WO1996021028A2 - Recepteurs de lymphocytes t heterodimeres solubles et leurs anticorps - Google Patents

Recepteurs de lymphocytes t heterodimeres solubles et leurs anticorps Download PDF

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WO1996021028A2
WO1996021028A2 PCT/US1995/016937 US9516937W WO9621028A2 WO 1996021028 A2 WO1996021028 A2 WO 1996021028A2 US 9516937 W US9516937 W US 9516937W WO 9621028 A2 WO9621028 A2 WO 9621028A2
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cells
tcr
cell receptor
heterodimeric
cell
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PCT/US1995/016937
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WO1996021028A3 (fr
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Julian Banerji
Sanjay Khandekar
Pamela Brauer
Jerome Naylor
Una Mckeever
Michael Jesson
Barry Jones
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Procept, Inc.
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Publication of WO1996021028A2 publication Critical patent/WO1996021028A2/fr
Publication of WO1996021028A3 publication Critical patent/WO1996021028A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the T cell receptor is a clonally expressed cell surface protein of T lymphocytes which mediates recognition of foreign antigens. It is composed of six polypeptide chains, two of which form a heterodi er and are unique to any given clonal T cell line.
  • Four polypeptides ( ⁇ , ⁇ , , S) form two different heterodimers (a: ⁇ and y. ⁇ ) ; the y. S heterodimer appears earlier than the a : ⁇ heterodimer in the development of an organism (Davis, M. M. and P. J.
  • variable (V) regions The amino terminal half of the ⁇ and ⁇ (or and ⁇ depending on the T cell subtype) chains which comprise the TCR are known as the variable (V) regions because the unique specificity of the TCR is a reflection of the marked amino acid sequence diversity of these regions.
  • This sequence diversity determines the specificity of the TCR, enabling recognition of a vast array of protein fragments, or epitopes, presented by the "restricting element", the major histocompatibility complex (MHC) (known in humans as the HLA complex) class I and class II proteins (Germain, R.N. , Cell 76:287-299 (1994)).
  • MHC major histocompatibility complex
  • TCR Recognition by the TCR of antigen in the context of MHC (or HLA) molecules triggers T cell activation, thus initiating the immune response.
  • the sequences of the TCR ⁇ - and /3-chain variable regions are encoded by gene segments that undergo somatic recombination to form complete transcriptional units during T cell development (Davis, M.M. and P. Bjorkman, Nature 334:395-402 (1988)). Because rearrangements of the V and J segments of the ⁇ -chain family and the V, D, and J segments of the 3-chain family occur independently in each developing T cell, the TCR repertoire of antigen-binding specificities is expressed clonally.
  • variable regions of the ⁇ //3 TCR (i) as variable regions connected by a polypeptide linker to create single chain (sc) TCR molecules; (ii) as fusions with immunoglobulin kappa light chains; and (iii) as phosphotidylinositol-linked heterodimers on the surface of cells in tissue culture.
  • This expression system offers efficient production of protein in high yields; however, much of the bacterially-derived scTCR is aggregated, improperly folded and insoluble. Refolding of purified and denatured recombinant proteins is often an inefficient process because the denatured scTCR is highly insoluble and prone to aggregation or precipitation when undergoing refolding.
  • the apparent low solubility of the scTCR as expressed in bacteria in aqueous solvents further reduces the yield following renaturation.
  • TCR ⁇ - and ⁇ - chains have been many attempts to express TCR ⁇ - and ⁇ - chains in eukaryotic cells (Traunecker, A., et al . , Immunol . Today 10 : 29 (1989).
  • TCR ⁇ / ⁇ heterodimers could not be obtained in the absence of coexpression of 7, S , e , and f chains; that is, the other members of the group of proteins that together form the CD3 complex.
  • both ⁇ and ⁇ chain extracellular domains can be synthesized as soluble chimeric molecules with carboxy-termini derived from immunoglobulin molecules (Gregoire, C. et al . Proc . Natl . Acad . Sci . USA 88:8077-8081 (1991); Gascoigne, N.R.J. et al . , Proc . Natl . Acad . Sci . USA 84:2936-2941 (1987) ; Weber, S. et al . , Nature 256:793-796 (1992)).
  • Phosphatidyl inositol membrane anchored ⁇ / ⁇ TCR heterodimers have been produced on the surface of CHO cells, and enzymatically released from the cell surface by phospholipase C treatment ((Lin, A.Y. et al . , Science 249:677 (1990); Slanetz A.E. and Bot well, A.L.M.,
  • the present invention concerns a polypeptide molecule comprising a heterodimeric T cell receptor (TCR) molecule containing a ⁇ subunit connected by disulfide bonds to an ⁇ subunit of the TCR.
  • TCR T cell receptor
  • the ⁇ and ⁇ subunits are generated as separate chimeric polypeptides, comprising an segment or a ⁇ segment in conjunction with either a f chain or an immunoglobulin constant region as a chimeric partner.
  • the ⁇ and j8 subunits can be generated as non-chimeric polypeptides comprising an o or a ⁇ segment.
  • the chimeric partners can be removed from the subunits through enzymatic cleavage.
  • Heterodimeric TCR from which the chimeric partners have been removed or are absent are soluble dual chain (dc) TCR.
  • the heterodimeric TCR of the current invention is a TCR protein, soluble in aqueous solvents containing simple buffers, with a conformation that is functionally indistinguishable, based upon reactivity with clonotype-specific antibodies, from the conformation which appears on the surface of T cells (referred to herein as a "native-like" or “native” conformation) .
  • This soluble TCR protein is biologically functional and does not require refolding or renaturation of the protein.
  • the invention also concerns nucleic acid molecules encoding heterodimeric TCR, as well as expression vectors which comprise nucleic acid molecules encoding the heterodimeric TCR, and also host cells containing such expression vectors.
  • the heterodimeric TCR molecules of the invention can be used to detect and analyze the peptide and MHC/HLA molecular constituents of TCR ligands.
  • the heterodimeric TCR can also be used for diagnostic purposes, such as for the detection of T cells with pathogenic properties.
  • the heterodimeric TCR can additionally be used in functional, cellular and molecular assays, and in structural analyses, including X-ray crystallography, nuclear magnetic resonance spectroscopy, and computational techniques, designed to identify TCR antagonists or agents that inhibit the interaction between TCR and MHC/HLA molecules complexed with antigenic peptides. Similar techniques can be performed to screen for agents capable of blocking the interaction of TCR with TCR specific antibodies.
  • the heterodimeric TCR can additionally be used in vivo , in order to compete with pathogenic T cells; or to immunize mammals, particularly humans, against TCR structures that occur on the surface of T cells which perform pathogenic or otherwise undesirable functions.
  • TCR-specific antibodies raised against heterodimeric TCR can be used in therapeutic strategies that are designed to regulate immune responses in vivo by either inhibiting or eliminating specific antigen-recognition by T cells. By selecting antibodies that recognize defined epitopes of the TCR, a restricted subset, or a clone of T cells involved in a disease or medically undesirable immune response, can be targeted.
  • the antibodies can be unmodified, or can also be linked to cytotoxic drugs, toxins, enzymes or radioactive substances.
  • Figure 1 depicts the coding regions unique to the ⁇ - chain (SEQ ID NO. 1) of the soluble, secreted form of the D10 dual chain TCR (dcTCR) .
  • Figure 2 depicts the coding regions unique to the ⁇ - chain (SEQ ID NO. 2) of the soluble, secreted form of the D10 dcTCR.
  • Figure 3 depicts baculovirus transfer vector p9/237 containing the D10 a/ ⁇ secreted TCR in pAcUW51.
  • Figure 4A depicts a schematic diagram of variable and constant domains of soluble D10 dcTCR, indicating predicted inter- and intra- chain disulfide bonds.
  • Figure 4B depicts a schematic diagram of variable and constant domains of the DlO-IgGl TCR construct, indicating predicted inter- and intra-chain disulfide bonds.
  • Figure 5 depicts baculovirus transfer vector plO/248 containing the BIO a/ ⁇ secreted TCR in pAcUW51.
  • Figure 6 depicts baculovirus transfer vector p2/246 containing the BIO TCR a/ ⁇ TCR-IgGl chimeras in pAcUW51.
  • Figure 7 depicts baculovirus transfer vector p7/243 containing the D10 dcTCR a/ ⁇ TCR-IgGl chimeras in pAcUW5l.
  • Figure 8 depicts the baculovirus transfer vector p3/598 containing the BDC 2.5 TCR 0-IgGl chimera in pVL941.
  • Figure 9 depicts the baculovirus transfer vector p7/599 containing the BDC 2.5 TCR ⁇ -IgGl chimera in pVL941.
  • Figure 10 depicts baculovirus transfer vector pl6/599 containing the BDC 2.5 a/ ⁇ TCR-IgGl chimeras in pAcUW51 (0/pH, ⁇ /plO).
  • Figure 11 depicts baculovirus transfer vector p20/599 containing the BDC 2.5 af ⁇ TCR-IgGl chimeras in pAcUW51 ( ⁇ /pH, 0/plO).
  • Figure 12 depicts nucleic acid (SEQ ID NO. 3) and deduced amino acid (SEQ ID NO. 4) sequences at the 5'-end of the V ⁇ -13.1 gene segment.
  • Figure 13 depicts baculovirus transfer plasmid pll/606 containing BDC 6.9 TCR ⁇ -lqGl downstream of the polyhedron promoter in pVL941 and BDC 6.9 TCR ⁇ -IgGl downstream of the P10 promoter in pAcUW51. Restriction sites used in the construction are indicated in bold.
  • Figure 14 depicts baculovirus transfer plasmid p33/606 containing BDC 6.9 TCR ⁇ -IgGl downstream of the polyhedron promoter in pVL941 and BDC 6.9 TCR 0-IgGl downstream of the P10 promoter in pAcUWSl. Restriction sites used in the construction are indicated in bold.
  • Figure 15 depicts baculovirus transfer plasmid pll/607 containing BDC 2.5 TCR / 3-IgGl downstream of the polyhedron promoter in pVL941 and BDC 2.5 TCR ⁇ -IgGl-hexahistidine downstream of the P10 promoter in pAcUWSl. Restriction sites used in the construction are indicated in bold.
  • Figure 16 depicts baculovirus transfer plasmid p2l/607 containing BDC 2.5 TCR 0-IgGl downstream of the polyhedron promoter in pVL941 and BDC 2.5 TCR ⁇ -IgGl-streptag downstream of the P10 promoter in pAcUWSl. Restriction sites used in the construction are indicated in bold.
  • Figure 17 depicts baculovirus transfer plasmid p41/607 containing BDC 2.5 TCR o-IgGl-hexahistidine downstream of the polyhedron promoter in pVL941 and BDC 2.5 TCR 0-IgGl downstream of the P10 promoter in pAcUWSl. Restriction sites used in the construction are indicated in bold.
  • Figure 18 depicts baculovirus transfer plasmid p51/607 containing BDC 2.5 TCR ⁇ -IgGl-streptag downstream of the polyhedron promoter in pVL941 and BDC 2.5 TCR /3-IgGl downstream of the P10 promoter in pAcUWSl. Restriction sites used in the construction are indicated in bold.
  • Figure 19 depicts baculovirus transfer plasmid pll/608 containing BIO TCR ⁇ -IgGl-hexahistidine downstream of the polyhedron promoter in pVL941 and BIO TCR ⁇ -IejGl downstream of the P10 promoter in pAcUWSl. Restriction sites used in the construction are indicated in bold.
  • Figure 20 depicts baculovirus transfer plasmid p31/608 containing BIO TCR ⁇ -IgGl-streptag downstream of the polyhedron promoter in pVL941 and BIO TCR 0-IgGl downstream of the P10 promoter in pAcUWSl. Restriction sites used in the construction are indicated in bold.
