EP1259591A2 - Polypeptides et methodes de vaccination thymique - Google Patents

Polypeptides et methodes de vaccination thymique

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Publication number
EP1259591A2
EP1259591A2 EP00983712A EP00983712A EP1259591A2 EP 1259591 A2 EP1259591 A2 EP 1259591A2 EP 00983712 A EP00983712 A EP 00983712A EP 00983712 A EP00983712 A EP 00983712A EP 1259591 A2 EP1259591 A2 EP 1259591A2
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Prior art keywords
thymocytes
polypeptide
antigens
disease
tcr
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German (de)
English (en)
Inventor
Ellis L. Reinherz
Tetsuro Sasada
Jia-Huai Wang
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • TCR types Recombinatorial diversity resulting from the joining of the various gene segments and association of diverse and ⁇ subunits coupled with junc tional diversity arising from N and P nucleotide additions gives rise to enormous diversity, approximately 10 TCR types. Many of these receptor specificities are useful to the organism in establishing protective cognate immune responses. On the other hand, some of the TCRs so created may be detrimental, including ones with self-reactive specificities able to mediate autoimmune diseases. It is the process of negative selection in the thymus that eliminates, in large part, unwanted autoreactive specificities (Nossal, G.J.V., Cell, 7(5:229-239 (1994)).
  • the present invention is drawn to methods of influencing the selection processes of T cell receptors (TCRs) in order to influence the T cell repertoire of an host.
  • TCRs T cell receptors
  • a polypeptide of interest or peptidomimetic is administered; the polypeptide of interest or peptidomimetic is a polypeptide that causes selection (either positive or negative) of thymocytes having a T cell receptor specificity in the thymus.
  • the methods of influencing the TCR selection processes can be used for thymic vaccination, which causes selection of thymocytes with TCR specificities that are designed to recognize disease antigens or foreign antigens, such as to treat or prevent cancers, autoimmune diseases, infections, or effects of biological warfare agents.
  • Polypeptides, vaccine compositions, expanded thymic cell populations produced according to the methods are described herein.
  • a synthetic thymus comprising stromal elements bearing relevant MHC molecules and loaded with the desired polypeptides suitable for carrying out the invention.
  • the invention is based on the discovery that the T cell repertoire of an individual can be influenced by targeting the selection processes of TCRs.
  • N15 TCR transgenic (tg) RAG-2-/- H-2b mice recognizing the vesicular stomatitis virus (VSV8) octapeptide RGYVYQGL bound to Kb were utilized in conjunction with VSV8 variants differing only at the central p4 position to probe the specificity of TCR selection.
  • V4I mutant octamer like VSV8, induces negative selection of immature double positive (DP) thymocytes on the ⁇ 2 M + /+ background and is a strong agonist for mature N15 T cells.
  • V4L or V4norvaline octamers promote positive selection in N15 tg ⁇ 2 M-/- RAG-2-/- H- 2b FTOC and are weak agonists for N15 T cells.
  • the absence of a p4 side chain ⁇ -methyl group results in positive selection of the N15 TCR.
  • Hydrophobicity of the p4 residues also modulates thymocyte fate: the positively selecting norvaline and leucine variants have one and two C ⁇ -methyl groups, respectively, while the weakly selecting ⁇ -methylleucine p4 contains three C ⁇ -methyl groups.
  • TCR D10 T cell receptor
  • pMHC peptide- MHC
  • selection process refers to positive or negative selection of thymocytes with a targeted TCR specificity in the thymus, to retain a desired specificity (positive) or to eliminate an undesired specificity (negative).
  • Thymic vaccination refers to administration of a polypeptide which influences the selection processes of TCRs while still in the thymus, thereby altering cognate antigens in order to create variants which positively select desired TCR specificities at the level of repertoire development, or which negatively select undesired TCR specificities at the level of repertoire development.
  • the invention provides a method for educating thymic cells to recognize disease or foreign antigens not previously recognized by the immune system (naive preselected double positive (CD4 + CD8 + ) thymic cells).
