 138: 3704-3712PubMed Google Scholar, 5Janeway Jr., C.A. Bottomly K. Cell. 1994; 76: 275-285Abstract Full Text PDF PubMed Scopus (777) Google Scholar). It is thought that pathogenic T cells home to the target tissue where autoantigen is present and, after local activation, selectively produce Th1 lymphokines (6Schwartz R.H. J. Exp. Med. 1996; 184: 1-8Crossref PubMed Scopus (456) Google Scholar). This cascade of events leads to the recruitment and activation of lymphocytes and monocytes that ultimately destroy the target tissue (7Weinberg A.D. Whitham R. Swain S.L. Morrison W.J. Wyrick G. Hoy C. Vandenbark A.A. Offner H. J. Immunol. 1992; 148: 2109-2117PubMed Google Scholar). Activation of CD4+ T cells in vivo is a multistep process initiated by co-ligation of the TCR and CD4 by the MHC1 class II-peptide complex present on APC (signal 1) as well as co-stimulation through additional T cell surface molecules such as CD28 (signal 2). Ligation of the TCR in the absence of co-stimulatory signals has been shown to disrupt normal T cell activation, inducing a range of responses from anergy to apoptosis (4Quill H. Schwartz R.H. J. Immunol. 1987; 138: 3704-3712PubMed Google Scholar, 5Janeway Jr., C.A. Bottomly K. Cell. 1994; 76: 275-285Abstract Full Text PDF PubMed Scopus (777) Google Scholar, 6Schwartz R.H. J. Exp. Med. 1996; 184: 1-8Crossref PubMed Scopus (456) Google Scholar). Within the context of this model of T cell activation, a direct approach toward Ag-driven immunosuppression would be to present the complete TCR ligand, Ag in the context of MHC, in the absence of costimulatory signals that are normally provided by specialized APCs. Toward the long term goal of targeted antigen-driven immunosuppression of pathogenic T cells, we have developed a family of novel recombinant TCR ligands (RTLs) that consist of the β1 and α1 domains of human MHC class II molecules. RTLs comprising the peptide binding/T cell recognition domain from rat MHC class II have been expressed as single exons (8Burrows G.G. Bebo Jr., B.F. Adlard K.L. Vandenbark A.A. Offner H. J. Immunol. 1998; 161: 5987-5996PubMed Google Scholar), and these RTLs loaded with soluble peptide Ag had potent inhibitory activity on encephalitogenic T cells (9Burrows G.G. Chang J.W. Bächinger H.-P. Bourdette D.N. Wegmann K.W. Offner H. Vandenbark A.A. Protein Eng. 1999; 12: 771-778Crossref PubMed Scopus (56) Google Scholar). The work on the rat system was recently extended when we described the in vivo andin vitro potency of single-chain constructs in which peptide Ag was genetically encoded at the 5′-end of the RTL exon, a design that favored specific loading and high occupancy of peptide Ag in the MHC binding cleft (10Burrows G.G. Adlard K.L. Bebo Jr., B.F. Chang J.W. Tenditnyy K. Vandenbark A.A. Offner H. J. Immunol. 2000; 164: 6366-6371Crossref PubMed Scopus (36) Google Scholar). We developed our rat RTL constructs for clinical studies, treating experimental autoimmune encephalomyelitis, a paralytic, inflammatory, and sometimes demyelinating disease mediated by CD4+ T cells specific for central nervous system myelin components, including myelin basic protein (MBP). Experimental autoimmune encephalomyelitis shares a number of immunological similarities with the human demyelinating disease multiple sclerosis (11Paterson P.Y. J. Chronic Dis. 1973; 26: 119-126Abstract Full Text PDF PubMed Scopus (43) Google Scholar) and has been a useful model for preclinical testing of therapies for the human illness (12Weiner H.L. Mackin G.A. Matsui M. Orav E.J. Khoury S.J. Dawson D.M. Hafler D.A. Science. 1993; 259: 1321-1324Crossref PubMed Scopus (549) Google Scholar, 13Vandenbark A.A. Hashim G. Offner H. Nature. 1989; 341: 541-544Crossref PubMed Scopus (567) Google Scholar, 14Howell M.D. Winters S.T. Olee T. Powell H.C. Carlo D.J. Brostoff S.W. Science. 1989; 246: 668-670Crossref PubMed Scopus (414) Google Scholar, 15Oksenberg J.R. Panzara M.A. Begovich A.B. Mitchell D. Erlich H.A. Murray R.S. Shimonkevitz R. Sherritt M. Rothbard J. Bernard C.C. Nature. 1993; 362: 68-70Crossref PubMed Scopus (411) Google Scholar, 16Yednock T.A. Cannon C. Fritz L.C. Sanchez-Madrid F. Steinman L. Karin N. Nature. 1992; 356: 63-66Crossref PubMed Scopus (1544) Google Scholar, 17Jameson B.A. McDonnel J.M. Marini J.C. Korngold R. Nature. 1994; 368: 744-746Crossref PubMed Scopus (145) Google Scholar, 18Vandenbark A.A. Vainiene M. Celnik B. Hashim G.A. Buenafe A.C. Offner H. J. Immunol. 1994; 153: 852-861PubMed Google Scholar). To extend this work toward eventual human therapy, we have engineered molecules consisting of the α1 and β1 domains of the human MHC class II HLA-DR2 molecule genetically linked into a single polypeptide chain with and without covalently coupled MBP-(85–99) peptide (Fig.1). We describe in this report the design, production, and purification of these HLA-DR2b-derived RTLs as well as biochemical and biophysical characterization demonstrating retention of properties required for TCR engagement. These findings anticipate the production of large amounts of these human recombinant TCR ligands for structural characterization and immunotherapeutic applications. Molecules with this design would be useful for studying binding specificity in vitro, for exploring primary TCR signaling events independent of costimulatory input, and for treating CD4+ T cell-mediated autoimmune disease in an epitope-specific manner. Sequence alignment of MHC class II molecules from human, rat, and mouse species provided a starting point for our studies, as previously described (9Burrows G.G. Chang J.W. Bächinger H.