  • Figure 21 depicts baculovirus transfer plasmid pl/258 containing the D10 ⁇ -chain downstream of the polyhedron promoter in pVL941.
  • Figure 22 depicts baculovirus transfer plasmid p7/258 containing the D10 S-chain downstream of the polyhedron promoter in pVL941.
  • Figure 23 depicts baculovirus transfer plasmid pl4/259 containing D10 dcTCR ⁇ -hexahistidine downstream of the polyhedron promoter in pVL941.
  • Figure 24 depicts baculovirus transfer plasmid p9/259 containing D10 dcTCR ⁇ -streptag downstream of the polyhedron promoter in pVL941.
  • Figures 25A and 25B are graphic depictions of the production of antibodies specific for the BDC 2.5 cell- surface TCR by NOD mice immunized with the BDC 2.5 TCR-IgGl protein.
  • Figure 25(A) is a series of three graphs depicting the following: T cells of the indicated type stained by indirect imrounofluorescence with (1) non-immune NOD serum (line) or (2) anti-BDC 2.5 TCR-IgGl antiserum (long broken line) followed by FITC-GaMIg antibody.
  • TCR expression was demonstrated by direct immunofluorescent staining with (3) FITC-anti-V ⁇ -4 mAb (short broken line) for BDC 2.5 and 6.9 cells, and (5) FITC-anti-TCR C ⁇ mAb for purified NOD splenic T cells (long-short broken line) .
  • Isotype matched controls (4) were FITC-anti-V ⁇ -6 for the V ⁇ -4-specific mAb, and FITC-anti-V 7 -3 for the TCR C ⁇ - specific mAb (dotted line) .
  • Figure 25(B) is a series of two graphs, depicting the following: BDC 2.5 cells preincubated with (6) buffer alone (line), (7) a 1:10 dilution of pooled NOD mouse non- immune serum (long broken line), or (8) a 1:10 dilution of NOD mouse anti-BDC 2.5 TCR-IgGl antiserum (short broken line) .
  • the cells were then stained with FITC-anti-V ⁇ -4 mAb, or FITC-anti-TCR C ⁇ as indicated.
  • Figures 26A and 26B are graphic depictions of the blocking of the antigen-specific response of the BDC 2.5 T cell clone in vitro by NOD mouse anti-BDC 2.5 TCR-IgGl antiserum.
  • Figure 27 is a graphic representation of cytofluorimetry demonstrating V/38-specific staining with antiserum from an SJL mouse immunized with D10 TCR-IgGl.
  • the present invention concerns a polypeptide molecule, or protein, comprising a heterodimeric T cell receptor (TCR) .
  • TCR T cell receptor
  • the heterodimeric TCR comprises ⁇ and ⁇ subunits joined by at least one disulfide bond; the term, "heterodimeric,” indicates that the TCR is a disulfide- 1inked heterodimer comprising separate ⁇ and ⁇ subunits.
  • the subunits of the heterodimeric TCRs can be chimeric polypeptides ("chimeras") comprising an ⁇ or ⁇ segment linked to a chimeric partner.
  • the chimeric partner is any polypeptide that allows or promotes heterodimer formation. In a preferred embodiment, the chimeric partner is an immunoglobulin domain.
  • the chimeric partner can be different for each of the ⁇ and ⁇ segments, provided that the different partners interact to allow formation of a heterodimer.
  • TCRs comprising chimeric polypeptides are referred to herein as "chimeric" TCR.
  • the subunits of the heterodimeric TCRs can include ⁇ and ⁇ segments that are not linked to a chimeric partner; TCRs comprising ⁇ and ⁇ segments without chimeric partners are referred to herein as "dual chain" TCRs (dcTCRs) .
  • dcTCRs dual chain TCRs
  • the heterodimeric TCRs are soluble when purified; moreover, they react with anti-clonotypic antibodies that are specific for the native conformation of the TCR. As described in detail below, soluble heterodimeric TCRs can be produced that are in a conformation that is functionally indistinguishable from the cell surface TCR determinant that is unique to a particular clonal line of T cells.
  • Several steps are taken to generate heterodimeric TCRs. First, nucleic acid fragments bearing gene sequences for the ⁇ and ⁇ segments of the TCR of interest are isolated.
  • the nucleic acid fragments can be DNA or cDNA that are isolated by known methods. For example, synthetic oligonucleotide primers corresponding to portions of the ⁇ and ⁇ gene sequences can be used in the polymerase chain reaction (PCR) to amplify DNA or cDNA prepared from T cells bearing the TCR of interest.
  • PCR polymerase chain reaction
  • soluble heterodimeric TCRs are generated through the use of ⁇ -f and ⁇ - ⁇ chimeras.
  • the nucleic acid fragments encoding the ⁇ chain and the ⁇ chain are each cloned into separate vectors comprising a nucleic acid fragment encoding a f chain, thereby generating a- ⁇ and ⁇ - ⁇ coding regions.
  • the ⁇ * is referred to herein as the "chimeric partner".
  • the ⁇ -f and ⁇ - ⁇ coding regions are then inserted into one combination vector with a different promoter for each of the coding regions, or into separate transfer vectors with the same promoter for both coding regions.
  • the combination transfer vector can comprise a single promoter for both the ⁇ -f and 0-f coding regions, or separate promoters for each of the coding regions.
  • soluble heterodimeric TCRs are generated through the use of ⁇ -IgGl and /3-IgGl chimeras.
  • nucleic acid fragments encoding the ⁇ chain and the ⁇ chain are each cloned into separate vectors comprising a nucleic acid fragment encoding the CH2 and CH3 regions of an IgGl molecule, thereby generating ⁇ -IgGl and ⁇ -IgGl coding regions.
  • the vector comprising a nucleic acid fragment encoding a portion of the IgGl molecule that encodes a nucleic acid fragment encoding the hinge, CH2 and CH3 domains of an IgGl heavy chain.
  • IgGl is referred to herein as the "chimeric partner".
  • the ⁇ -IgGl and 3-IgGl coding regions are then inserted into one combination vector with a different promoter for each of the coding regions, or into separate transfer vectors with the same promoter for both coding regions.
  • soluble dcTCRs are generated through the use of nucleic acid fragments encoding ⁇ -f and 0-f chimeras, or ⁇ -IgGl and j ⁇ -IgGl chimeras, into which "stop" codons have been inserted near the transmembrane domains of both the ⁇ - and 0-chains preceding the f chain gene, or between the ⁇ - and 3-chains and the IgGl coding regions.
  • the nucleic acid sequences encoding the chimeras are generated as described above; "stop" codons are then inserted in the appropriate place.
  • the coding regions are then inserted into a single combination vector with a different promoter for each of the coding regions, or into separate transfer vectors with -li ⁇ the same promoter for both coding regions, as described above.
  • soluble dcTCRs are generated through the use of nucleic acid fragments encoding the ⁇ and ⁇ segments of the TCR of interest, without chimeric partners.
  • the coding regions are then inserted into a single combination vector with a different promoter for each of the coding regions, or into separate transfer vectors with the same promoter for both coding regions, as described above.
  • the combination vector, or transfer vectors are expressed in an appropriate vector and host system.
  • a host cell is transformed or transfected with the combination vector or the transfer vectors for replication, transcription and translation.
  • the host cell can be prokaryotic.
  • Gram negative bacterial strains such as Escherichia coli
  • gram positive bacterial strains such as Staphylococcus aureus
  • eukaryotic cells of mammalian or insect origin, or yeast such as Saccharomyces cerevisiae or Schizosaccharomyces pombe can be used.
  • the host cell is an insect cell, such as the Sf9 cell line derived from pupal ovarian tissue of Spodoptera frugipoda .
  • the combination transfer vectors can be introduced into host cells by various methods known in the art. For example, transfection of host cells with combination transfer vectors can be carried out by electroporation. Other methods can also be employed for introducing fusion protein vectors into host cells; calcium phosphate, calcium chloride or ruthenium chloride mediated transfection, or other techniques, some involving membrane fusion, can be used.
  • the combination transfer vector is cotransfected with linearized baculovirus DNA into insect cells, such as Spodoptera frugipoda (Sf9) cells (Pharmingen, San Diego, CA) or High 5 Trichoplusia ni (SB1-) cells (Invitrogen, San Diego, CA) .
  • ⁇ -f and 0-f subunits ⁇ -IgGl and /3-IgGl subunits, or ⁇ and ⁇ segments are expressed, ⁇ -f and ⁇ - ⁇ subunits interact to form ⁇ - ⁇ / ⁇ - ⁇ complexes on the cell surface, known as "f chimeric TCR"; ⁇ -IgGl and 3-IgGl subunits interact to form soluble ⁇ -IgGl/ ⁇ -IgGl complexes, known as "IgGl-chimeric TCRs"; and ⁇ and ⁇ segments interact to form soluble ⁇ / ⁇ complexes, known as "dual chain TCRs" (dcTCRs) .
  • dcTCRs dual chain TCRs
  • Soluble dcTCRs can be generated from the chimeric TCRs by removal of the chimeric partners. For example, soluble dcTCRs are released from ⁇ - ⁇ / ⁇ - ⁇ complexes by digestion with an enzyme that cleaves the subunits near the ⁇ region. Dual chain TCRs are released from ⁇ -IgGl/3-IgGl complexes in a similar manner.
  • the heterodimeric TCRs can be purified to homogeneity from host cell lysates by known methods, such as by affinity chromatography and standard biochemical techniques.
  • carboxy-terminal extensions can be attached to the ⁇ chain region. Representative extensions include hexahistidine or streptag tails. Hexahistidine tails can be used to complex with metal, such as through nickel affinity purification. Streptag tails bind to biotin to take advantage of biotin- streptavidin binding.
  • the heterodimeric TCR can be further purified to maximize yield.
  • the heterodimeric TCRs can be assayed immunologically using conformation sensitive immunoassays, either before or after proteolytic digestion to liberate free dcTCRs.
  • the heterodimeric TCR can be tested for the presence of the native conformation utilizing the methods described by Engel et al. (Science 256:1318 (1992)). These workers transfected the rat basophilic leukemia line RBL-2H3 with recombinant genes encoding the TCR extracellular domains linked to the transmembrane segment and cytoplasmic tail of the f chain. The transfected cells expressed heterodimeric TCR on the cell surface.
  • This TCR could appropriately recognize the stimulatory peptide bound to the I-E MHC class II molecule, resulting in MHC-restricted activation of the RBL cells.
  • variable regions of TCR may provide drug targets that could potentially be specific for T cells involved in pathological mechanisms.
  • T cell-mediated pathology in human diseases examples include: pancreatic /3-cell destruction in insulin-dependent diabetes mellitus (IDDM) , demyelination within the central nervous system in multiple sclerosis, pathology in rheumatoid arthritis, and graft rejection following allografting between HLA incompatible individuals.
  • IDDM insulin-dependent diabetes mellitus
  • Production of the variable region of the TCR in soluble form is a prerequisite for determining the structure of the TCR involved in disease, and for constructing receptor-ligand assays for screening for TCR antagonists.
  • the heterodimeric TCR of the invention can be used to derive TCR structures for identification of TCR antagonists or agents that inhibit the interaction between the TCR and MHC/HLA molecules complexed with antigenic peptides.
  • TCR structures can be applied in rational drug design using computational techniques.
  • TCR structural information derived from one heterodimeric TCR can be used to deduce general rules concerning the whole class of TCR proteins or certain subsets thereof, thereby aiding in the identification of inhibitory compounds.
  • Structural information concerning one particular heterodimeric TCR can be used to devise highly specific inhibitors for a particular T cell clone.
  • Structural information from one heterodimeric TCR can be obtained by standard methods, including information obtained from X-ray diffraction, nuclear magnetic resonance (NMR) spectroscopy, or biochemical or biophysical investigation of the interaction of the heterodimeric TCR with ligands such as MHC/HLA molecules complexed with antigenic peptide or superantigen, or TCR-specific antibodies.