  • naive thymic cells (functional thymus or synthetic thymus) are contacted with a polypeptide of interest that causes selection of a thymocytes with TCR receptor specificities in the thymus capable of recognizing disease antigens or foreign antigens.
  • the so produced "educated" thymic cells can then be induced to leave the thymus and be expanded into a population of antigen recognizing thymic cells that is sufficient to elicit an immune response against the disease antigens or foreign antigens.
  • the methods described herein are applicable to both Class I and Class II MHC complex formation.
  • the antigens can be components such as bacterial, viruses and macro components of cells and soluble antigens such as proteins, peptides, glycoproteins and carbohydrates.
  • Antigens of particular interest are viral or bacterial antigens, allergens, tumor-associated antigens, oncogene products, parasite antigens, fungal antigens or fragments of these.
  • the peptides and methods described herein can be used to treat cancer tumors and infections in an individual such as, but not limited to, infections caused by bacteria, viruses, fungus and parasites.
  • the polypeptide of interest is a short polypeptide, having between approximately 6 and 16 amino acids, preferably between 8 and 12 amino acids.
  • the polypeptide of interest is a polypeptide which influences the TCR selection process in the thymus to select TCR specificities (either positive or negative).
  • the polypeptide can include natural amino acids, artificially created amino acids, and/or amino acid analogs, and can also be modified, such as by substituted linkages, glycosylations, acetylations, carboxylations, phosphorylations, ubiquitination, labeling (e.g., with radionuclides), enzymatic modifications, or other modifications known in the art, both naturally and non-naturally occurring.
  • a carrier molecule such as another polypeptide or other agent, can be used in conjunction with the polypeptide of interest.
  • a polypeptide of interest or peptidomimetic which influences the selection processes of thymocytes with TCRs of targeted specificities, is administered to the host animal.
  • the polypeptide of interest or peptidomimetic is administered by a means which exposes it to the immune system in the host animal.
  • the polypeptide of interest can be administered in a pharmaceutical composition.
  • a polypeptide or peptidomimetic can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • compositions of this invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, oral and intranasal.
  • Other suitable methods of introduction can also include gene therapy, rechargeable or biodegradable devices, particle acceleration devises ("gene guns") and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
  • compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages.
  • the agents can be separated, mixed together in any combination, present in a single vial or tablet.
  • Agents assembled in a blister pack or other dispensing means is preferred.
  • unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
  • the invention is further illustrated by the following references, which are not intended to be limiting in any way. The teachings of all references cited herein are incorporated by reference in their entirety.
  • mice N15 TCR tg (N15+/+) RAG"/- (H-2b) and N15 TCR tg (N15+/+) RAG-/- ⁇ 2 M-/- mice were generated as previously described (Ghendler et al, Eur. J. Immunol, 27:2279-2289 (1997); Ghendler et al, J. Exp. Med., 757:1529- 1536 (1998)). The lack of RAG-2 or ⁇ 2 M gene expression in knockout animals were identified based on the fluorescence-activated cell sorter (FACS) analysis on peripheral blood cells (Ghendler et al, Proc. Natl. Acad. Sci. USA, 95:10061-10066 (1998)).
  • FACS fluorescence-activated cell sorter
  • TAP-/- mice were purchased from Taconic (NY). All lines were maintained and bred under sterile barrier conditions at the animal facility of Dana-Farber Cancer Institute. Peptides synthesis VSV8 variant peptides were synthesized by standard solid phase methods on an Applied Biosystems 430A synthesizer (Foster City, CA) at the Biopolymers Laboratory of Massachusetts Institute of Technology. Norvaline (nV) and ⁇ - methylleucine (mL) were obtained from Bachem Biosicence Inc.
  • PEC Peritoneal exudate cells
  • TAP-/- mice 129/Ola X C57BL/6 mice (Taconic) mice (Taconic), induced 5 days previously with 2ml of 3% thioglycollate, were suspended in AEVI-V medium (Life Technologies) and plated at 1 x 106 per well in a 96-well microtiter plate. After adherence for 2 hr, monolayers were washed with ATM-V medium four times. Thymocytes (5 x 105) from 4- to 6-week-old N15 tg RAG-/-/ ⁇ 2 M-/- were co-cultured with the PEC for 18 hr at 37°C, and stained for the expression of CD4 and CD8 ⁇ .
