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WO2006016113A1 - Procédé d’essai de ligand du tcr cellulaire - Google Patents

Procédé d’essai de ligand du tcr cellulaire Download PDF

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Publication number
WO2006016113A1
WO2006016113A1 PCT/GB2005/003002 GB2005003002W WO2006016113A1 WO 2006016113 A1 WO2006016113 A1 WO 2006016113A1 GB 2005003002 W GB2005003002 W GB 2005003002W WO 2006016113 A1 WO2006016113 A1 WO 2006016113A1
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Prior art keywords
tcr
tcrs
cells
cell
tcr ligand
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PCT/GB2005/003002
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English (en)
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Marco Atul Purbhoo
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Avidex Ltd.
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Publication of WO2006016113A1 publication Critical patent/WO2006016113A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex

Definitions

  • the invention relates to an assay method for estimating the average number per cell of molecules of a given TCR ligand on a sample of test cells.
  • the assay relies on the use of ⁇ T cell receptors (TCRs) which specifically recognise and bind to said TCR ligand, said TCRs having a Kd for their interaction with the said TCR ligand of 50 nM or less.
  • TCRs ⁇ T cell receptors
  • pMHC molecules are one class of cell-bound TCR ligand. Almost all nucleated cells of higher vertebrates present MHC molecules. Such MHC expressing cells are known as antigen presenting cells (APCs). The MHC molecules of these APCs form a complex with peptide antigens so that the peptides are presented on the surface of the APCs as pMHCs. pMHCs are recognised by T cells via T cell receptors (TCRs) and a co-receptor expressed on the surface of the T cell. Binding of the MHC-peptide complex with the TCR and coreceptor transduces signals in the T cell that activate the cell, leading to a cellular immune response.
  • TCRs T cell receptors
  • MHC molecules are divided into MHC Class I and MHC Class II molecules.
  • the former require the CD8 co-receptor for T cell activation, and the latter require the CD4 coreceptor for T cell activation.
  • TCRs mediate the recognition of specific Major Histocompatibility Complex (MHC)-peptide complexes by T cells and, as such, are essential to the functioning of the cellular arm of the immune system.
  • MHC Major Histocompatibility Complex
  • Antibodies and TCRs are the only two types of molecules which recognise antigens in a specific manner, and thus the TCR is the only receptor for particular peptide antigens presented in MHC, the alien peptide often being the only sign of an abnormality within a cell.
  • T cell recognition occurs when a T-cell and an antigen presenting cell (APC) are in direct physical contact, and is initiated by ligation of antigen-specific TCRs with pMHC complexes.
  • APC antigen presenting cell
  • the native TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.
  • TCRs exist in ⁇ and ⁇ forms, which are structurally similar but have quite distinct anatomical locations and probably functions.
  • the MHC class I and class II ligands are also immunoglobulin superfamily proteins but are specialised for antigen presentation, with a highly polymorphic peptide binding site which enables them to present a diverse array of short peptide fragments at the APC cell surface.
  • the extracellular portion of native heterodimeric ⁇ and ⁇ TCRs consist of two polypeptides each of which has a membrane-proximal constant domain, and a membrane-distal variable domain. Each of the constant and variable domains includes an intra-chain disulfide bond.
  • the variable domains contain the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies. CDR3 of ⁇ TCRs interact with the peptide presented by MHC, and CDRs 1 and 2 of ⁇ TCRs interact with the peptide and the MHC.
  • the diversity of TCR sequences is generated via somatic rearrangement of linked variable (V), diversity (D), joining (J), and constant genes
  • ⁇ and ⁇ chain polypeptides are formed by rearranged V-J-C regions, whereas ⁇ and ⁇ chains consist of V-D-J-C regions.
  • the extracellular constant domain has a membrane proximal region and an immunoglobulin region.
  • TRAC and TRDC single ⁇ and ⁇ chain constant domains
  • TRBCl and TRBC2 IMGT nomenclature
  • TRBCl and TRBC2 N 4 K 5 - ⁇ -K 4 N 5 and F 37 ->Y (IMGT numbering, differences TRBC1->TRBC2), the final amino acid change between the two TCR ⁇ chain constant regions being in exon 3 of TRBCl and TRBC2: Vi->E.
  • the constant ⁇ domain is composed of one of either TRGCl, TRGC2(2x) or TRGC2(3x).