  • ligands such as MHC/HLA molecules complexed with antigenic peptide or superantigen, or TCR-specific antibodies.
  • superantigens are proteins that share the ability to bind to human and mouse HLA/MHC Class II proteins to form a ligand complex for the V ⁇ segment of the TCR.
  • a superantigen-HLA/MHC Class II complex can stimulate many more T cells than a complex of a particular Class II molecule and an antigenic peptide.
  • Superantigens are represented by the Staphylococcal enterotoxins and Streptococcal toxins
  • the heterodimeric TCR of the invention can additionally be utilized in assays to screen for agents that inhibit the interaction of TCR with: 1) complexes formed between MHC/HLA molecules and antigenic peptides or superantigens (referred to herein collectively as antigens) , and 2) TCR specific antibodies, including but not limited to anti-clonotypic antibodies.
  • agents include TCR blockers or antagonists, MHC/HLA blockers or antagonists, and molecular mimics of the TCR ligands.
  • a sample of isolated and purified heterodimeric TCR is incubated with the MHC/HLA molecules and antigenic peptides or superantigens of interest, under conditions that allow the heterodimeric TCR to interact with the MHC/HLA molecules and antigenic peptides/superantigens.
  • This sample is the control sample.
  • a second sample identical to the control sample except that it is exposed to the agent to be tested, is also incubated under the same conditions.
  • Both the control sample and the test sample are then evaluated to determine the level of interaction of TCR with the complexes formed between the MHC/HLA molecules and antigenic peptides or superantigens of interest. If less interaction occurs in the presence of the agent to be tested (in the test sample) than in the absence of the agent to be tested (in the control sample) , then the agent is an inhibitor of the interaction between TCR and the complexes formed between the MHC/HLA molecules and antigenic peptides or superantigens of interest.
  • an assay similar to that described above is conducted, using a sample of isolated and purified heterodimeric TCR that is incubated with the TCR specific antibody of interest as the control sample. Less interaction between the heterodimeric TCR and the antibody in the presence of the agent to be tested, than in the absence of the agent to be tested is indicative that the agent is an inhibitor of the interaction between TCR and the TCR specific antibody of interest.
  • the heterodimeric TCR of the invention can also be used to detect the MHC/HLA molecular constituents of TCR ligands using molecular assays.
  • Recombinant, soluble forms of MHC/HLA molecules can be immobilized on a solid support.
  • Synthetic and/or naturally occurring peptides can be incubated with the MHC/HLA molecules to form complexes that can be investigated for their ability to bind heterodimeric TCR added in the solvent phase. Binding of the receptor proteins can be detected utilizing TCR-specific antibodies and standard ELISA, or by surface plasmon resonance using the BIAcoreTM (Pharmacia, Piscataway, NJ) biosensor system (Fagerstam, L. , Tech . Prot .
  • Such assays would be conducted in a similar manner to the assays described above: a sample of isolated and purified heterodimeric TCR of interest (i.e., heterodimeric TCR that has a native-like conformation that is functionally indistinguishable from that present on the pathogenic T cells, generated by the methods described above) and its ligand is incubated under conditions that allow interaction between the heterodimeric TCR and its ligand; a second sample of heterodimeric TCR and ligand is exposed to the agent to be tested and incubated in a similar manner.
  • heterodimeric TCR of interest i.e., heterodimeric TCR that has a native-like conformation that is functionally indistinguishable from that present on the pathogenic T cells, generated by the methods described above
  • a second sample of heterodimeric TCR and ligand is exposed to the agent to be tested and incubated in a similar manner.
  • the level of interaction between the heterodimeric TCR and ligand is then examined; a lower level of interaction in the presence of the agent than in the absence of the agent is indicative of the ability of the agent to block activation of the heterodimeric TCR, and thus to block activation of the pathogenic T cells.
  • Agents that could block activation of pathogenic T cells include antibodies to T cell receptors, such as those described below.
  • the heterodimeric TCR of the invention can also be used to generate antibodies, either monoclonal or polyclonal, using standard techniques.
  • the term "anti ⁇ body”, as used herein, encompasses both polyclonal and monoclonal antibodies, as well as mixtures of more than one antibody reactive with heterodimeric TCR (e.g., a cocktail of different types of monoclonal antibodies reactive with heterodimeric TCR) .
  • the term antibody is further intended to encompass whole antibodies and/or biologically functional fragments thereof, chimeric antibodies comprising portions from more than one species, humanized antibodies and bifunctional antibodies.
  • Biologically functional antibody fragments which can be used are those fragments sufficient for binding of the antibody fragment to heterodimeric TCR. Once the antibodies are raised, they are assessed for the ability to bind to heterodimeric TCR. Conventional methods can be used to perform this assessment.
  • the chimeric antibodies can be derived from two different species (e.g., a constant region from one species and variable or binding regions from another species) .
  • the portions derived from two different species can be joined together chemically by conventional techniques or can be prepared as single contiguous proteins using genetic engineering techniques.
  • DNA encoding the proteins of both the light chain and heavy chain portions of the chimeric antibody can be expressed as contiguous proteins.
  • Monoclonal antibodies (mAb) reactive with heterodimeric TCR can be produced using somatic cell hybridization techniques (Kohler and Milstein, Nature 256 : 495-497 (1975)) or other techniques.
  • a crude or purified heterodimeric TCR protein, or peptide derived from the heterodimeric TCR protein can be used as the immunogen.
  • An animal is immunized with the immunogen to obtain anti-heterodimeric TCR antibody-producing spleen cells.
  • the species of animal immunized will vary depending on the specificity of mAb desired.
  • the antibody producing cell is fused with an immortalizing cell (e.g., a myeloma cell) to create a hybridoma capable of secreting anti-heterodimeric TCR antibodies.
  • the unfused residual antibody-producing cells and immortalizing cells are eliminated.
  • Hybridomas producing desired antibodies are selected using conventional techniques and the selected hybridomas are cloned and cultured.
  • Polyclonal antibodies can be prepared by immunizing an animal in a similar fashion as described above for the production of monoclonal antibodies. The animal is maintained under conditions whereby antibodies reactive with heterodimeric TCR are produced. Blood is collected from the animal upon reaching a desired titer of antibodies. The serum containing the polyclonal antibodies (antisera) is separated from the other blood components. The polyclonal antibody-containing serum can optionally be further separated into fractions of particular types of antibodies (e.g., IgG, IgM) .
  • the antibodies of the invention can be used to detect T cells with pathogenic.properties in mammals, including humans.
  • lymphocytes To detect pathogenic T cells, a sample of lymphocytes is incubated with antibodies to the heterodimeric TCR of interest (the heterodimeric TCR that has a native-like conformation that is functionally indistinguishable from that present on the pathogenic T cells, generated by the methods described above) . Interaction between the lymphocytes and the antibodies is assessed; the presence of interaction between the lymphocytes and the antibodies is indicative of the presence of pathogenic T cells.
  • the lymphocytes can be obtained, using standard techniques, from peripheral blood, bodily fluids (including cerebrospinal fluid and synovial fluid) , and lymph nodes, or spleen or other tissue biopsy specimens. Analysis of the lymphocytes can be performed before or after in vitro culture of the lymphocytes.
  • the antibodies of the invention can also be used to deplete T cells or inhibit T cell activation in vivo in mammals, including humans.
  • Therapeutic regimens can be designed in which antibodies are administered, using standard methods, in order to inhibit antigen recognition, by binding to T cell surface TCR and thereby sterically blocking the interaction between the variable region of the TCR and the specific complex of antigenic peptide and MHC molecule.
  • the complexes formed between the TCR-specific antibodies and the cell surface TCR can deplete T cells by utilizing accessory elements of the immune system that destroy the antibody- bound T cell.
  • T cell depletion can be enhanced by administering TCR-specific antibodies that are covalently conjugated to a cytotoxic or anti-metabolic agent, such as toxins of microbial or synthetic origin, including peptide toxins or polypeptides related to toxins (Frankel, A.E., J. Biol . Response Mod . 4:437-446 (1985)); enzymes; radioactive substances; or cytotoxic drugs (Hawkins, R.E., et al .
  • a cytotoxic or anti-metabolic agent such as toxins of microbial or synthetic origin, including peptide toxins or polypeptides related to toxins (Frankel, A.E., J. Biol . Response Mod . 4:437-446 (1985)); enzymes; radioactive substances; or cytotoxic drugs (Hawkins, R.E., et al .
  • TCR- specific antibodies in vivo as immune response modifiers, immunoregulators or immunosuppressors, the selection of antibodies with defined specificity allows targeting of either the whole T cell population, or a defined T cell sub-population, within an individual animal or human.
  • antibodies specific for a clonotypic epitope would target only the members of a single T cell clonotype, whereas antibodies specific for a V ⁇ family-specific epitope would target all the T cell clones bearing TCR utilizing Vj8-segments belonging to that particular family.
  • Antibodies can be administered directly; alternatively, they can be administered indirectly, such as by maternal transmission (transplacental transmission to offspring of a mammal during gestation, or by transmission during nursing) .
  • the pregnant or nursing female is immunized with the TCR of interest, and maternally generated antibodies are passively transmitted to the offspring before and/or during birth, and/or after birth during nursing.
  • the antibodies to the TCR are administered to a mammal in a therapeutically effective amount, which is the amount of the antibody that is necessary to inhibit the activation of, deplete or eliminate the pathogenic T cells.
  • the heterodimeric TCR of the invention can also be used in vivo in mammals, including humans, to compete with pathogenic T cells for their specific MHC/HLA class II associated peptide antigen. In this manner, the heterodimeric TCR can be used to deplete antigen such that the activation of the pathogenic T cells would be reduced or eliminated in vivo.
  • Pathogenic T cells of interest include those which are involved in pancreatic /3-cell destruction in insulin-dependent diabetes mellitus (IDDM) , demyelination within the central nervous system in multiple sclerosis, pathology in rheumatoid arthritis, and graft rejection following allografting between HLA incompatible individuals.
  • IDDM insulin-dependent diabetes mellitus
  • the heterodimeric TCR are administered to a mammal in a therapeutically effective amount, which is the amount of the heterodimeric TCR that is necessary to inhibit the activation of, deplete or eliminate the targeted T cells.
  • the heterodimeric TCR of the invention can also be used to immunize mammals, including humans, against TCR antigenic structures that occur on the surface of T cells which perform pathogenic or otherwise undesirable functions (the "targeted T cells") , such as graft rejection following transplantation.
  • T cells can be identified in samples of peripheral blood, or in biopsy specimens taken from lymphoid organs or sites of inflammation. Lymphocytes in a sample are purified and investigated in vitro for their ability to make a T cell dependent proliferative response to the relevant MHC/HLA associated antigenic epitope.
  • T cells that undergo cell division can be established in vitro as lines or clones from which TCR genes can be cloned and used to produce heterodimeric TCR by the recombinant DNA technology described herein.
  • TCR antigenic structures include clonotypic epitopes, V ⁇ or V ⁇ family-specific epitopes, conformational epitopes, and linear epitopes. Immunization against TCR antigenic structures that occur on the surface of the targeted T cells inhibits the activity of the targeted T cells, thereby reducing the pathogenic or undesirable effects of the targeted T cells.
  • the heterodimeric TCR are administered to a mammal in an effective amount, which is the amount of the heterodimeric TCR that is necessary to inhibit the activity of the targeted T cells.
  • heterodimeric TCR can be in the form of a single dose, or a series of doses separated by intervals of days or weeks.
  • single dose can be a solitary dose, and can also be a sustained release dose.