-P. Bourdette D.N. Wegmann K.W. Offner H. Vandenbark A.A. Protein Eng. 1999; 12: 771-778Crossref PubMed Scopus (56) Google Scholar). Graphic images were generated with the program Sybyl (Tripos Associates, St. Louis, MO) and an O2 work station (IRIX 6.5, Silicon Graphics, Mountain View, CA) using coordinates deposited in the Brookhaven Protein Data Bank was on the coordinates of human J. L. K.W. J. Exp. Med. 1998; PubMed Scopus Google Scholar, H. R. J. 2000; PubMed Scopus Google Scholar) as well as J.C. 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Med. 1998; PubMed Scopus Google Scholar), the were and the peptide was as a structural using local production of the human was CA) from cells from R. and St. Louis, MO) in was using II the reaction as the of the and were by polymerase with an of The to β1 were and The to α1 were and The were and the were The at the 5′-end of and and at the of The two were linked in a the of were to a reaction and at an of reaction containing the and was to the reaction and at an of polymerase was in The reaction was and the was The was with the CA) and into an was to a single from which was was with and and the was The was and into were on and protein was from and with the and a single was for of the peptide acid was genetically into the and of the β1 The peptide Ag and was at of the in a reaction polymerase the was as in two the the from the of the of the to the point of the was with the and the the from the point of the to the of the was with the and reaction was and the were the of was to a reaction and at an of the a containing the and was to the and the was using a The of the the and the from the point of The resulting with the produced in the reaction the and of polymerase extended from the of the which as in the The reaction was using and the was The was and with a and into a coli were for protein and the of the with a was from and single was for of the peptide of and constructs the same to single at and and had been previously for number which in an in the and molecules We to the to the number Two were using the and as the and were the and were The two resulting were and and as described on the and of of the The were and the were The were to the RTL constructs into for into cells and were for of the empty HLA-DR2 molecule and the molecule coli cells were with the were in to in containing at protein production was by the of for the cells were by and at of the cells were at The cell were in and for with the cell in a The cell was the was and the cell was and in and in for the containing the recombinant protein of was and at The recombinant proteins of were and by protein using in an using a with The proteins was which the and of the recombinant This was were required for of the RTL constructs to after which could be into at and by with purification to a was using on in an The of protein and of dichroism were on a with an and cells of and on peptide Data are as was with acid to of and at and 1973; PubMed Scopus Google Scholar). were the of from to at a of with a Data were at were and using the of the and were was with the program S.W. J. PubMed Scopus Google Scholar). were at a of from to were generated with a and and were and was in the cell with a and and on an and on The were to the of the peptide using the where represent the for the and and is the observed at protein RTLs derived from the α1 and β1 domains of rat MHC class II (9Burrows G.G. Chang J.W. Bächinger H.-P. Bourdette D.N. Wegmann K.W. Offner H. Vandenbark A.A. Protein Eng. 1999; 12: 771-778Crossref PubMed Scopus (56) Google Scholar) provided a starting point for and human RTLs. of the MHC class II protein HLA-DR2 and, the α1 and β1 of the molecule that the antigen binding domain were on the of human J. L. K.W. J. Exp. Med. 1998; PubMed Scopus Google Scholar, H. R. J. 2000; PubMed Scopus Google Scholar, J.C. 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A. 1996; PubMed Scopus Google Scholar). The β1 domain of MHC class II molecules a that covalently the to the of the β-sheet platform by the This is MHC class II molecules from rat, human, and mouse and is the domain of MHC class It to a as a that primary in the molecule a of G.A. Science. PubMed Scopus Google Scholar) appear to the RTLs predicted the antigen binding domain would in the absence of the and were by the the amino of HLA-DR2 α1 domain to the the of the β1 We the RTL TCR for proteins with this G. Burrows, this we present that the empty RTL with the native a that the human MBP-(85–99) antigenic peptide and of these molecules containing that had a single to that activity work demonstrated that the of could be from the protein after and purification in containing (9Burrows G.G. Chang J.W. Bächinger H.-P. Bourdette D.N. Wegmann K.W. Offner H. Vandenbark A.A. Protein Eng. 1999; 12: 771-778Crossref PubMed Scopus (56) Google Scholar). of the RTLs was and by the protein was which the and of the recombinant This was were required for of the RTL constructs to after which could be into at for in vivo The of empty and antigenic RTLs was of and amino acid to chain residues and to the native was demonstrated by a in which with without the were the absence of are and proteins a through to of this are shown for the empty and and the and molecules of the RTL molecules produced showed this the of the native These represent a primary of the of the molecules. demonstrated the of RTL and and with the of class II molecules by J. L. K.W. J. Exp. Med. 1998; PubMed Scopus Google Scholar, H. R. J. 2000; PubMed Scopus Google Scholar, J.C. Nature. 