  • Norvaline4, L4 and ⁇ -methylleucine4 all lack a C ⁇ methyl group but differ in having 1, 2 and 3 C ⁇ - methyl groups, respectively.
  • Cyclohexylglycine4 is the most hydrophobic of all p4 side chain R groups. Given that the p3, p5 and p8 anchor residues have not been modified in these APL, it is not su ⁇ rising that the Kb binding affinity for these variants relative to VSV8 are unaltered (Ghendler et al, Proc. Natl. Acad. Sci. USA, 95:10061-10066 (1998)). Activation of mature N15 splenic CD8 + T cells by p4 variants
  • norvaline4 and L4 are weak agonists, differing by * > 1000 fold from 14 and VSV8.
  • ⁇ -methylleucine4 and cyclohexylglycine4 are even weaker with detectable stimulating activity present only at a peptide concentration of 10-4 M.
  • N15 tg Rag-2-/- ⁇ 2M-/- thymocytes were cultured in vitro for 18 h with peritoneal exudate cells (PEC) from TAP-/- mice at varying peptide concentrations.
  • PEC peritoneal exudate cells
  • TCR interaction with pMHC ligand is detected as a reduction in the intensity of CD4 and CD8 expression on the surface of the DP thymocytes.
  • Alterations in the expression of CD4 and CD8 ⁇ on DP thymocytes were detected by flow cytometry after gating on 10,000 live cells.
  • the DP dulling assay results detected the interactions of N15tg thymocytes with VSV8 and altered peptide ligands of VSV8.
  • a negative control was used that contained thymocytes plus PEC cultured in the absence of any exogenous peptide additions; for the negative control, in the absence of peptide, 90% of the cells are DP thymocytes whose CD4 and CD8 expression falls in an expected range.
  • 90% of the cells are DP thymocytes whose CD4 and CD8 expression falls in an expected range.
  • the VSV8 exposed cultures only 10-26% of thymocytes fall in this range at peptide concentrations from 10 ⁇ M to 1 nM. The remaining thymocytes have clearly reduced co-receptor expression levels. Even at a VSV8 concentration of 10 pM, there is dulling of a fraction (-5%) of DP thymocytes. An identical result was observed with 14.
  • the % of DP thymocytes is 81. This number is virtually the same for L4, norvaline4, ⁇ -methylleucine4 and cyclohexylglycine4 (64, 72, 75 and 84%, respectively). By contrast, with VSV8 and 14, the % of DP thymocytes was diminished (14 and 18, respectively).
  • Prior studies showed that the majority of DP thymocytes were deleted by a caspase-dependent apoptotic mechanism during this time (Ghendler et al, J. Exp. Med., 757:1529-1536 (1998); Clayton et al, EMBO J., 7(5:2282-2293 (1997)).
  • thymocytes were released from the lobes by passing through a steel mesh and cell numbers were counted by flow cytometry to detect CD4 versus CD8 ⁇ staining profiles of total thymocytes after FTOC.
  • VSV8 in 14 only 1% of CD8 SP thymocytes were generated compared to 9% for the control culture
  • the total number of CD8+ SP thymocytes (mean ⁇ SD of four different lobes) after FTOC are shown for the indicated culture conditions.
  • the numbers of CD8 SP thymocytes were calculated by quantifying the total numbers of thymocytes and percentages of CD8+ SP subsets determined above by FACS. Number of CD8 SP thymocytes positive selected by L4 and norvaline4 are quite comparable and each ⁇ 10 fold more than the "peptide minus" control.
  • the subsequent immune response of the harvested thymocytes to the VSV8 cognate peptide is dramatically increased as judged by cellular proliferation of VSV8 at 1 or 10 nM.
  • the proliferation of thymocytes obtained from the L4 or norvaline4 culture at FTOCs is comparable to that of adult N15 tg Rag-2-/- H-2b thymocyte controls.