  • the heterodimeric TCR can be administered subcutaneously, intravenously, intramuscularly, intraperitoneally, orally, by nasal spray or by inhalation, opthamologically, topically, via a slow- release compound, or via a reservoir in dosage formulations containing conventional, physiologically-acceptable carriers and vehicles.
  • a DNA fragment encoding the heterodimeric TCR can be administered.
  • the formulation in which the heterodimeric TCR is administered will depend in part on the route by which it is administered, and the desired effect.
  • D10 clonotype-specific monoclonal antibody (mAb) 3D3 was provided by A. Bothwell (Yale Medical School, New Haven, CT) .
  • H28-710-16 (H28) and H57-597 (H57) are C ⁇ - and C3-specific monoclonal antibodies, respectively (Kubo, R. T. et al . , J. Immunol . 142:2736-2742 (1989); Becker, M.L. et al . , Cell 58:911-921 (1989)); they were obtained from E. Reinherz (Dana Farber Cancer Institute, Boston, MA) .
  • V ⁇ 2- specific mAb B20.1.1 Piereau, H. et al .
  • anti-V ⁇ ll mAb Jameson, S.C. et al . , J. Immunol . 146:2010 (1991)
  • anti-V33 V03; Sugihara et al. J. Jjiununol. 150:683 (1993)
  • anti-V38.1/8.2 V08; Kanagawa, 0. , J. Exp. Med . 170:1513 (1989)
  • Anti TCR V-region, family specific monoclonal antibodies were purchased from Pharmingen, Inc. , (San Diego, CA) apart from the V ⁇ ⁇ family specific antibody which was purchased from Harlan Bioproducts for Science (Indianapolis, IN) .
  • the plasmid ph3C3f containing a human CD3 f-chain cDNA, was obtained from the laboratory of E. Reinherz (Dana Farber Cancer Institute, Boston, MA) .
  • a BamHI-EcoRI restriction fragment spanning the transme brane and cytoplasmic coding regions of the CD3 f-chain was excised from the plasmid and cloned between the BamHI and EcoRI restriction sites within the polylinker of the vector pAcC5 (Luckow, V.A. and M.D. Summers, Bio /Technology 6:47-55 (1988)).
  • the PCR reactions were done using oligonucleotides that were designed to amplify the extracellular V, J and C regions of the ⁇ chain, and V, D, J and C regions of the ⁇ chain, from the methionine initiation codons of the signal sequences to the codons for the first amino acid after the conserved cysteines involved in the inter-chain sulfhydryl bond of the a/ ⁇ TCR.
  • the primers used to amplify the ⁇ - chain were 5'-
  • PCR was performed using Taq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT) under conditions recommended by the supplier for 30 cycles at 95°C/45", 55°C/45", 72°C/90".
  • TCR genes were determined using Sequenase (U.S.B., Cleveland, OH) according to the manufacturer's recommendations. They were found to encode the wild type amino acid sequences ( Figures 1 and 2) except for a mutation creating a glutamine codon in the /3-chain C-region. The mutation was discovered in the cloned cDNA, and was not due to infidelity of the Taq polymerase. A fragment with the correct codons for the erroneous region was substituted to revert the mutation so as to encode the wild-type arginine. The sequences of the cloned genes were verified by sequence analysis. The recombinant plasmids were named pl/226 ( ⁇ ) and p6/227 ( ⁇ ) .
  • the D10 ⁇ gene excised from pl/226 as a Ncol-Bglll fragment
  • the D10 jS-gene excised from p6/227 as a
  • pll/231 encodes the ⁇ -f chimera under control of the polyhedron promoter, and the ⁇ - ⁇ chimera under control of the P10 promoter.
  • Sf9 cells a subclone of the IPLB-Sf21-AE line, or High 5 cells, were propagated at 27°C, either in suspension or as monolayer cultures in either TMN-FH media (JRH Biosciences, Lawrence, KS) supplemented with 10% heat- inactivated FCS, 10 units/ml penicillin, 100 ⁇ g/ml streptomycin, and 0.25 ⁇ g/ml amphotericin B (Sigma, St. Louis, MO) or in serum-free media, SF900II (Gibco, Life Technologies, Bethesda, MD) or Excell 400 (JRH Biosciences, Lawrence, KS) , supplemented with antibiotics.
  • TMN-FH media JRH Biosciences, Lawrence, KS
  • FCS 10 units/ml penicillin
  • streptomycin 100 ⁇ g/ml streptomycin
  • 0.25 ⁇ g/ml amphotericin B Sigma, St. Louis, MO
  • Transfer plasmid and linearized BaculoGold viral DNA were co-transfected into Sf9 cells according to the manufacturer's recommendations (Pharmingen, San Diego, CA) . Briefly, 2 x 10° Sf9 cells were seeded onto a 60 mm tissue culture plate and the cells were allowed to attach for l hour at 27 ⁇ C. 0.5 ⁇ g of linearized BaculoGold viral DNA was mixed with 2-3 ⁇ g of transfer plasmid DNA, including the gene of interest, in a sterile microfuge tube. After five minutes at room temperature, l ml of transfection buffer B (25 mM HEPES pH 7.1, 140 mM NaCl, 125 mM CaCl 2 ) was added to the DNA.
  • transfection buffer B 25 mM HEPES pH 7.1, 140 mM NaCl, 125 mM CaCl 2
  • transfection buffer A (Grace's medium containing 10% FCS) was added.
  • transfection buffer B/DNA solution was added drop-wise to the plate while it was gently rocked.
  • FCS fetal calf serum
  • the virus stock was diluted in TMN-FH medium with 10% FCS in dilutions of 10"*, 10' 5 , IO" 6 , and 10 "7 .
  • the culture medium was aspirated from the cells and 0.5 ml of each virus dilution was added to triplicate sets of plates. The infection was rocked gently at room temperature for 1 hour to allow the virus particles to attach to the cells. The supernatant was then aspirated from the cells and 4 ml of 0.5% agarose, SEAKEM ME grade (FMC Bio-Products, Rockland, ME) in TMN-FH medium with 10% FCS was added as an overlay to each plate.
  • SEAKEM ME grade FMC Bio-Products, Rockland, ME
  • the agarose was allowed to harden and the plates were then incubated at 27°C for 4 days, after which the plates were stained with 10P ⁇ g/ml neutral red (Sigma, St. Louis, MO) in 0.5% agarose in PBS. The plates were incubated overnight at 27°C and the plaques, which appeared as clear circular areas against a dark red background, were then counted to determine the titer of the virus preparation (pfu/ml) . Neutral red stains live cells.
  • Sf9 cells were seeded at 3 x 10 s cells/ml in 800 ml of TMN-FH media with 10% fetal calf serum and grown in suspension in a 1 liter spinner flask. In an initial experiment the cells were counted at various times to determine when the cells were in log phase. In the virus amplification step, the cells were infected when in early to mid log phase (8 x 10 5 cells/ml) with the recombinant virus of interest at a multiplicity of infection (MOI) of 0.1 plaque forming virus particles per cell. The infection was allowed to incubate at 27°C for 5 days post- infection (pi) .
  • MOI multiplicity of infection
  • the resulting high titer virus supernatan was then harvested and cleared of cells and debris by centrifugation for 20 minutes at 1500 rpm and filtered through 0.22 micron filter unit. The supernatant was stored at 4°C and titered as described above to determine the number of pfus/ml. Virus was amplified no more than two times before use.
  • 6- to 8-L cultures of Sf9 or High 5 cells in serum-free media were grown (Rice, J.W. et al . , Biotechniques 15:1052-1059 (1993)) in 8-L spinner flasks equipped with overhead drive systems (Bellco Glass, Vineland, NJ) to a density of 0.8 t 1.2 x 10* cells/ml and infected at an MOI of 5.
  • Media were harvested three days post infection by centrifugation for 30 minutes at 4.5K rpm and filtration through a 0.2-micron filter.
  • tissue culture plate was seeded with 5 x 10 6 Sf9 cells in TMN-FH medium with 10% FCS, and infected with recombinant baculovirus at an MOI of 10.
  • Three days post infection, cells or media were harvested and assayed by FACS (Becton Dickinson, San Jose, CA) or ELISA (see below, Example 4) after treatment with thrombin.
  • the resuspended cells were incubated at room temperature for one hour in the presence of from 0 - 20 units of thrombin (Calbiochem, San Diego, CA) .
  • 500 units of thrombin were resuspended in 300 ⁇ l 50 mM Tris-HCl (pH 8.0), 2 mM CaCl 2 .
  • the cells were spun down at 1000 rpm for 5 minutes, rinsed in PBS with 2% Fetal calf serum (FCS) and stained with
  • FITC/mAbs recognizing the cell surface TCR either mAb 3D3, or V ⁇ 2- or V38-specific mAbs.
  • V ⁇ 2- and V ⁇ -specific mAbs react with the V-regions of the D10 dcTCR.
  • V ⁇ ll and V03 family- specific mAbs were used as controls.
  • Bright staining of Sf9 insect cells infected with recombinant virus vll/231 (derived from transfer plasmid pll/231 encoding both the ⁇ - f and ⁇ - ⁇ chimeras) was observed with mAb 3D3, the D10 clonotype-specific mAb.
  • stop codons were introduced into pll/231 (described above) at the ends of both TCR ⁇ - and ⁇ - genes immediately following the codons encoding the first amino acid after the conserved inter-chain disulfide- forming cysteine residues and before the beginning of the f-chain genes.
  • GGCCGAGGCCTGGGGCCGAGCAGACTGTGGGTGATAACCATGGTAC-3' SEQ ID NO. 11
  • 5'-CATGGTTATCACCCACAGTCTGCTCGGCCCCAGGCCTC'-3' SEQ ID NO. 12
  • the transfer plasmid pl/258 ( Figure 21) , encoding only the D10 ⁇ -chain, was constructed by cloning into the BamHI site of pVL941 (Pharmingen, San Diego, CA) , two ⁇ -chain- encoding fragments, a Bglll-Hindlll fragment excised from 1/226, and a Hindlll-Bglll fragment from pBDC 6.9 ⁇ (f4) .
  • the transfer plasmid p7/258 ( Figure 22) , encoding only the D10 ⁇ -chain, was similarly constructed by using two ⁇ - chain-encoding fragments, a BglII-Bpull02I fragment from p9/237 and a Bpull02I-BglII fragment from pCR-BDC 6.9 j8(f4).
  • pCR-BDC 6.9 ⁇ (f4) and pCR-BDC 6.9 0(f4) contain BDC 6.9 TCR ⁇ - and /3-chain genes truncated prior to the transmembrane regions at the same locations described here for the DIO dcTCR. Bglll sites occur immediately 3' of the stop codons.
  • plO/248 ( Figure 5) , encoding a secretable form of the BIO TCR
  • DIO V ⁇ - and V/3-encoding sequences within the plasmid p9/237 were sequentially replaced with appropriate restriction fragments encoding the V ⁇ - and V3-regions of the BIO TCR.
  • a plasmid pl/244 was constructed by replacing the DIO V ⁇ -encoding sequence within the plasmid p3/236, between the BamHI site immediately downstream of the polyhedrin promoter and the Ncol site within the constant region, with a BIO V ⁇ - encoding BamHI-Ncol restriction fragment from the plasmid pB10 ⁇ 2 (Fink, P.J. et al .
  • plO/248 was constructed by cloning both a BIO V3-encoding Xbal-EagI fragment from p2/246 (described below; see Figure 6) and a Eagl-Bglll fragment from p9/237 containing the stop codons after the 3-gene between the Xbal and Bglll sites of pl/244.
  • plO/248 contains the BIO ⁇ -gene downstream of the polyhedrin promoter and the BIO 3-gene downstream of the P10 promoter, both followed by stop codons.