  • TRGC2 constant domains differ only in the number of copies of the amino acids encoded by exon 2 of this gene that are present.
  • TCR extracellular domains The extent of each of the TCR extracellular domains is somewhat variable. However, a person skilled in the art can readily determine the position of the domain boundaries using a reference such as The T Cell Receptor Facts Book, Lefranc & Lefranc, Publ. Academic Press 2001.
  • TCRs The production of recombinant TCRs is beneficial as these provide soluble TCR analogues suitable for the following purposes:
  • Single-chain TCRs are artificial constructs consisting of a single amino acid strand, which like native heterodimeric TCRs bind to MHC-peptide complexes.
  • scTCRs Single-chain TCRs
  • attempts to produce functional alpha/beta analogue scTCRs by simply linking the alpha and beta chains such that both are expressed in a single open reading frame have been unsuccessful, presumably because of the natural instability of the alpha-beta soluble domain pairing.
  • TCR heterodimers which include the native disulphide bridge which connects the respective subunits (Garboczi, et al, (1996), Nature 384(6605): 134-41; Garboczi, et al. (1996), J Immunol 157(12): 5403- 10; Chang et al, (1994), PNAS USA 91: 11408-11412; Davodeau et al., (1993), J. Biol Chem. 268(21): 15455-15460; Golden et al, (1997), J. Imm. Meth. 206: 163- 169; US Patent No. 6080840).
  • TCRs can be recognised by TCR-specific antibodies, none were shown to recognise its native ligand at anything other than relatively high concentrations and/or were not stable.
  • a soluble TCR which is correctly folded so that it is capable of recognising its native ligand, is stable over a period of time, and can be produced in reasonable quantities.
  • This TCR comprises a TCR ⁇ or ⁇ chain extracellular domain dimerised to a TCR ⁇ or ⁇ chain extracellular domain respectively, by means of a pair of C-terminal dimerisation peptides, such as leucine zippers.
  • This strategy of producing TCRs is generally applicable to all TCRs.
  • Class I MHC molecules are presented by almost all nucleated cells of higher vertebrates. These molecules generally present peptides that are derived from intracellular polypeptides and proteins.
  • Class I MHC is a dimeric protein complex consisting of a variable heavy chain and a constant light chain, ⁇ 2 -microglobulin ( ⁇ 2 m).
  • the heavy and light chains of Class I MHC molecules are made by ribosomes on the rough endoplasmic reticulum and then translocated to the lumen of the endoplasmic reticulum (ER).
  • the peptides that are loaded by Class I MHC molecules are generated in the cytosol by proteosomes, before being transported into the lumen of the ER. Within the ER the MHC heavy and light subunits and a peptide combine to form a stable pMHC molecule which is then transported to the cell surface via the Golgi.
  • the pMHC molecules are anchored in the cell membrane by the MHC heavy chain.
  • Class I MHC presented peptides are usually 8-11 amino acids in length, depending on the degree of arching introduced in the peptide when bound in the MHC molecule.
  • the binding cleft which is formed by the membrane distal ⁇ l and ⁇ .2 domains of the MHC heavy chain, has "closed" ends, imposing quite tight restrictions on the length of peptide which can be bound.
  • ⁇ 2 m is a polypeptide found free in serum, which is non-covalently associated with MHC Class I molecules at the cell surface and which can exchange in the MHC complex with other free ⁇ 2 m molecules (Bernabeu, et al Nature 308: 642-5 (1984); Cook, et al. J Immunol 157: 2256-61 (1996); Horig, et al. Proc Natl Acad Sci USA 94: 13826-31 (1997); Hyafil & Strominger, Proc Natl Acad Sci USA 76: 5834-8
  • Class I pMHC molecules are recognised by CD8 + cyto-toxic T cells (CTLs). This recognition leads to a cyto-toxic response by the T cell which leads to the killing of the APC and clonal expansion of the CTL.
  • the CD8 coreceptor of CTLs is expressed as either a ⁇ homodimer, or occasionally as an ⁇ heterodimer protein consisting of extracellular immunoglobulin, membrane- proximal stalk, transmembrane and cytoplasmic domains.
  • the native dimers have a molecular weight of 47 & 45 kDa respectively (The Leucocyte Antigen Factsbook, 2 nd Ed., Barclay et al, (1997) Academic Press).