  • little proliferation is observed to VSV8 by thymocytes harvested from FTOC lacking exogenous peptide addition.
  • pi and p6 residues of the VSV8 octamer participate in the selection process as well.
  • a pi Arg Lys mutation in VSV8 results in a peptide with neither the ability to negatively or positively select in the N15 TCR tg system (Ghendler et al, Proc. Natl. Acad. Sci. USA, 95:10061-10066 (1998)).
  • the L4, norvaline4 and ⁇ -methylleucine4 peptides are the only ones able to induce positive selection.
  • the adaptive immune response is dependent on the specific recognition function of ⁇ T lymphocytes (1).
  • Each T cell detects a protein fragment (i.e. peptide) of a self-protein or cell-associated pathogen derived from either viral, bacterial, fungal, parasitic or tumor cell origin bound to a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • pMHC major histocompatibility complex
  • T lymphocytes release cytotoxic molecules and/or inflammatory cytokines which destroy the infected or otherwise altered cells through various effector mechanisms.
  • immune recognition is mediated via a clonotypic ⁇ heterodimeric structure (Ti) non-covalently associated with the monomo ⁇ hic CD3 signaling components.
  • each of the ⁇ and ⁇ chains consists of canonical immunoglobulin (Ig)-like variable and constant domains with the hypervariable complementarity-determining regions (CDRs) from the two variable domains (V ⁇ and V ⁇ ) forming the ligand binding site for pMHC within the immunorecognition module.
  • Ig canonical immunoglobulin
  • CDRs hypervariable complementarity-determining regions
  • Class I and class II MHC molecules have evolved to facilitate T cell detection of pathogens residing in distinct intracellular compartments (6-8).
  • TCR-pMHC ⁇ interaction was structurally defined.
  • the first x-ray crystal structures of a TCR- pMHCII ternary complex are described herein.
  • the complex contains the V module of the D10 TCR [single chain (sc) D10] derived from AKR J (H-2k) mouse T cell clone D10.G4 and a fragment of conalbumin (CA) bound to the self-I-Ak molecule (17, 18).
  • sc single chain
  • CA conalbumin
  • Crystal structure of the scDlO-CA I-Ak complex was determined with molecular replacement and alternative cycles of model building and refinement. Crystals of the ternary complex were grown using the conventional hanging droplet vapor diffusion method at room temperature.
  • the scDIO TCR constructed by PCR, consists of 237 residues and was organized from - to C-terminus as follows: V ⁇ 8.2 (residues 3-110)-linker (GSADDAKKDAAKKDG)-V ⁇ 2 (AV225) (residues 1-112) with a Cys235Ser mutation.
  • This linker (SEQ ID NO:2) was modified from that previously utilized for NMR studies (20) since the longer linker failed to give rise to I-Ak co-crystals of diffraction quality.
  • the T7 promoter expression vector pET-1 la was used for bacterial expression.
  • refolding buffer 50 mM Tris-HCl, pH 8.0, 400 mM arginine, 2 M urea, 2 mM EDTA, 4 mM reduced glutathione and 0.4 mM oxidized glutathione.
  • the refolded material was then applied to a 3D3 affinity column followed by gel filtration on Superdex 75 and exchanged to crystallization buffer (20 mM sodium acetate, pH 5.0 with 0.025% sodium azide).
  • the undeglycosylated CA/I-Ak from CHO Lec3.2.8.1 cells were prepared as follows: a 13 residue hen egg CA peptide (residues 134-146) which is recognized by D10 TCR was fused (48) to the N-terminus of the mature I-Ak ⁇ chain via a flexible linker.
  • the 37 residue leucine zipper (LZ) sequences (49) were attached to both the ⁇ and ⁇ chains, with ACID-pl to the ⁇ chain and BASE-pl to the a chain via flexible thrombin-cleavable linkers.
  • the cDNA constructions were subcloned into the pEE14 vector and expressed in Lec3.2.8.1 CHO cells.
  • the screening of secreted recombinant protein in the culture supernatant was performed by both sandwich ELISA and BIAcore using antibodies specific for I-Ak (10.2.16) and the LZ epitope (2H11 or 13A12). The yield was -0.7 mg of I-Ak/1 supernatant.