  • RNA was prepared from approximately 1 x IO 7 3D3 cells using RNazol B (Tel- Test, Friendswood, TX) according to the manufacturer's recommendations. One ⁇ g of RNA was incubated with
  • GAAGATCTCATTTACCAGGAGAGTGGGAGAGGCTCTTCTC-3' (SEQ ID NO. 14) to yield a single product of the expected size.
  • Triplicate reactions were pooled, purified, and cloned into the vector pCRII (Invitrogen, San Diego, CA) .
  • the IgGl-encoding sequence contained in this plasmid was determined to be identical to the published sequence (French, D.L. et al . , J . Immunol . 146:2010 (1991)).
  • BIO TCR as IgGl chimera
  • a plasmid, p7/245, containing the BIO TCR ⁇ -IgGl chimeric gene downstream of the polyhedrin promoter was constructed.
  • pl/242 was an intermediate in the construction of p7/243 and contains the DIO TCR ⁇ -IgGl gene downstream of the polyhedrin promoter.
  • the BIO TCR ⁇ gene was first removed from the plasmid pBlOjSl (Fink, P.J. et al . , Nature 321:219-223 (1986) , provided by S. Hedrick, University of California, San Diego, CA) , and cloned into the vector pFRSV-SR ⁇ DlOp 1 GPI-TCR (described above) so as to replace the DIO V3- encoding region with the V3-encoding region of the BIO TCR.
  • BIO TCR 3-IgGl gene was created and cloned into the plasmid p7/245 to generate p2/246. This was accomplished by cloning two fragments, a BamHI-Ncol fragment from pl/245, and a Ncol- Bglll fragment from p7/243, into the Bglll site of p7/245 immediately downstream of the P10 promoter.
  • RNA was prepared from approximately 5 million BDC 2.5 cells (K. Haskins, Barbara Davis Center for Childhood Diabetes, Denver, CO) by an RNazol B (Tel-Test, Friendswood, TX) procedure (Chomczynski et al . , Anal . Biochem . 262:156 (1987)).
  • RNA was reverse transcribed with 20 ng of oligo (dT) at 42°C in a 20 ⁇ L volume using Superscript reverse transcriptase (BRL, Bethesda, MD) according to the manufacturers recommendations.
  • the TCR ⁇ and ⁇ genes were amplified from 1 ⁇ L of this cDNA preparation under the following conditions: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.001% gelatin, 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT) , and 270 nM each of either the V34 and 3' D10 ⁇ - oligonucleotide (5'-CCTCTAGAAGATCTCCATGGGCTCCATTTTCCTCAGTT- 3'; SEQ ID NO.
  • Triplicate PCRs were pooled and purified using the PCR Purification Spin Kit (Qiagen Inc., Studio City, CA) and cloned into the vector pCRII using the TA Cloning Kit (Invitrogen, San Diego, CA) . The resulting white colonies were used to prepare plasmid DNA for sequencing and plasmid construction. Restriction digestion indicated that the plasmids p ⁇ lO/597 and p/34/597 contained full-length PCR products of the TCR a and ⁇ genes respectively. These cloned PCR products were completely sequenced using SP6, T7, and the primers Mus C ⁇ seq (5'- TCTCAGCTGGTACACG-3' ; SEQ ID NO.
  • p3/598 was constructed by ligating into the BamHI site of pVL941 (Pharmingen, San Diego, CA) both a Bglll to Kpnl BDC 2.5 TCR ⁇ containing fragment from pj84/597 and a Kpnl to Bglll IgGl containing fragment from pIGGl (la)/584.
  • p7/599 was constructed by ligating into the BamHI site of pVL941 (Pharmingen, San Diego, CA) both a Bglll to Kpnl BDC 2.5
  • TCR ⁇ containing fragment from p ⁇ lO/597 and a Kpnl to Bglll IgGl containing fragment from pIGGl(la) /584.
  • p5/598 and p7/598 were constructed, both of which contain the BDC 2.5 TCR ⁇ -IgGl chimera downstream of either the polyhedrin or P10 promoters respectively.
  • p5/598 and p7/598 were constructed by ligating into either the BamHI or Bglll sites of pAcUW51 (Pharmingen, San Diego, CA) both a Bglll to Kpnl BDC 2.5 TCR ⁇ containing fragment from p/34/597 and a Kpnl to Bglll IgGl containing fragment from pIGGl(la)/584.
  • Kpnl to PvuII and PvuII to Bglll BDC 2.5 TCR ⁇ fragments from p ⁇ lO/597 and a Bglll to Hindlll BDC 2.5 TCR 0-IgGl-containing fragment from p5/598 were ligated into p7/598 between the Kpnl and Hindlll sites to construct pl6/599.
  • p20/599 was constructed by ligating between the EcoRI and Kpnl sites of p5/598 an EcoRI to BamHI BDC 2.5 TCR /3-IgGl containing fragment from p7/598 and Bglll to PvuII and PvuII to Kpnl BDC 2.5 TCR ⁇ fragments from p ⁇ lO/597.
  • P3/598, p7/599, pl6/599 and p20/599 have been cotransfected into Sf9 cells along with linearized BaculoGold DNA (Pharmingen, San Diego, CA) following the manufacturers protocol, as described above.
  • the infected cell supernatant contains a protein, as expected, that reacts with a V34 specific mAb as a detection antibody in a sandwich ELISA that uses the mAb H57 (directed against C ⁇ ) as a capture antibody in a protocol that is essentially identical to that described previously to assay soluble DIO dcTCR.
  • Recombinant baculoviruses were constructed for expression of soluble BDC 6.9 TCR.
  • BDC 6.9 TCR As in the case of the BDC 2.5 TCR, described above, two viruses were designed to secrete the BDC 6.9 TCR as an IgGl chimera.
  • the genes for the a and ⁇ chains of the BDC 6.9 TCR were amplified from RNA by PCR using conditions identical to those previously described above, except that RNA was prepared from BDC 6.9 T-cells (K. Haskins, Barbara Davis Center for Childhood Diabetes, Denver, CO) and different primer pairs were used in the PCRs.
  • the 5'-primers used to amplify the ⁇ and ⁇ Chains were 5'-CCTCTAGAAGATCTCCATGGGCTCCATTTTCCTCAGTT-3' (V/34; SEQ ID NO. 21) and 5'-
  • CCTCTAGAAGATCTTCATGAAAACATACGCTCCTACATTA-3' (MusV ⁇ 13.1; SEQ ID NO. 22) , respectively, both of which are complementary to the 5'-end of the signal peptide sequence.
  • Primers were designed based on published sequences. As the published sequence of the murine V ⁇ l3.1 gene segment is incomplete (Yague et al., Nuc. Acids Res . 16:11355-11364 (1988)) the 5'-terminal coding sequence needed to be determined.
  • PCR products were cloned into a plasmid vector, pCRII, as previously described above to yield four plasmids, pCR-BDC 6.9 ⁇ (Z), pCR-BDC 6.9 ⁇ (f4), pCR-BDC 6.9 /3(Z), and pCR-BDC 6.9 ⁇ * (f4).
  • the TCR genes within these plasmids were sequenced as previously described above.
  • pCR-BDC 6.9 ⁇ (Z) was found to contain two mutations, one of which introduced a stop codon within the coding sequence and another which altered a conserved cysteine codon to an arginine codon.
  • pCR-BDC 6.9 ⁇ (f4) was found to contain one mutation in which the second stop codon had been altered to an arginine codon.
  • TCR nonmutated BstXI to Aatll
  • pCR-BDC 6.9 ⁇ (Z) was found to have a one-base pair deletion near the 5'-end of the TCR gene, which did not need to be corrected.
  • pCR-BDC 6.9 ⁇ (f ⁇ ) was found to contain no mutations.
  • pAcUW51-based plasmids containing genes encoding both the ⁇ - and 0-chains of the BDC 6.9 TCR as fusions to the IgGl CH2 and CH3 domains
  • two intermediate plasmids were first constructed.
  • p51/604 was built by ligating both the Kpnl to Agel (IgGl-vector) fragment from pl6/599 ( Figure 10) and the Sail to Kpnl (BDC 6.9 8) fragment from pCR-BDC 6.9 0(Z) between the Sail and Agel sites of pl6/599.
  • p32/604 was built by ligating both the Sail to Agel (BDC 2.5 3-vector) fragment from p7/598 and the Sail to Kpnl (BDC 6.9 /3) fragment from pCR-BDC 6.9 ⁇ ( Z) between the Kpnl and Agel sites of P20/599.
  • pll/606 Figure 13
  • pll/606 Figure 13
  • p33/606 ( Figure 14), containing the BDC 6.9 TCR ⁇ -IgGl gene downstream of the polyhedrin promoter and the BDC 6.9 TCR 3-IgGl gene downstream of the P10 promoter, was constructed by ligating both the Kpnl to Hindlll (IgGl) fragment from p20/599 and the Bglll to Kpnl (BDC 6.9 ⁇ ) fragment from p9/603 between the BamHI and Hindlll sites of p32/604.
  • Hexahistidine (HH) and StrepTag (ST) affinity tails were added to the 3'-end of the BDC 2.5 TCR ⁇ -IgGl genes contained in both plasmids pl6/599 and p20/599.
  • HH Hexahistidine
  • ST StrepTag
  • two complementary synthetic oligonucleotides 5'- CTGGTAAACATCACCATCACCATCACTCACCCGGGAAGTAATGACTCGAG-3' (IGG1HHA; SEQ ID NO. 23) and 5'-
  • GATCCTCGAGTCATTACTTCCCGGGTGAGTGATGGTGATGGTGATGTTTACCAGGAGA- 3' (IGG1HHB; SEQ ID NO. 24) were annealed in a 50 ⁇ L volume containing approximately 5 ⁇ g of each by heating to 90 C C for 15' and allowing the mixture to cool to room temperature.
  • a streptag coding sequence with BstXI and BamHI complementary ends, was generated using oligonucleotides 5'- CTGGTAAAGCATGGCGACATCCGCAATTCGGGGGGTAATGACTCGAG-3' (IGG1STA; SEQ ID NO. 25) and 5'-
  • pll/607 containing the BDC 2.5 TCR ,3-IgGl gene downstream of the polyhedrin promoter and the BDC 2.5 TCR ⁇ -IgGlr ⁇ gene downstream of the P10 promoter was constructed by ligating both the Sail to BstXI (BDC 2.5 TCR ⁇ -IgGl) fragment from pl6/599 and the BstXI to BamHI hexahistidine-encoding double stranded oligonucleotide between the Sail and Bglll sites of pl ⁇ /599.
  • p21/607 ( Figure 16) was created by using the Streptag-encoding double- stranded oligonucleotide.
  • p41/607 ( Figure 17) , containing the BDC 2.5 TCR 3-IgGl gene downstream of the P10 promoter and the BDC 2.5 TCR ⁇ -IgGl HH gene downstream of the polyhedrin promoter was constructed by ligating both the Sall to BstXI (BDC 2.5 TCR ⁇ -IgGl) fragment from p20/599 and the BstXI to BamHI hexahistidine-encoding double stranded oligonucleotide between the Sail and BamHI sites of p7/598.
  • p5l/607 ( Figure 18) was created by using the Streptag-encoding double-stranded oligonucleotide.
  • p31/608 ( Figure 20) , containing the BIO TCR ⁇ -IgGl s ⁇ gene behind the polyhedrin promoter and the BIO TCR j8-IgGl gene behind the P10 promoter, was constructed by using the Kpnl to Hindlll (IgGlyr) fragment from p51/607.
  • Two pVL941 based plasmids, pl4/259 and p9/259 ( Figures 23 and 24) , containing either the hexahistidine or streptag affinity tails respectively, behind the D10 dcTCR ⁇ -gene were also constructed.