  • Class II MHC molecules are only presented by specialised APCs including the interdigitating dendritic cells found in the T cell areas of the lymph nodes and spleen in large numbers; Langerhans cells in the skin; follicular dendritic cells in B cell areas of the lymphoid tissue; monocytes, macrophages and other cells of the monocyte/macrophage lineage; B cells and T cells; and a variety of other cells such as endothelial cells and fibroblasts which are not classical (specialised?) APCs but can act as such.
  • Class II MHC molecules generally present peptides derived from polypeptides and proteins in the extracellular spaces. (The HLA Factsbook, Marsh et al, (2000), Academic Press)
  • Class II MHC is a membrane-bound 61-65kDa ⁇ heterodimeric protein complex consisting of two similar non-covalently associated chains (The Leucocyte Antigen Factsbook, 2 nd Ed., Barclay et al, (1997) Academic Press).
  • Class II HLA heavy and light chains are made by ribosomes on the rough endoplasmic reticulum and then translocated to the lumen of the endoplasmic reticulum (ER).
  • the Class II MHC molecules are then assembled with a polypeptide, known as the invariant chain.
  • the Class II MHC-invariant chain complexes are then transported via the Golgi to endocytic vesicles called the MHC Class II compartment.
  • MIIC Within this compartment the invariant chain is degradable and replaced by a peptide derived from endocytosed material.
  • the peptides presented by Class II MHC molecules are generally 12-24 amino acids in length.
  • the binding cleft is formed by the membrane distal ⁇ l and ⁇ l domains of the MHC chains (The HLA Factsbook, Marsh et al, (2000) Academic Press).
  • Class II pMHC molecules are recognised by CD4 + helper T cells. This recognition leads to either the production of cytokines which directly act on macrophages to enhance their killing rate or the stimulation of B cells to produce antibodies specific for the Class II MHC-loaded peptide.
  • the CD4 coreceptor of T helper cells is expressed as monomeric protein consisting of extracellular region made up of four immunoglobulin superfamily domains, transmembrane and cytoplasmic domains.
  • the native protein has a molecular weight of 48.4 kDa (The Leucocyte Antigen Factsbook, 2 nd Ed., Barclay et al, (1997) Academic Press).
  • CDl antigens are also capable of functioning as cell-bound TCR ligands.
  • CDl antigens are MHC class I-related molecules whose genes are located on a different chromosome from the classical MHC class I and class II antigens.
  • CDl molecules are capable of presenting peptide and non-peptide (e.g. lipid, glycolipid) moieties to T cells in a manner analogous to conventional class I and class II-MHC-pep complexes. See, for example (Barclay et al, (1997) The Leucocyte Antigen Factsbook 2 nd Edition, Academic Press) and (Bauer (1997) Eur J Immunol 27 (6) 1366-1373))
  • APCs present pMHCs whether they are "healthy” or not.
  • the MHC molecules of healthy cells will present "self peptides and such complexes are "scanned” by T cells but do not normally elicit a T cell response.
  • T cells T cells
  • the transformation of a healthy cell to a cancerous include mutations within genes involved in the regulation of cell cycle and/or translocation events that lead to the up or down-regulation of gene expression.
  • Viruses such as influenza, hepatitis B virus (HBV), Epstein -Barr virus (EBV) and human immunodeficiency Virus (HIV)
  • Bacteria such as mycoplasmas • Protozoa, such as plasmodium and trypanosomes.
  • extracellular pathogen including, but not limited to bacteria, such as Staphylococcus aureus and Pseudomonas aeruginosa which are capable of causing disease in humans without invading cells.
  • bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa
  • the presence of such extracellular pathogens generally provokes a humoral (antibody) based response. This response is facilitated by the presentation of peptides derived from the pathogen on the Class II MHC molecules of specialised APCs.
  • T cells mature in the thymus where they undergo at least two selection mechanisms, generally referred to as positive and negative selection.
  • the structures of most, or all, TCRs are believed to share certain general architectural features (Chothia, et al, Embo J(1988) 7: 3745-55) that provide a framework suitable for MHC/peptide binding by the variable complementarity determining regions (CDRs).
  • CDRs variable complementarity determining regions
  • T cells with high affinity for one of the self MHC molecules will be negatively selected (Amsen & Kruisbeek. (1998). Immunol Rev 165: 209-29. Sebzda, et al (1999). Annu Rev Immunol 17: 829-74).