  • the production supernatant was applied to 2H11 affinity column and I-Ak protein was eluted by 50 mM citrate, 20 mM Tris, 0.5 M NaCl, 10% glycerol, pH 4.0. The eluted protein was then exchanged to 50 mM Tris-HCl, pH 8.0 and cleaved by thrombin (2u/50 ⁇ g I- Ak) at 4°C for 4 h.
  • Thrombin was then removed by passage through benzamidine Sepharose 6B beads and gel filtration on Superdex 75. Subsequently, the purified I- A was exchanged to 10 mM HEPES, pH 7.0, 0.025% sodium azide for crystallization.
  • Crystals were stepwisely transferred to cryoprotectant solution that contains 30% glycerol in addition to the crystallization buffer before freezing.
  • One data set was collected at the SBC-CAT of Advanced Photon Source (APS) at the Argonne National Laboratory with an APS1 mosaic 3x3 CCD detector under 100°K. The wavelength used was 1.069A. Data were processed using programs DENZO and SCALEPACK (54). The structure was solved with molecular replacement using AMoRe (55). The refined structure of CA/I-Ak (56) was taken as the search model. At the beginning, only one of the CA/I-Ak pMHC (molecule A) was identified. The CA/I-Ak molecule B was located only after the first one was rigid body refined and fixed.
  • the rigid body refinement of the two I-Ak molecules was then carried out, each of the Ig-like domains and the bound peptide being treated as one rigid body. A few degrees of rotations were seen for the ⁇ 2 and ⁇ 2 domains. After positional and individual B-factor refinement, the Rfree dropped, and the Ig-like domains of scDIO, especially the one in complex -A, were already visible in the calculated 2F - F c difference map. Cycles of model building and refinement gradually improved the density, allowing the correct sidechain assignment and eventually the completion of the model building and refinement. All the refinement was done using the program X-PLOR (57), and model building with program O (50). Ten percent of reflections were set aside for Rfree calculation.
  • each complex contains residues 1-182 and 2-190 of I-Ak ⁇ and ⁇ chains, respectively, all 16 residues of the bound peptide (3 leader derived and 13 CA-derived), as well as residues 2-117 and 3-116A of D10 V ⁇ and V ⁇ domains, respectively.
  • Ten carbohydrate moieties were modeled in three potential glycosylation sites in I-Ak molecules. At this resolution no water molecules were included.
  • the final 2F Q -F C map is of excellent quality, particularly in the TCR regions and the interface between TCR and pMHC. There are very few density breaks, mainly in the BC loops of the I-Ak ⁇ 2 domains.
  • Rmerge ⁇ ( ⁇ Ii(hkl)- ⁇ H(hkl)> ⁇ )/ ⁇ H(hkl) *in working set.
  • the complex -A and D10-B pack together to form layers pe ⁇ endicular to the longest Y axis, whereas the I-Ak molecules B connect layers in a fashion analogous to pillars between different floors in a building, thus leaving spaces filled with large amounts of solvent.
  • the structures of the two complexes are very similar.
  • the root-mean-square deviation (Rmsd) value of C ⁇ supe ⁇ osition is only 0.8A for the whole complex. Consequently, only complex A is discussed further.
  • An omit map in the bound peptide region showed the core of CA (P- 1 to P8) to be involved in TCR-based immune recognition.
  • a sigma A weighted 2F 0 -F C omit electron density map contoured at l.O ⁇ was generated using the program O (50) and prepared using a cover radius around the atoms. The omit map was generated by omitting the CA peptide entirely and after a round of torsion angle dynamics calculation.
  • the human class I HLA-A2/Tax-specific B7 TCR is by far the most structurally similar to the murine class II- specific scDIO described herein. Virtually the entire V module of these two TCRs can be superimposed.
  • the Rmsd values of the supe ⁇ osition for the entire V ⁇ domain's 110 C ⁇ atoms (excluding the first residue which is not seen in the density map of our scDIO structure) and 107 C ⁇ atoms of the V ⁇ domain (excluding part of the CDR3) are only 0.98 A and 0.12k, respectively.