  • oligonucleotide encoding the streptag sequence and two stop codons, was created by annealing oligonucleotides 5'- CCCCTGTGCATGGCGACATCCGCAATTCGGGGGGTAATGAGGTACCTCGAG-3' (STIIIA; SEQ ID NO. 29) and 5'-
  • pl4/259 was constructed by ligating into the dephosphorylated BamHI site of pVL94l both the Bglll to Aatll (DIO dcTCR ⁇ ) fragment from p4/224 and the Aatll to Bglll hexahistidine-encoding double- stranded oligonucleotide just described. In an otherwise identical manner, p9/259 was constructed using the streptag-encoding oligonucleotide.
  • a thrombin cleavage site was engineered between each TCR chain and the IgGl domains in the DlO-lgGl TCR chimera. To determine the accessibility of these sites, 5 ⁇ g of purified DIO TCR-IgGl chimera was digested with 2 ⁇ g of thrombin overnight at 37 ⁇ C. Following digestion, the material was centrifuged at 4000 rpm for 10 minutes and the supernatant was subjected to SDS-PAGE under non-reducing conditions. An undigested control sample was prepared under the same conditions.
  • An immunoaffinity matrix was prepared by covalently coupling 16 mg of 3D3 mAb to immobilized protein A beads (Repligen, Cambridge, MA) using dimethylpimelimidate as described (Harlow, E. and D. Lane, Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988)).
  • Supernatants from baculovirus-infected insect cell cultures were concentrated approximately five-fold using a Pellicon concentrator with a 10-kDa membrane filter (Millipore, Bedford, MA), and filtered through a 0.2 ⁇ filter prior to passage over a 3D3 immunoaffinity column (XK16, Pharmacia, Piscataway, NJ) .
  • samples were applied overnight at 4°C with a flow rate of 1-2 ml/min.
  • PBS phosphate buffered saline
  • bound material was eluted with 50 mM citrate (pH 3.0).
  • Eluted fractions were neutralized with 1 M Tris-HCl (pH 8.8) and dialyzed against PBS.
  • Protein concentrations were determined by the method of Bradford, using BSA as a standard (Bradford, M.M. , Anal. Biochem. 72:248-254 (1976)). Material purified by immunoaffinity chromatography was analyzed by SDS-PAGE under non-reducing and reducing conditions.
  • the protein migrated as a monomer with an Mr of approximately 30 kDa (data not shown) , confirming that the di er is linked by an intermolecular disulfide bond.
  • small amounts of covalently aggregated species, and a species with an Mr of 30 kDa can be observed (data not shown) .
  • Their presence varied from preparation to preparation, and never exceeded 1P% of the total yield of immunoaffinity purified protein.
  • the aggregated material does not appear to be a contaminant derived from the insect cell, nor does it appear to contain homodimers of ⁇ - or J-chains.
  • the 3 kDa Mr species could be derived from noncovalently linked heterodimers that dissociated in the presence of SDS.
  • D10 ⁇ and D10 ⁇ chains were purified from recombinant baculovirus-infected insect cell supernatants using H28 an H57 mAb immunoaffinity resins, respectively.
  • SDS-PAGE analysis indicated the presence of monomeric and disulfide linked polymeric species in both preparations (data not shown) .
  • the presence of unpaired cysteines in each of the chains cause the disulfide-linked aggregation.
  • the ⁇ -chain appeared to be significantly more aggregated than the 3-chain.
  • Two-dimensional SDS-PAGE was performed on purified DIO TCR-IgGl, DIO dcTCR, and the individual ⁇ - and ⁇ - chains of the DIO dcTCR, under both equilibrium and non-equilibrium IEF conditions.
  • DIO TCR-IgGl For purified DIO TCR-IgGl, analysis was carried out using equilibrium pH gradient electrophoresis in the first, and SDS-PAGE in the second dimension, as recommended by the manufacturer (BioRad, Hercules, CA) . Two distinct, closely spaced spots, each migrating around 50 kDa in a neutral pH range, were observed (data not shown) .
  • the theoretical molecular masses and pi of DIO TCR ⁇ -IgGl are 47.985 kDa and 6.0, and those of DIO TCR ⁇ -IqGl are 51.672 kDa and 7.4, respectively. Although the two chains were not fully resolved, the results are consistent with the SDS-PAGE and IEF analyses, and further confirm the presence of a- and ⁇ - chains in the DIO TCR-IgGl chimera.
  • D10 dcTCR Two sets of IEF conditions were used to separate the ⁇ - and ⁇ - chains of purified D10 dcTCR, because of difficulties in forming a linear 3-10 pH gradient on a single gel.
  • D10 dcTCR migrated as a single heterogeneous species with a molecular mass of about 30 kDa and an acidic pi (data not shown) .
  • the 3-chain which has a pi in the range of 8.6-8.8, could not be focused.
  • D10 dcTCR migrated as a single heterogeneous species with a similar molecular mass of about 30 kDa and a basic pi (data not shown) .
  • this species corresponds to the /3-chain.
  • the ⁇ -chain which has a pi in the range of 4.8-5.0, failed to focus.
  • the two-dimensional standards provided by the manufacturer (BioRad, Hercules, CA) also behaved similarly in both formats (data not shown) .
  • V ⁇ 2-specific mAb to purified DIO dcTCR and DIO TCR-IgGl was investigated. Protein samples subjected to SDS-PAGE under nonreducing or reducing conditions were electroblotted onto PVDF membranes using a semi-dry blotting system (Hoefer, San Francisco, CA) .
  • V ⁇ 2 mAb can recognize the ⁇ chain of each construct after exposure to SDS and subsequent transfer to PVDF membrane (data not shown) , but not after reduction of disulfide bonds under otherwise similar conditions (data not shown) .
  • the integrity of the intramolecular disulfide bond in the vicinity of the variable region of the ⁇ -chain is critical for formation of the epitope recognized by the V ⁇ 2-specific mAb.
  • the epitope does, however, appear to be resistant to SDS, at least under these assay conditions.
  • DIO dcTCR and DIO TCR- IgGl preparations were immunoprecipitated with H28 and H57 mAb coupled to immobilized protein A beads.
  • the purified proteins contained both ⁇ - and jS-chains; moreover, both chains appeared to be present in equivalent amounts. These results were confirmed by nonreducing SDS- PAGE on immunoprecipitated samples (data not shown) .
  • the size range is consistent with the calculated values of molecular mass for the truncated D10 ⁇ - and 3-chains (22.758 and 26.574 kDa, respectively) . The higher Mr and the heterogeneity seem to be due to N-linked glycosylation.
  • D_ Conformationallv-Sensitive Slot Blot Assay _ P1Q qcTCR The presence of native-like material in the purified preparations of DIO dcTCR was confirmed by performing conformationally-sensitive ECL slot-blot analysis utilizing V ⁇ 2- and V08- specific mAbs.
  • Purified DIO dcTCR and DIO TCR-IgGl were serially diluted (1 ⁇ g-8 ng) in PBS (pH 7.4) and applied to an Immunodyne activated membrane (Pall
  • Bio TCR-IoGl Chimera RR8/H57/8G2 affinity purified B1P TCR-IgGl chimera was subjected to a conformationally-sensitive ECL assay as described previously. The results indicated that the eluted material shows strong reactivity to all three monoclonal antibodies 8G2, H57, and H28, further indicating the presence of correctly folded heterodimeric BIO TCR-IgGl in the IA eluted fractions (data not shown) .
  • the theoretical pi for the heterodimer is 6.45. These values appear to be in close agreement with those observed, and further confirm that 3D3 immunoaffinity purified preparations of D10 dcTCR are heterodimeric. Purified D10 TCR-IgGl chimera under nondenaturing conditions were observed to have a pi of 6- 6.5 (data not shown). This is in close agreement to the value of 6.5 predicted from the primary sequence of the D10 TCR-IgGl chimera. Importantly, species with the pi values predicted for ⁇ or ⁇ homodimers were not observed. These results demonstrate the absence of homodimeric material in the 3D3 IA purified preparations of DIP dcTCR and D10 TCR- IgGl.
  • the apparent native molecular weights of purified D10 dcTCR and D10 TCR-IgGl were determined by chromatography on a Superdex 200 PG 10/30 size exclusion column (Pharmacia, Piscataway, NJ) equilibrated with PBS (pH 7.4) and calibrated with the following molecular mass standards: thyroglobulin (666 kDa), 7-globulin (158 kDa), ovalbumin (43 kDa), myoglobin (17 kDa), and vitamin B12 (1 kDa).
  • a 200 ⁇ g aliquot of purified protein was injected and eluted at a flow rate of 0.5 ml/min, and 0.5-ml fractions were collected.
  • the native molecular mass of purified soluble DIO dcTCR was determined to be approximately 58 kDa (data not shown) . This value is higher than that predicted from the primary sequence of DIO dcTCR, and this appears to be due to glycosylation of the protein in insect cells.
  • the dimeric species Mr - 120 kDa
  • N-glycanase digestions were performed according to the manufacturer's instructions (Genzyme Corp., Framingham, MA). Briefly, 50 ⁇ g of purified D10 dcTCR was incubated in 40 mM sodium phosphate buffer (pH 8.0) containing 0.5% SDS, 50 mM 2- mercaptoethanol and 1.5% NP40. The sample was boiled for five minutes to fully denature the protein, which was then incubated in the presence of N-glycanase at 37°C, and analyzed by SDS-PAGE.
  • mAb 3D3 for DIO dcTCR and DIO TCR- IgGl chimera was evaluated using the BIAcore" biosensor system (Pharmacia, Piscataway, NJ) .
  • the technique can detect binding of soluble analytes to a ligand immobilized on a dextran-coated chip in real time (Fagerstam, L. , Tech . Prot . Chem . 2:65-71 (1991); Malmqvist, M. , Current Biology 5:282-286 (1993)).
  • 0.6 ⁇ g of pure mAb 3D3 was coupled to dextran surface by standard amine chemistry (Johnsson, B. et al . , Anal .
  • HBS HEPES buffered saline
  • Soluble two-domain CD4 provided by M. van Schravendijk, Procept, Inc., Cambridge, MA
  • BDC 6.9 TCR- and BDC 2.5 TCR-IgGl chimera proteins were used as specificity controls.
  • D10 dcTCR at 0.3 ⁇ M gave a significant binding signal (1384) , indicating the formation of the binary complex (data not shown) . This signal was reproducible (data not shown) .
  • DIO dcTCR did not bind to the dextran matrix alone (data not shown) .
  • Binding of the purified a- and purified /S-chain of DIO to immobilized 3D3 was also monitored. Neither purified DIO dcTCR ⁇ - nor ⁇ - chains at 1.5 ⁇ M and 3 ⁇ M bound to immobilized 3D3. These concentrations were in 5- and 10-fold excess of that of the DIO dcTCR.
  • the clone specific characteristics of the mAb 3D3 were further examined by analyzing the BIO TCR-IgGl, 2-domain CD4, and BDC 6.9 TCR. None of these proteins showed any binding to the immobilized 3D3. These experiments confirm the anticlonotypic properties of mAb 3D3 for DIO dcTCR. The results also show that purified DIO and DIO TCR-IgGl preparations are heterodimeric, since purified ⁇ and ⁇ chains did not interact with the immobilized 3D3.
  • NOD mice were purchased from Taconic Farms (Germantown, NY) and were housed in a conventional animal facility. The cumulative incident of diabetes at 27 weeks was 70-80% for females and 40-50% for males.
  • NOD/Lt- Tg(RIPTag)lLt hereafter referred to as NOD/Lt RIP-Tag
  • transgenic mice described by Hamaguchi et al. (Diabetes 40:842 (1991)) were bred in our colony from breeding pairs obtained from Dr. E. Leiter of The Jackson Laboratory. These mice subsequently became available from the Animal Resources Unit of The Jackson Laboratory.
  • the transgene is a recombinant simian virus 40 (SV40) oncogene in which the rat insulin 5' promoter has been inserted immediately upstream of the SV40 early region (Hanahan, D., Nature 315:115 (1985)); consequently, transgene expression is restricted to the ⁇ cell, resulting in the transformation of this cell type exclusively.