  • TCRs in the cellular immunity can be considered to be analogous to antibodies in the humoral immunity.
  • Antibodies have been successfully used, either as therapeutic agents in their own right (e.g. Herceptin) or as targeting agents (e.g. mylotarg), and interest in this area continues to grow. Similar strategies could be devised using T cell receptors.
  • soluble TCRs are useful, not only for the purpose of investigating specific TCR-pMHC interactions, but also as a diagnostic tool to detect infection, or to detect autoimmune disease markers, or to detect the efficacy of T cell vaccines.
  • Soluble TCRs also have applications in staining, for example to stain cells for the presence of a particular cancer or viral antigen presented in the context of the MHC.
  • soluble TCRs can be used to deliver a therapeutic agent, for example a cytotoxic compound or an immunostimulating compound, to cells presenting a particular antigen.
  • TCRs Two factors have hindered the exploitation of TCRs. Firstly, a generally applicable method for the production of soluble (i.e. non-membrane bound) T cell receptors have only been made available in the last few years. Secondly, the affinity of the T cell receptor for its specific pMHC ligand is much lower (KQ in the ⁇ M range) than for antibodies (K D in the nM range). This lower affinity of the TCR is thought to be a result of negative selection during development, and it is therefore probably not possible to find TCRs with high affinity for self-MHC-peptide complexes (Salzmann & Bachmann, Molecular Immunology, 1998, 35:65-71).
  • phage particles are caused to display a diverse library of mutated ⁇ TCRs which contain a non-native disulfide interchain bond.
  • the phage-displayed d TCR library is the subjected to one or more round(s) of selection against a given pMHC in order to identify high affinity TCRs clones specific for the given pMHC.
  • Yeast has also been investigated as a system for displaying scTCR and also for selecting stable and high affinity scTCR mutants.
  • US6,759,243 Holler, et al. (2000) Proc. Natl. Acad. Sci. 97, 5387-5392; Shusta, et al. (2000) Nature Biotechnology 18, 754-759; Shusta, et al. (1999) J. MoI. Biol. 292, 949-956; Kieke, et al. (1999) Proc. Natl. Acad. Sci. 96, 5651-5656)).
  • TCRs can exhibit higher affinities for allogenic pMHCs than syngeneic pMHCs.
  • WO 2004/044004 discloses generally applicable proteinaceous particle (preferable phage particle) display-based methods for the production of high affinity ⁇ TCRs.
  • pMHC-specific antibody Fab monomers for labelling cells transfected to express the Telomerase catalytic subunit (hTERT) and tumour cells which naturally expressed this protein.
  • the level of pMHC expression was determined by FACS using an anti -human Fab labelled with fluorescein isothiocyanate (FITC) to provide the fluorescent signal.
  • FITC fluorescein isothiocyanate
  • the number of FITC molecules which bind to a given Fab fragments may vary across a population of such labelled molecules, making the enumeration of a specific pMHC on a single cell using a FITC labelling method unreliable.
  • FITC is not a strong enough fluorophore to allow microscopy-based detection of a single FITC molecule.
  • US patent application no. US 2001/0006782 discloses methods for the diagnosis of Endometriosis based on comparing the HLA A/B/C complex expression levels of healthy and potentially diseased cells. The disclosed methods depend on contacting the healthy and potentially diseased cells with HLA A/B/C specify antibodies, of fragments thereof.
  • Soluble TCRs are useful, not only for the purpose of investigating specific TCR- pMHC interactions, but also potentially as a diagnostic tool to detect infection, or to detect autoimmune disease markers. Soluble TCRs also have applications in staining, for example to stain cells for the presence of a particular peptide antigen presented in the context of the MHC".
  • “In tetrameric TCR formed using biotinylated heterodimers, fluorescent streptavidin (commercially available) can be used to provide a detectable label. A fluorescently- labelled tetramer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the TCR is specific.”
  • the present invention provides an assay method comprising providing a plurality of test cells, which present a specific TCR ligand, contacting the test cells with an excess of ⁇ T cell receptors (TCRs) which specifically recognise and bind to said TCR ligand, said TCRs having a Kd for their interaction with the said TCR ligand of 50 nM or less, said TCRs being labelled, or adapted to be labelled, with a detectable signal; separating non-cell-bound TCRs from the cells, in the case where the TCRs were adapted to be labelled rather than labelled, labelling the cell-bound TCRs with the detectable signal, then detecting and quantifying the label signals from one or a plurality of the cells, and estimating from the resultant signal quantity(ies) the average number of said TCR ligands presented per cell.