  • the orientations of two V ⁇ domains differ only by a 3.7° rotation, indicating that V ⁇ -V ⁇ dimerization is very similar for these two TCRs as well.
  • the orientation angle of a TCR on MHC is defined as the angle between two vectors determined for the orientation of the TCR and pMHC, respectively.
  • the vector representing the TCR direction is drawn from the mass center of V ⁇ to the mass center of V ⁇ .
  • the vector representing the pMHC complex direction is drawn from the N-terminal C ⁇ atom to the C-terminal C ⁇ atom of the peptide in the case of MHC class I.
  • MHC class ⁇ the vector is drawn from the PI residue to the P9 residue of the peptide.
  • twist and tilt angles are two projections of the orientation angle more accurately defined here. While the twist is a top view from the TCR towards the MHC, the tilt is a side view, pe ⁇ endicular to the bound peptide.
  • the hydrogen-bonding pattern between the CA peptide and the I-Ak molecule which is conserved in other pMHC class II structures was analyzed. Ten hydrogen bonds between the CA and I-Ak are conserved in known pMHCII structures.
  • the P-3 to P-l segment is an extension. This extension plays a unique role in the orthogonal docking mode.
  • the peptide binding groove is much wider in the middle relative to its tapered ends so that the MHC class II molecule needs to use sidechains of multiple conserved residues from ⁇ l and ⁇ 1 helical regions to reach the peptide mainchain atoms.
  • the residues include asparagine and glutamine which form bidentate hydrogen bonds to the peptide backbone.
  • This H-bonding pattern determines the peptide binding polarity in the class II MHC system (9-12).
  • An important characterization of class ⁇ MHC molecules is that the ⁇ l helix is two turns shorter in the N-terminus than the corresponding class I MHC molecule ⁇ l helix. In particular, from Arg52 ⁇ to Glu55 ⁇ , the helix is replaced by an extended strand that reaches close enough to the N-terminal extension segment of the bound peptide to form a mini parallel ⁇ -sheet using mainchain atoms.
  • the pair of mainchain-mainchain hydrogen bonds between Arg53 ⁇ of the MHC class II molecule and the P-2 and PI residues at the N-terminal part of the peptide are conserved among all known pMHCII structures.
  • the scD10-CA/I-Ak structure shows that a diagonal TCR docking would result in a collision between the V ⁇ domain of TCR and the pMHCII on the "left" side.
  • the tilt angle of a TCR relative to an MHC molecule (see Table 3 legend for the definition of tilt angle) exacerbates this potential clash by maintaining the V ⁇ domain in close proximity to MHC.
  • the TCR- pMHC class I docking may have more variation in terms of the orientation angle as demonstrated in Table 3, the topology of TCR binding to pMHC class II may be more closely restricted to an orthogonal mode due to the "ridge" described above.
  • the protrusion of the peptide's N-terminus has been suggested as a site for disruption by DM in the process of exchanging CLIP for an antigenic peptide in the MHC class II molecule (21).
  • V ⁇ accounts for 519A while V ⁇ accounts for 338A of the TCR buried surfaces. This result is consistent with the notion that V ⁇ dominates in the interaction which is generally true for the class I system as well.
  • the calculations showed that the buried surface areas of V ⁇ and V ⁇ are 480A and 430A 2 for the 2C-dEV8/H-2Kb complex, 576A 2 and 319A 2 for A6-Tax/HLA-A2, and 555A and 260A for B7-Tax/HLA-A2, respectively.
  • V ⁇ 8.2 recognizes the I-Ak a l helical residues Lys39, Gln57 and Leu ⁇ O via CDR2, and Gln ⁇ l via CDRl . Given that these two docking interactions are to highly conserved MHC class I and to highly conserved class II residues, respectively, it appears that the V ⁇ domain plays a major role in MHC recognition by both classes of TCRs and perhaps in pre-TCRs as well (29).
  • the peptide in the ternary structure is 16 residues in length, designated as from P-3 to PI 3, the TCR interaction is restricted to the P-l to P8 segment.