1993; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus (145) Google Scholar, J. 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Protein Eng. 1999; 12: 771-778Crossref PubMed Scopus (56) Google Scholar). which the covalently coupled showed a in with This in was by a in thermal stability of with demonstrating the of the RTL molecule and, by the antigen platform of MHC class II molecules that peptide this is to has been observed using rat RTL molecules (9Burrows G.G. Chang J.W. Bächinger H.-P. Bourdette D.N. Wegmann K.W. Offner H. Vandenbark A.A. Protein Eng. 1999; 12: 771-778Crossref PubMed Scopus (56) Google Scholar), the by the covalently coupled peptide is for the human RTLs. The molecule showed a in and a in thermal stability with which had the and the covalently coupled showed in thermal was relative to and and the could be from the and for the thermal stability from using the coordinates from and MHC class II and J. L. K.W. J. Exp. Med. 1998; PubMed Scopus Google H. R. J. 2000; PubMed Scopus Google Scholar). These demonstrated that is a central the of the RTL of a of that to the that is in human class II molecules and is rat, and human MHC class The β-sheet to a central of polypeptide two of the α1 The structural is the α1 domain and the α1 domain at the of the peptide binding where the amino of the of residues of residues are as shown in with the number in is to of on chain of the is the antigenic is of the chain of DR2. The were using coordinates from J. L. K.W. J. Exp. Med. 1998; PubMed Scopus Google Scholar). The residues are as shown in with the number in is to of on chain of the is the antigenic in a new is of the chain of DR2. The were using coordinates from J. L. K.W. J. Exp. Med. 1998; PubMed Scopus Google Scholar). We have soluble single-chain RTL molecules derived from the antigen binding β1 and α1 domains of human MHC class II molecule DR2. The RTLs the the domain to bind to and the and The of the RTLs the to and the molecules from in high of cell The RTLs into and had in The that the human DR2-derived and structural and with the native MHC class II MHC class II molecules a that and antigenic to the T cell receptor is structural and to this model J.C. Nature. 1993; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus (145) Google Scholar, J. Science. 1996; PubMed Scopus Google Scholar, H. Nature. 1993; PubMed Scopus Google Scholar, Immunol. PubMed Scopus Google Scholar), the that provided by the MHC class II molecule in antigen T cell and T cell activation to be approach protein to structural from with recombinant technology to design and produce single-chain TCR ligands on the MHC class II peptide binding/T cell recognition the native molecule this domain is derived from of the and polypeptide that to a described as a β-sheet platform which two to an antigen binding is by a single exon the α1 and domains of MHC class with the that the peptide binding of MHC class II is the of constructs that the peptide binding/T cell recognition domain and an antigenic peptide H. J. J. J. Nature. 1994; PubMed Scopus Google Scholar). a and design this molecule a major of the human RTL molecules described in this report for the the peptide binding domains from the platform of biochemical and properties from and from the of that at the cell surface APC and T cell with the T cell a approach to T cell-mediated diseases is in of human RTL molecules described in this report of the specific by a TCR independent from the platform domains of MHC class with Th1 cell in the absence of APC costimulatory initiated a of through the These chain and activity as well as The of T cell with RTLs with APC and peptide had a to and and production These for the the signaling events by direct of the TCR in the absence of signals by molecules. The and immunological of RTL treatment on T cell behavior are described in a recently G. Burrows, J. Chang, D. and H.-P. Bächinger, for have a number of the the and The α1 and β1 domains showed an and a and The RTL of the α1 and β1 have the to as a single independent from the at this be required for of the α1 and β1 domains for of peptide in this surface a in the MHC class molecules with to the of the primary of amino acid residues two of the as well as of the β-sheet platform the a number of of the the high the peptide binding of the and β-sheet the of the of residues and 1) is in rat, human, and mouse class II and an as using the novel RTL molecules described in this report of the specific by of these are in biochemical The an and a is by circular a is additional the is to to of of at was to a for RTL The was as the of the in from the of of at a that peptide binding the α1 and β1 domain of MHC class II molecules in and a for binding to a of the T cells in the T cells in the to the point where are to the is to that empty RTL molecules bind to the TCR on these cells. of and biochemical RTLs as a platform technology for of protein with engineered specificity for target cells and primary in these molecules was for T cell behavior and that bind T cell in an antigen-specific would have a number of including the to antigen-specific T cells, to T cells in with and to mediated by antigen-specific T cells.