  • TCRs ⁇ T cell receptors
  • the invention provides an assay method comprising providing a plurality of test cells, which present a specific TCR ligand, contacting the test cells with an excess of ⁇ T cell receptors (TCRs) which specifically recognise and bind to said TCR ligand, said TCRs having a Kd for their interaction with the said TCR ligand of 50 nM or less, said TCRs being labelled, or adapted to be labelled, with a detectable signal; separating non-cell-bound TCRs from the cells, in the case where the TCRs were adapted to be labelled rather than labelled, labelling the cell-bound TCRs with the detectable signal, then detecting and quantifying the label signals from one or a plurality of the cells, and estimating from the resultant signal quantity(ies) the average number of said TCR ligands presented per cell.
  • TCRs ⁇ T cell receptors
  • the ⁇ TCR has a Kd for their interaction with the said TCR ligand of 20 nM or less, more preferably 1OnM or less, or most preferably 5 nM or less.
  • SPR Surface Plasmon Resonance
  • Example 3 herein provides a detailed description of a Biacore ® -based method for carrying out such determinations.
  • TCR ligand is a Class I pMHC, or a Class II pMHC or a CDl -antigen.
  • the label signals are detected and quantified cell by cell.
  • the detectable signal may be produced by the spontaneous or excited emission of any form of electromagnetic wave including, but not limited to, gamma rays, visible or ultraviolet light.
  • the detectable signal may be produced by the emission of a particle including, but not limited to ⁇ or ⁇ particles.
  • the detectable signal is fluorescence.
  • fluorescent molecules that can be of use in the present invention, including, but not limited to, phycobiliproteins.
  • Phycoerythrin (PE) is a particularly preferred phycobiliprotein for use in the present invention.
  • the fluorescent molecules utilised comprise a known number of fluorophores and have extinction coefficients and/or quantum yields in the order of 1.96x 10 6 - 2.4 x 10 6 M "1 cm "1 and 0.84 - 0.98 respectively.
  • the label signals may be detected by 3-dimensional fluorescence microscopy and quantified by counting individual fluorescent signals.
  • 3-dimensional fluorescence microscopy involves subjecting the specimen under investigation to light of the appropriate wavelength to excite the fluorophores utilised. This excitation of the fluorophores causes them to emit light of a different, longer, wavelength. The microscope will reveal any fluorescence that occurs on, or very near to, the focal plane as a bright focussed spot, which is then recorded photographically. This process of excitation and emission is repeated through a range of focal planes until the entire specimen has been imaged.
  • the images produced by this process can then be assessed in order to provide a count of the total number of fluorescent signals present on the specimen.
  • (Li et al, Nature Immunology 8 (5) 791-799) provide details relating to the 3-dimensional fluorescence microscopy of APCs.
  • test cells For contacting with the TCR, the test cells may be in suspension or adhered to a substrate.
  • the required estimated average may adjusted by subtracting a number representing the average number of TCRs bound to the cell otherwise than specifically to the TCR ligand.
  • the average number of TCRs bound to the cell otherwise than specifically to the TCR ligand may be derived from one or more control assays wherein the method of the invention is repeated except that
  • soluble ⁇ dTCRs constructs containing a non-native disulfide interchain bond as disclosed in WO 03/020763 are particularly preferred and the preferred method for generating high affinity variants of TCRs specific for a given TCR ligand is selection from a diverse library of phage particles displaying such TCRs as disclosed in WO 2004/044004.
  • the high affinity TCR utilised in the present invention may either be labelled, or adapted to be labelled, with a detectable signal.
  • a detectable signal there are many suitable methods, known to those skilled in the art of antibody labelling, by which such a detectable signal can be associated with said high affinity TCR. These include, but are not limited to, the following:
  • Biotin / streptavidin -mediated labelling - these methods rely on the high affinity of these two bacterial molecules for each other in order to facilitate the association of the high affinity TCR and detectable label.
  • the high affinity TCR is biotinylated, using for instance the methods disclosed in WO 03/020763, and then contacted with a streptavidin-detectable label conjugate. These methods of association can be employed either before or after the high affinity TCR is contacted with the APCs.