  • Table 2 lists all the contacts to the peptide. It is noteworthy that of 27 atomic contacts with the peptide, 23 involve V ⁇ and only four involve V ⁇ . This dominance of the V ⁇ domain in peptide recognition was not appreciated previously, although early molecular modeling efforts correctly suggested that an orthogonal TCR docking mode was possible (Davis & Bjorkman, ref. 2). The spiral conformation of bound peptide (12) dictates that of the deeply buried peptide residues, only those at positions P2, P5 and P8 are accessible to the TCR molecule.
  • the T ⁇ at the P7 position is an exception due to its bulky indole ring, which is partially exposed on the TCR binding surface.
  • the backbone of the P-2 residue is engaged in a mini parallel ⁇ -sheet with the MHC molecule as discussed above, while the P-3 and the C-terminal three residues (PI 1- P13) have no contacts with either MHC or TCR whatsoever, though well defined by unambiguous densities.
  • the P2 residue is an Arg. It forms multiple salt bridges with both Asp30 ⁇ in the D10 CDRl ⁇ and I-Ak Glu74 ⁇ , respectively. The same TCR Asp30 ⁇ also interacts with I-Ak Arg70 ⁇ .
  • the upward-pointing P2-Arg is within van der Waals contacts to backbone of CDR3 ⁇ Gly99 ⁇ and CDRl ⁇ Thr28 ⁇ (see Table 2).
  • This knitted local structure packs closely onto the sidechain of lie at the P5 position from the N-terminal side of the peptide.
  • the P5 residue is important structurally and biologically. Alteration of this residue adversely affects D10-TCR recognition of CA/I-Ak (17).
  • the sidechain of He at P5 fits extremely well into a hydrophobic pocket.
  • the neutralized network discussed above on the C- terminal peptide side is the indole ring of the T ⁇ at P7 stacking onto the isobutyl group of the P5-Ile.
  • the P5-Ile contacts the backbone of the tip of CDR3 ⁇ which consists of Gly99 ⁇ -Serl00 ⁇ -Phel01 ⁇ .
  • the phenolic ring of PhelOl bends towards the P7-T ⁇ position.
  • the exposed tip of the indole ring of P7-T ⁇ makes contacts with the PhelOl ⁇ aromatic ring.
  • the other peptide residue engaged in recognition is the P8-Glu residue.
  • P8-Glu forms bifurcated hydrogen bonds to sidechains of Tyr ⁇ O ⁇ and Tyr67 ⁇ of the I-Ak molecule.
  • peripheral T cells are able to recognize allogeneic MHC molecules to which they were never exposed (38).
  • the precise molecular basis of alloreactivity is yet to be fully defined.
  • the complex of scD10-CA/I-Ak is informative since the D10 TCR not only recognizes the antigenic CA peptide bound to I-Ak but also responds to all MHC class II molecules whose I- A ⁇ chain contains the sequence "PEI" at positions 65-67, including I-A v ' p q ' (17).
  • MHC class II molecules having a Tyr at this position such as I-A ' r ' s ' "' 6 , cannot stimulate D10 cells in the absence of the CA peptide.
  • the aliphatic sidechain of the Ile67 in I- Ad can replace the aromatic ring on the sidechain of Tyr67 in I-Ak, forming van der Waal's contacts with the V ⁇ CDR3 loop.
  • side chains from residue Arg99 of D10 V ⁇ and residue Glu66 of I- Ad ⁇ l are rotated and the mainchain conformation around PEI on I- Ad is slightly modified.
  • the backbone NH vectors of residues directly adjacent to Arg99 are among the most mobile in scDIO (20).
  • Glu66 can form multiple potential interactions with CDRl and CDR2 of D10 V ⁇ . Additionally, the hydrogen bond between Gln64 and Arg99 from CDR3 of V ⁇ is preserved. Therefore, despite loss of one hydrogen bond of Tyr67 to D10 V ⁇ Gly98, these potential additional contacts between the CDR loops of D10 V ⁇ and the inserted PEI residues can enhance the affinity between MHC and D10. Other TCR allo-pMHC ⁇ interactions cannot be excluded. Consistent with this view, it has also been suggested that in the case of the 2C allo-MHCI response (Ld), allorecognition results from increased interaction between the 2C TCR V ⁇ domain and the allostimulus (39).