  • SV40 simian virus 40
  • the animals develop ⁇ cell tumors at 10-16 weeks of age which cause hypoglycemia resulting in rapid onset of death.
  • the first litters from these mice were perpetuated by brother-sister mating. Later, in order to avoid the loss of litters due to death of mothers that succumb to tumors before their offspring are weaned, transgenic males were mated to NOD females. After weaning, the young animals were provided with 5%
  • Islet cells were isolated from NOD mice according to methods outlined previously (Haskins, K. , et al . ,Diabetes 37:1444-1448 (1988); Haskins, K. et al . , Proc . Natl . Acad . Sci . USA 86:8000-8004 (1989)) apart from the following modifications: after the islets were hand-picked from the exocrine tissue they were then put through a 70- ⁇ m nylon mesh screen (cell strainer, Falcon, Oxnard, CA) without any further digestion. This cell suspension was then frozen in liquid nitrogen until required. Before use as islet antigen in proliferation assays or for propagation of cell lines, cells were rapidly thawed at 37°C.
  • pancreata from tumor and non tumor bearing mice were irradiated with at least 4000R prior to the collagenase digestion step.
  • Islet tumors were harvested from tumor-bearing NOD/Lt Rip-Tag or (NOD x NOD/Lt RIP-Tag)Fl pancreata by the method of Bergman and Haskins (Diabetes 43:197 (1994)) with the modification that immediately after death the pancreas was infused with 5 a collagenase (Boehringer Mannheim, Indianapolis, Indiana) solution (0.2 mg/ml) via the common bile duct.
  • the pancreas was excised and incubated in vitro for 15 min. at 37°C.
  • the encapsulated tumors were dissected from exocrine tissue and forced through a 70- ⁇ m nylon mesh screen to
  • IP produce a single cell suspension.
  • the cells were examined microscopically to ensure viability prior to freezing at -70°C.
  • Tumor size varies but can yield approximately twenty times the number of cells that can normally be isolated from the pancreas of a NOD mouse.
  • I-A ,7 -restricted, CD4 + , islet antigen specific T- cell clones have been described (Haskins, K. , et al . , Diabetes 37:1444-1448 (1988); Haskins, K. et al . , Proc .
  • Staining buffer for immunofluorescence was prepared by supplementing PBS-D with 0.1% sodium azide and 5% FCS.
  • a total of 0.5 x IO 6 to 1 x 10° cells were resuspended in 10 ⁇ l of the appropriate dilution of FITC-conjugated, biotin conjugated or PE conjugated antibody in staining buffer in 96 well U-bottom microtiter plates, and incubated for 30 min. at 4°C FITC- streptavidin (Pierce, Rockford, IL) was added where needed and plates were incubated for another 30 min.
  • the cells were washed twice and finally resuspended in PBS containing 1% paraformaldehyde as previously described (Jones, B. et al . , J . Immunol . 136:348-356 (1986)). The number of cells analyzed by flow cytometry using a FACScan (Becton
  • Dickinson, CA varied from 5000 to 30000. Cells were gated according to forward and side scatter parameters.
  • the NOD mouse T cells were purified by applying a whole spleen cell population to T cell columns according to the manufacturers' instructions (R & D Systems, Minneapolis, MN) .
  • T cell proliferation assays were performed in Click's medium (EHAA: Irvine Scientific, Santa Ana, CA) supplemented with 5% FCS (Hyclone Labs, Logan, UT) .
  • Cultures were set up in triplicate in 200 ⁇ l volumes in 96- well round-bottomed plates. Irradiated (3000R) syngeneic mouse splenocytes were routinely used as antigen presenting cells (APCs) . T cell proliferation was assessed by pulsing each culture with 1 ⁇ Ci 3 H-thymidine for the final 12-16 hours of incubation. The cells were harvested on a Tomtec harvester (Tomtec, Orange, CT) and radioisotope incorporation measured using a beta-plate scintillation counter (Wallac, Gaithersburg, MD) .
  • Tomtec harvester Tomtec, Orange, CT
  • beta-plate scintillation counter Allac, Gaithersburg, MD
  • NOD mice were primed with the BDC 2.5 TCR-IgGl protein in complete Freund's adjuvant. Boosting injections without adjuvant were given 14-21 days after priming and repeated after a further 7-14 days. Indirect immunofluorescence with sera collected 4 days after the second boosting injections revealed that 18 out of 32 immunized animals made antibodies that bound the surface of the BDC 2.5 T cell clone. Positive antisera were pooled and further analysis revealed that the antibodies were apparently specific for the BDC 2.5 clone. Typical data are shown in Figure 25(A). The antiserum stained BDC 2.5 T cells but not BDC 6.9 or normal splenic NOD T cells, suggesting that the antibodies recognized clonotypic epitopes of the BDC 2.5 TCR.
  • Antibodies recognizing TCR variable region epitopes have been found to inhibit the antigen-specific responses of T cell clones in vitro (White, J. et al . , J . Immunol . 130 (3) :1033-1037 (1983)).
  • the inhibitory activity of the BDC 2.5 specific antiserum was investigated in in vitro cultures of BDC 2.5 or 6.9 cells, stimulated by irradiated NOD mouse spleen cells and pancreatic islet cells as sources of APC and antigen.
  • BDC 2.5 or 6.9 rested T cells were preincubated with or without the indicated dilutions of antisera raised against BDC 2.5 TCR-IgGl or D10 dcTCR proteins, and then tested for their response to NOD/RIP-Tag islet cells and NOD APC in the standard proliferation assay.
  • the anti-DIO dcTCR antiserum contained antibodies specific for the D10 T cell clone, and was used as a control. Unstimulated control cultures received APC without islet cells.
  • the data are shown in Figure 26. Values represent the mean 3 H-thymidine incorporated during a 15 hr. pulse after a 72 hr. culture period.
  • AKR/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) , housed in a conventional animal facility and used between 6 and 8 weeks of age.
  • T cell clones The pancreatic 0-cell specific NOD T cell clones, BDC 2.5 and BDC 6.9 are described above under Example 5A.
  • the I-A k -restricted, CD4+ hen-egg conalbumin peptide specific T cell clone DIO has been described (Kaye, J., et al., J. Exp. M d. 158:836-856 (1983)). It was derived from the lymph nodes of conalbumin immunized AKR mice. Bl was derived by a similar procedure at Procept from the lymph nodes of NOD mice and is an autoreactive (anti-I-A
  • Red cells were removed from the spleen cell population and the T cells isolated by negative selection on mouse T cell enrichment columns (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions.
  • All of the DIO TCR-IgGl immunized mothers made antibodies specific for the cell surface clonotype of the DIO TCR, as indicated by immunofluorescent staining of DIO cells but not of Bl clone T cells which, like the DIO clone, utilize TCR V0-8.1/2 and V ⁇ -2 segments.
  • both BDC 2.5 and BDC 6.9 T cells induced the early onset of diabetes. Disease occurred between 10 to 25 days after the first injection of cells in 30-50% of pups delivered by non-immunized mothers, as opposed to the natural time of onset, which is usually at >3 months of age in unmanipulated NOD mice. Immunizations of mothers that successfully resulted in a BDC 2.5 TCR clonotype-specific antibody response appeared to completely protect their pups from the induction of diabetes by the adoptive transfer of the BDC 2.5 T cell clone.
  • the islet-specific BDC 6.9 T cell clone is clonotypically distinct from BDC 2.5.
  • Injection of the BDC 6.9 clone into pups from mothers producing BDC 2.5 TCR clonotype-specific antibodies resulted in accelerated diabetes with an incidence similar to that in control pups from non- immunized mothers.
  • the protection against adoptively transferred disease afforded by maternal immunization therefore appeared to be immunologically specific.
  • the immunological specificity of the maternally transferred protection was further demonstrated by the failure of immunization against the D10 clonotype to provide protection.
  • the BDC 2.5 TCR clonotype-specificity of the maternally-transferred protection strongly suggests that it was mediated via the transfer of specific antibodies from the mother, either transplacentally or in milk, or by both routes. That is, protection resulted from the transfer of TCR-specific antibodies with the properties described under Example 5B.
  • This interpretation was supported by the demonstration that 3-4 week old pups delivered by mothers successfully immunized with TCR-IgGl protein contained titers of clonotype-specific antibodies of >1/100.
  • the data support the concept that immunization with soluble TCR can induce an immune response of therapeutic value in protection against autoimmune diseases caused by T cell specifically recognizing tissue-specific autoantigens: in this example, a pancreatic islet /3-cell antigen.
  • AKR/J and SJL/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) , housed in a conventional animal facility and used between 6 and 10 weeks of age.
  • the I-A k -restricted, CD4+ hen-egg conalbumin peptide specific T cell clone DIO has been described (Kaye, J. , et al . , J. Exp. Med. 158:836-856 (1983)). It was derived from the lymph nodes of conalbumin immunized AKR mice. Bl was derived by a similar procedure from the lymph nodes of NOD mice and is an autoreactive (anti-I-A* 7 ) CD4+ T cell clone.
  • Monoclonal antibodies (MAb) specific for mouse TCR C ⁇ , V3-8.1/2, and V/3-8.3, were obtained from Pharmingen (San Diego, CA) as fluorescein conjugates.
  • Spleen cell cultures 5 x 10* spleen cells were cultured in the wells of 24- well, flat-bottomed plates (Corning, Corning, NY) in 2 ml cultures in Click's medium (Irvine Scientific, Santa Ana, CA) , supplemented with 5% fetal calf serum (Hyclone Laboratories, Logan, UT) .
  • Staphylococcal enterotoxin (SEB) purchased from Toxin Technologies (Sarasota, FL) was added to the culture medium at a final concentration of 1 ⁇ g/ml.
  • V/3 family-specific antibodies provide one approach to V/3- targeted therapy.
  • the presence of V/3 family-specific antibodies in vivo should deplete the pool of mature T cells bearing members of the Vj8 family recognized by the antibodies, while T cells expressing unrelated V/3 segments should be unaffected.
  • the allele of the SJL mouse inbred strain has deleted V/3 5, V/3 8, V/3 9, V/3 11, V/3 12, and
  • V/3 13 structural genes consequently, mice of this strain are not tolerant to V/3-8 antigenic epitopes.
  • Immunization of C57L/J mice, which have a similar deletion of V/3 genes, with V/8-8-expressing T cells have been shown to stimulate the production of V/3-8 family-specific antibodies (Staerz et al . , J. Immunol . 134:3994-4000). Immunizations of SJL mice with soluble DIO TCR have been performed in order to investigate the immunotherapeutic potential of an antibody response specifically directed towards a particular TCR variable region family.
  • the DIO TCR contains a V3-segment encoded by a member of the V/3-8.2 gene family.
  • the ability of immunizations with the D10 TCR-IgGl protein to stimulate an antibody response specific for TCR V/3-8 segments was tested by immunizing SJL mice.
  • SJL mice were each primed by subcutaneous injection in the hind limbs with 20-25 ⁇ g D10 TCR-IgGl protein emulsified in complete Freund's adjuvant. Intraperitoneal boosting injections without adjuvant were administered 14-21 days after priming, and repeated after a further 7-14 days.
  • AKR mice possess V/3-8 family structural genes, they are tolerant of V/8-8.1/2 family-specific epitopes. Therefore, in this mouse strain, D10 TCR-IgGl immunization does not stimulate the production of antibodies recognizing these antigens. Instead, the antisera collected from AKR mothers were specific for D10 clonotypic epitopes, as indicated by indirect immunofluorescent staining of D10 cells but not of the Bl cloned T cells, which, similarly to the D10 clone, use TCR V/3-8.1/2 and V ⁇ -2 genes. The clonotype-specific antibodies were also transferred to the (AKR x SJL)F1 offspring. The data are presented in Table III.