  • Example 2 herein provides details of a method for the formation of such a biotin-streptavidin mediated conjugate post contacting the high affinity TCR with the APCs.
  • streptavidin analogues such as extravidin, neutravidin and avadin which may be used to replace streptavidin.
  • Direct polypeptide fusion - these method rely on the production of a fusion protein comprising one chain of a high affinity dTCR or a high affinity scTCR and the polypeptide utilised as the detectable signal.
  • a subsequent refolding is required in order to form the dTCR-detectable label.
  • This step involves the in- vitro refolding of the TCR chain-detectable label fusion protein with the remaining TCR chain.
  • Disulfide bond-mediated detectable label association These methods rely on using a disulfide bond to facilitate the association between the high affinity TCR and the detectable label.
  • WO 03/020763 details a method for the production of a soluble dTCR incorporating an unpaired cysteine residue at the C-terminus of either of the TCR chains.
  • the polypeptide utilised as the detectable signal is then similarly altered to incorporate an unpaired cysteine reside at the C or N-termini.
  • the modified high affinity TCR and polypeptide are then contacted in-vitro under conditions suitable for the formation of the disulfide bond-mediated association.
  • These methods of association are only suited to the formation of an association between the high affinity TCR and the polypeptide utilised as the detectable signal prior to contacting the high affinity TCR with the APCs.
  • tags containing multiple histidine residues can be added to the termini of polypeptides and proteins in order to facilitate the association of metal ions thereto.
  • Hexa-histidine tags are the most commonly used example of such tags and these are generally used to facilitate the association of divalent metal cations such as Ni 2+ or Co 2+ with the polypeptides.
  • multiple histidine tags may be utilised to facilitate the association of a radioactive metal cation to the high affinity TCR. These methods of association are only suited to the formation of an association between the high affinity TCR and the metal ion utilised as the detectable signal prior to contacting the high affinity TCR with the APCs.
  • Metal chelates As is also known to those skilled in the art it is possible to attach chelating agents to polypeptides which can "trap" metal ions such as lanthanides. These lanthanide ions can have fluorescent and/or radioactive properties. Methods of estimating TCR ligands on the surface of a cell
  • Example 2 herein details a preferred 3-dimensional fluorescence microscopy-based method of estimating the per cell average number of a given TCR ligand on the surface of a cell.
  • the quantification of the signal from the labelled TCR ligand-bound TCRs provides a quantity related to the number of TCRs bound per cell, and therefore the required estimate is obtainable from that quantity. Quantification of course depend on the nature of the signal, but two alternatives to the microscopy-based methods are:
  • FACs Fluorescence-activated cell sorting
  • TCRs may be detected, and which also allows the estimation of the average number of a given TCR ligand presented per cell for a population of cells.
  • Figure Ia details the DNA sequence encoding the soluble Clone 134 A6 TCR ⁇ chain.
  • This DNA sequence comprises an introduced non-native cysteine codon to facilitate disulfide bond-mediated interchain pairing of the expressed soluble TCR, and the native A6 TCR ⁇ variable domain.
  • the inserted cysteine codon is shaded.
  • Figure Ib details the amino acid sequence of the soluble Clone 134 A6 TCR ⁇ chain.
  • This amino sequence comprises an introduced non-native cysteine to facilitate disulfide bond-mediated interchain pairing of the soluble dTCR, and the native A6 TCR ⁇ variable domain.
  • the inserted cysteine is shaded.
  • Figure 2a details the DNA sequence encoding the soluble Clone 134 A6 TCR ⁇ chain.
  • This DNA sequence comprises an introduced non-native cysteine codon to facilitate disulfide bond-mediated interchain pairing of the expressed soluble dTCR and a mutated A6 TCR ⁇ variable domain.
  • the inserted cysteine codon is shaded and the variable domain mutations are shown in bold.
  • Figure 2b details the amino acid sequence of the soluble Clone 134 A6 TCR ⁇ chain.
  • This amino sequence comprises an introduced non-native cysteine to facilitate disulfide bond-mediated interchain pairing of the soluble dTCR and a mutated A6 TCR ⁇ variable domain.
  • the inserted cysteine is shaded and the variable domain mutations are shown in bold.
  • Figure 3 illustrates the imaging of individual Streptavidin-PE/TCR complexes on APCs (J82 cancer cells transfected with a minigene for the Tax ⁇ -19 epitope) stained with the high affinity Clone 134 A6 TCR.