  • the ability of exposed MHC helical polymo ⁇ hic residues to permute the number and nature of contacts with the TCR is a feature of other class II MHC-restricted allogeneic responses (40, 41).
  • the naturally occurring I-Ab mutant H-2bml2 generates a strong alloresponse in H-2b mice. This molecule differs from I-Ab a t only three position: 67 ⁇ , 70 ⁇ and 71 ⁇ .
  • Superantigens are a family of immunostimulatory and disease-causing proteins derived from bacterial or endogenous retroviral genes which are capable of activating a large fraction of the T cell population (43). In general, the activation appears to require a bridging interaction between the V ⁇ domain of the TCR and an MHC class ⁇ molecule.
  • crystal structures (44) showing the detailed interactions between SEB, a representative bacterial SAG, and a TCR V ⁇ 8.2 chain or SEB and the HLA-DR1 class ⁇ MHC molecule have been determined, the physiologically relevant tripartite TCR-SAG-pMHC complex has not yet been characterized.
  • TCR-SAG-pMHC complex A structural model of TCR-SAG-pMHC complex was previously generated (44) based on least squares supe ⁇ osition of 1) the 14.3. d V ⁇ C ⁇ -SEB complex, 2) the SEB-HLA-DR1 complex and 3) the 2C TCR ⁇ ⁇ heterodimer.
  • the docking mode of TCR on the class II MHC was structurally unknown and presumed to be similar to the observed diagonal mode of TCR on class I MHC, it was noted that the rotational orientation of the TCR and MHC molecules in the predicted TCR-SEB-pMHC complex was substantially different (-40°) from the 2C-dEV8/Kb complex.
  • the superantigen wedges itself between V ⁇ and the MHC class ⁇ ⁇ l helix, forcing the MHC to swing away from V ⁇ and toward V ⁇ while preserving the direct interaction between the V ⁇ domain of the TCR and MHC class II ⁇ 1 helix.
  • This latter interaction has been proposed to be critical in stabilizing the TCR-SAG-pMHC complex.
  • T cell activation by SAG is believed to be dependent upon the interaction between a given TCR V ⁇ domain and the MHC class II ⁇ l helix (45).
  • Thymus Given that there are no intrinsic structural differences between class I vs. class II MHC restricted TCR V modules as shown above, it is questioned what directs expression of a TCR to the proper CD4 or CD8 subset.
  • progenitor cells transit from a CD4-CD8- double negative (DN) stage through a CD4+CD8+ double positive (DP) stage and then into a CD4+CD8- or CD4-CD8+ single positive (SP) stage (46). Selection for maturation occurs upon the interaction of thymocytes with stromal cells expressing self-pMHCI or self- pMHCII ligands within the thymus, beginning at the DP stage where the TCR first appears.
  • DN CD4-CD8- double negative
  • DP CD4+CD8+ double positive
  • SP CD4+CD8+ single positive
  • CA/I-Ak s crystallized with the hanging-droplet vapor diffusion method.

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Abstract

L'invention concerne des méthodes de vaccination thymique, dans lesquelles un polypeptide d'intérêt est administré et permet la sélection positive ou négative d'une spécificité du récepteur de l'antigène des lymphocytes TCR dans le thymus, de sorte qu'une spécificité (positive) voulue soit conservée ou qu'une spécificité indésirable (négative) soit éliminée au niveau de l'établissement du répertoire des caractères de TCR, que des TCR conçus pour reconnaître des antigènes de maladies ou des antigènes étrangers soient générés et que cela permette le traitement des cancers, des maladies auto-immunes, des infections ou des effets d'agents de guerre biologiques.
EP00983712A 1999-11-24 2000-11-16 Polypeptides et methodes de vaccination thymique Withdrawn EP1259591A2 (fr)

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