  • Soluble TCR Mating (female x Maternal Titer of anti-V/3- immunization of male) antibodies 8.1/.2 antibodies mother produced in (SJL x AKR) FI offspring
  • Soluble TCR Mating FI %" of purified 2 ' splenic T cells expressing immunization (female x antiserum V/3-8 segments in FI mice at age: of mother male) specificity
  • Splenic T cells were purified by immuno-affinity chromatography to yield lymphocyte preparations that contained 86-95% ⁇ //3 TCR expressing cells and >3% B cells as determined by direct immunofluorescent staining of the cell surface.
  • the splenic T cells of (SJL x AKR)FI mice derived from mice derived from unimmunized SJL mothers expressed the V/3-8.2 and V/3-8.3 members of the TCR V/3-8 as expected.
  • the FI animals received maternal antibodies which could be shown by indirect immunofluorescence to recognize V/8-8.2 family specific epitopes.
  • Splenic T cells in these FI mice failed to express TCR containing V/3-8.2 segments and the expression of V/3-8.3 segments was greatly reduced.
  • peripheral T cells utilizing V/3-8 family genes appeared to result specifically from immunization of the SJL mothers with DIO TCR-IgGl, because in (SJL x AKR)FI mice born of mothers immunized with BDC 2.5 TCR-IgGl containing a V/3-4 segment, the expression of V/3-8 family genes occurred normally in the splenic T cell population.
  • Staphylococcal enterotoxin B acts as a superantigen and in vitro activates all mature T cells utilizing V/3-segments: 3, 7, 8.1/2/3, and 17 (Marrack, P. and J. Kappler, Science 248 : 705-711) .
  • SEB Staphylococcal enterotoxin B
  • the presence of functional peripheral T cells with V/3-8 TCR was tested by stimulating spleen cells with SEB in vitro .
  • the cell population from each spleen was cultured separately for 4 days with SEB and then recombinant IL-2 was added twice, 2 days apart during a further 5 day incubation period.
  • the total cell population from each culture was analyzed individually by direct immunofluorescent staining of the cell surface with antibodies specific for V/3-8.1/2, V/3-8.3, TCR a/ ⁇ and immunoglobulin. The data are presented in Table V.
  • Soluble TCR Mating FI anti ⁇ %" of SEB-activated spleen cells from 5-7 week immuni ⁇ (female x serum old mice expressing 2 ': zation of male) speci ⁇ mother ficity V/3-8.2 V/3-8.3 TCR ⁇ //3 ig TCR non- SJL none 13.8 ⁇ 0.7 15.3 ⁇ 1.0 99.6 ⁇ 0.2 0.7 ⁇ 0.3 immunized AKR (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10)
  • V/3-8.1/2-bearing T cells were virtually absent from the SEB stimulated populations, and the proportion of V/3-8.3- bearing cells was reduced to less than 50% of that in cultures of spleen cells from (SJL x AKR)F1 mice derived from non-immunized SJL mothers.
  • SJL x AKR spleen cells from (SJL x AKR)F1 mice derived from non-immunized SJL mothers.
  • the reduction in the proportion of V/8-8-bearing T cells stimulated by SEB was immunologically specific.
  • the data of the Table V indicate that DIO TCR-IgGl maternal immunization has a greater inhibitory effect on the population of T cells bearing the V/3-8.1/2 common epitope, recognized by the MAb MR5-2, than V/3-8.3 bearing T cells.
  • One interpretation of the data is that when SJL mice are immunized with DIO TCR-IgGl, the V/3-8.2 epitopes stimulated a stronger antibody response than epitopes that are common to 8.1/2 and 3 V/3-segments.
  • the data indicate that specific maternal immunization against a V/3 family can result in the transfer to the offspring of serum antibodies that deplete the peripheral lymphocyte population of T cells bearing V/3 segments of the family against which immunization is performed.
  • the level of depletion is sufficient to diminish significantly the contribution of these T cells to a superantigen response.
  • similar immunization strategies designed to stimulate an antibody response against the relevant family-specific epitopes could reduce the severity of disease by clonally depleting the pathogenic T cells.
  • ADDRESSEE HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
  • AGA CAA AGT CCC CAA TCT CTG ACA GTC TGG GAA
  • MOLECULE TYPE protein

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Abstract

L'invention concerne des récepteurs protéiques de lymphocytes T hétérodimères, comprenant des sous-unités α et β réunies par au moins une liaison disulfure. Ces sous-unités α et β sont générées en tant que polypeptides chimères utilisant des chaînes z ou des régions constantes d'IgG1 en tant que partenaires chimères. Elle concerne également des molécules de récepteurs de lymphocytes T hétérodimères solubles, dont la conformation ne peut se distinguer de celle qui apparaît sur la surface de lymphocytes T. L'invention concerne encore l'ADN codant les récepteurs de lymphocytes T (TCR) hétérodimères, des vecteurs de transfert comprenant l'ADN codant les TCR hétérodimères et des cellules hôtes contenant ces vecteurs de transfert. Elle concerne, de plus, différentes utilisations des TCR hétérodimères. On peut utiliser ces protéines dans des techniques de dosage moléculaire servant à quantifier leur fixation à des ligands, y compris des complexes antigène peptidique-MHC/HLA ou des anticorps spécifiques de TCR. Ces techniques sont efficaces pour détecter des agents bloquant l'interaction TCR-ligand. On peut également utiliser les TCR hétérodimères afin d'immuniser des animaux, y compris l'homme, dans le but de produire des anticorps spécifiques de TCR. De plus, on peut utiliser ces protéines dans leur conformation native ou dénaturée, afin de vacciner des animaux, y compris l'homme, de manière à supprimer la réaction immune de lymphocytes T portant des TCR, qui partagent des déterminants antigéniques avec la protéine de vaccination.
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Cited By (8)

* Cited by examiner, † Cited by third party
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WO1997008303A1 (fr) * 1995-08-30 1997-03-06 University Technologies International Inc. Animaux transgeniques exprimant des transgenes de recepteurs de cellules t diabetogenes
WO1999060120A2 (fr) * 1998-05-19 1999-11-25 Avidex Limited Recepteur de lymphocyte t soluble
WO2002094860A3 (fr) * 2001-05-18 2003-06-26 Univ Arizona Composition
WO2004048410A2 (fr) * 2002-11-22 2004-06-10 Isis Innovation Limited Recepteurs des lymphocytes t
WO2004074322A1 (fr) * 2003-02-22 2004-09-02 Avidex Ltd Recepteur des lymphocytes t soluble modifie
US7323174B1 (en) 2000-06-12 2008-01-29 Arizona Board Of Regents On Behalf Of The University Of Arizona Modulation of immune response and methods based thereon
US7329731B2 (en) 2001-08-31 2008-02-12 Medigene Limited Soluble T cell receptor
EP2328934A1 (fr) * 2008-08-26 2011-06-08 MacroGenics, Inc. Anticorps des récepteurs des lymphocytes t et leurs méthodes d utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001715A1 (fr) * 1990-07-19 1992-02-06 The Board Of Trustees Of The Leland Stanford Junior University Proteines dimeres solubles de surface cellulaire
WO1993012814A2 (fr) * 1991-12-24 1993-07-08 The Immune Response Corporation Vaccination et procedes contre des maladies resultant de reponses pathogenes de populations de lymphocytes t specifiques
WO1994012648A2 (fr) * 1992-11-25 1994-06-09 Institut National De La Sante Et De La Recherche Production de sous-unites de recepteurs t solubles par co-transfection, utilisations des produits ainsi obtenus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001715A1 (fr) * 1990-07-19 1992-02-06 The Board Of Trustees Of The Leland Stanford Junior University Proteines dimeres solubles de surface cellulaire
WO1993012814A2 (fr) * 1991-12-24 1993-07-08 The Immune Response Corporation Vaccination et procedes contre des maladies resultant de reponses pathogenes de populations de lymphocytes t specifiques
WO1994012648A2 (fr) * 1992-11-25 1994-06-09 Institut National De La Sante Et De La Recherche Production de sous-unites de recepteurs t solubles par co-transfection, utilisations des produits ainsi obtenus

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
IMMUNOLOGIST (1995), 3(2), 59-66 CODEN: INOLEG;ISSN: 1192-5612, March 1995, XP000575900 WULFING, CHRISTOPH ET AL: "Soluble T - cell receptor fragments. Guidance of folding and assembly" *
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 21, 25 July 1993, MD US, pages 15455-15460, XP002008366 F.DAVODEAU ET AL: "Secretion of disulfide-linked human T-cell receptor gamma-delta heterodimers" *
NATURE, vol. 356, 30 April 1992, pages 793-796, XP002008364 S. WEBER ET AL: "Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor" cited in the application *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 (24). 1994. 11408-11412. ISSN: 0027-8424, XP002008362 CHANG H-C ET AL: "A general method for facilitating heterodimeric pairing between two proteins: Application to expression of alpha and beta T - cell receptor extracellular segments." *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 89, August 1992, WASHINGTON US, pages 6871-6875, XP002008365 D. EILAT ET AL: "Secretion of a soluble, chimeric gamma-delta T-cell receptor-immunoglobulin heterodimer" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 91, August 1994, WASHINGTON US, pages 8462-8466, XP002008360 J. KAPPLER ET AL: "Binding of a soluble alpha-beta T-cell receptor to superantigen/major histocompatibility complex ligands" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 91, December 1994, WASHINGTON US, pages 12862-12866, XP002008363 K. MATSUI ET AL: "Kinetics of T-cell receptor binding to peptide/I-Ek complexes" *
SCIENCE, vol. 256, 29 May 1992, pages 1318-1321, XP002008361 I. ENGEL ET AL: "High-efficiency expression and solubilization of functional T cell antigen receptor heterodimers" *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997008303A1 (fr) * 1995-08-30 1997-03-06 University Technologies International Inc. Animaux transgeniques exprimant des transgenes de recepteurs de cellules t diabetogenes
WO1999060120A2 (fr) * 1998-05-19 1999-11-25 Avidex Limited Recepteur de lymphocyte t soluble
WO1999060120A3 (fr) * 1998-05-19 2000-02-03 Avidex Ltd Recepteur de lymphocyte t soluble
US7323174B1 (en) 2000-06-12 2008-01-29 Arizona Board Of Regents On Behalf Of The University Of Arizona Modulation of immune response and methods based thereon
WO2002094860A3 (fr) * 2001-05-18 2003-06-26 Univ Arizona Composition
US7998926B2 (en) 2001-05-18 2011-08-16 The Arizona Boad of Regents on Behfl of the University of Arizona Dimerized T-cell receptor fragment, its compositions and use
US7329731B2 (en) 2001-08-31 2008-02-12 Medigene Limited Soluble T cell receptor
US7763718B2 (en) 2001-08-31 2010-07-27 Immunocore Limited Soluble T cell receptors
WO2004048410A3 (fr) * 2002-11-22 2004-11-11 Isis Innovation Recepteurs des lymphocytes t
WO2004048410A2 (fr) * 2002-11-22 2004-06-10 Isis Innovation Limited Recepteurs des lymphocytes t
US7666604B2 (en) 2003-02-22 2010-02-23 Immunocore Limited Modified soluble T cell receptor
WO2004074322A1 (fr) * 2003-02-22 2004-09-02 Avidex Ltd Recepteur des lymphocytes t soluble modifie
EP2328934A1 (fr) * 2008-08-26 2011-06-08 MacroGenics, Inc. Anticorps des récepteurs des lymphocytes t et leurs méthodes d utilisation
EP2328934A4 (fr) * 2008-08-26 2013-04-03 Macrogenics Inc Anticorps des récepteurs des lymphocytes t et leurs méthodes d utilisation

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