  • the fluorescent images represents the z- stacks corresponding to the bottom plasma membrane (in contact with the chamber- slide).
  • Figure 4 illustrates the quantification of HLA/Taxn.ig complexes on peptide pulsed cells using high affinity TAXwtcl34 TCR. T2 cells were pulsed with indicated concentrations of Taxn-i 9 peptide.
  • Figures 5a and 5b provide the amino acid sequences of the ⁇ and ⁇ chains of the high- affinity c58c61 1G4 TCR containing an introduced non-native disulfide interchain bond respectively The high affinity inducing mutated amino acids are underlined and the introduced cysteine codon are highlighted.
  • Figures 6a and 6b provide DNA sequences encoding the ⁇ and ⁇ chains of the high- affinity c58c61 1G4 TCR containing an introduced non-native disulfide interchain bond respectively.
  • the high affinity inducing mutations are in bold and the introduced cysteine codon are highlighted.
  • Figure 7 illustrates the average number of SLLMWITQC-HLA-A*0201 complexes detected on the surface of individual SK-Mel-37, Mel 624 and Mel 526 tumour cells.
  • the HLA-A2-Tax specific soluble clone 134 A6 TCR containing a non-native disulfide interchain bond was produced.
  • the DNA and amino acid sequences of this soluble dTCR are provided in Figures 1 and 2 respectively.
  • WO 2004/044004 discloses in detail the production of this particular high affinity soluble TCR.
  • the TCR biotinylation methods disclosed in WO 03/020763 are appropriate for producing this soluble biotinylated TCR.
  • Example 2 Quantification of cell surface TCR ligands by fluorescence microscopy using high affinity monoclonal TCR
  • the number of HLA-A2 complexed TaXn -19 antigens on target cells was determined (on the assumption that one fluorescence signal relates to a single labelled TCR bound to its cognate pMHC ligand on the surface of the target cell) by single molecule fluorescence microscopy using the high-affinity Clone 134 A6 TCR.
  • T2 Cells were pelletted by centrifugation for 5 min at 14000 rpm (Megafuge 1.0, Hereaus). Where appropriate, pelleted cells were resuspended at 10 5 -10 6 cells ml "1 in RlO medium (RPMI 1640, 10% FCS, 2 mM L- glutamine, penicillin/streptomycin) and pulsed with antigen (10 "5 - 10 ⁇ 10 M "1 ) for at least 90 min at 37°C.
  • RlO medium RPMI 1640, 10% FCS, 2 mM L- glutamine, penicillin/streptomycin
  • Fluorescence microscopy Fluorescent microscopy was carried out using an Axiovert 200M (Zeiss) microscope with a 63x Oil objective (Zeiss).
  • a Lambda LS light source containing a 300W Xenon Arc lamp (Sutter) was used for illumination, and light intensity was reduced to optimal levels by placing a 0.3 and a 0.6 neutral density filter into the light path.
  • Excitation and emission spectra were separated using a TRITC/Dil filter set (Chroma).
  • Cells were imaged in three dimensions by z-stack acquisition (21 planes, 1 ⁇ m apart). Image acquisition and analysis was performed using Metamorph software (Universal Imaging) as described (Irvine et ah, Nature (419), p845-9, and Purbhoo et al, Nature Immunology (5), p524-30.).
  • Example 3 - BIAcore surface plasmon resonance characterisation of a high affinity A6 TCR binding to HLA-A2 Tax.
  • a surface plasmon resonance biosensor (BIAcore 3000TM) was used to analyse the binding of the high affinity clone 134 A6 TCR to the HLA- A2 Tax ligand. This was facilitated by producing pMHC complexes (described below) which were immobilised to a streptavidin-coated binding surface in a semi-oriented fashion, allowing efficient testing of the binding of a soluble T-cell receptor to up to four different pMHC (immobilised on separate flow cells) simultaneously. Manual injection of HLA complex allows the precise level of immobilised class I molecules to be manipulated easily.
  • Biotinylated class I HLA- A2 tax complexes were refolded in vitro from bacterially- expressed inclusion bodies containing the constituent subunit proteins and synthetic peptide, followed by purification and in vitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • HLA-heavy chain was expressed with a C- terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains of the protein in an appropriate construct.
  • Inclusion body expression levels of -75 mg/litre bacterial culture were obtained.
  • the HLA light-chain or ⁇ 2-microglobulin was also expressed as inclusion bodies in E.coli from an appropriate construct, at a level of -500 mg/litre bacterial culture.
  • E. coli cells were lysed and inclusion bodies were purified to approximately 80% purity. Protein from inclusion bodies was denatured in 6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA, and was refolded at a concentration of 30 mg/litre heavy chain, 30 mg/litre ⁇ 2m into 0.4 M L-Arginine-HCl, 100 mM Tris pH 8.1, 3.7 mM cystamine, mM cysteamine, 4 mg/ml peptide (e.g. tax 11 - 19), by addition of a single pulse of denatured protein into refold buffer at ⁇ 5°C. Refolding was allowed to reach completion at 4°C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 ⁇ m cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCl gradient. HLA-A2 -peptide complex eluted at approximately 250 mM NaCl, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged HLA- A2 complexes were buffer exchanged into 10 mM Tris pH
  • Biotinylated HLA-A2 complexes were purified using gel filtration chromatography. A Pharmacia Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min. Biotinylated HLA- A2 complexes eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated HLA- A2 complexes were stored frozen at -2O 0 C. Strep tavidin was immobilised by standard amine coupling methods.
  • PerBio Coomassie-binding assay
  • SPR measures changes in refractive index expressed in response units (RU) near a sensor surface within a small flow cell, a principle that can be used to detect receptor ligand interactions and to analyse their affinity and kinetic parameters.
  • the probe flow cells were prepared by immobilising the individual HLA- A2 peptide complexes in separate flow cells via binding between the biotin cross linked onto ⁇ 2m and streptavidin which have been chemically cross linked to the activated surface of the flow cells.
  • the assay was then performed by passing sTCR over the surfaces of the different flow cells at a constant flow rate, measuring the SPR response in doing so. Initially, the specificity of the interaction was verified by passing soluble A6 TCR at a constant flow rate of 5 ⁇ l min-1 over four different surfaces; one coated with -1000 RU of HLA- A2 Tax complex, the second coated with -1000 RU of HLA- A2 NY-ESO complex, and two blank flow cells coated only with streptavidin.
  • the Kd for the interaction between the high affinity Clone 134 A6 TCR and its cognate HLA-A2-Tax pMHC was determined to be approximately 5 nM.
  • the Kd for the same interaction using a wild-type soluble A6 TCR is 1-2 ⁇ M.
  • the number of HLA-A*0201 complexes loaded with the NY-ESO-I derived SLLMWITQC peptide on the surface of SK-Mel-37, Mel 624 and Mel 526 tumour cells was determined (on the assumption that one fluorescence signal relates to a single labelled TCR bound to its cognate pMHC ligand on the surface of the target cell) by single molecule fluorescence microscopy using the high-affinity biotinylated c58c61 1G4 TCR containing an introduced non-native disulfide interchain bond.
  • tumour cells Staining of tumour cells.
  • the tumour cells were plated into chamber well slides and allowed to adhere overnight in incubator. (37°C, 5% CO 2 ) Media was removed and replaced with fresh RlO. Media was removed, and cells washed twice with 500 ⁇ l of PBS supplemented with 400 ⁇ M MgCl 2 (PBS/Mg). Cells were incubated in 200 ⁇ l of TCR solution (10 ⁇ g ml "1 TCR in PBS/Mg containing 0.5% BSA) for 30 min at room temperature. The TCR solution was removed, and cells were washed three times with 500 ⁇ l of PBS/Mg.
  • interferon ⁇ interferon ⁇
  • a number of control stainings were also carried out. Firstly, an irrelevant mTCR staining (the 134 A6 TCR used in Example 2) was carried out using the same concentration (10 ⁇ g ml "1 TCR in PBS/Mg containing 0.5% BSA) of this TCR as used for the cognate high affinity c58c61 1G4 TCR. Secondly, 500 ⁇ g/ml non-biotinylated soluble c58c61 1G4 TCR was added into the TCR staining solution in order to study the ability of this solule TCR to compete for binding to the cognate SLLMWITQC- HLA-A2 complexes. Finally, a "soluble TCR-free" control staining using only streptavidin-PE was carried out.
  • Fluorescence microscopy Fluorescent microscopy was carried out using an Axiovert 200M (Zeiss) microscope with a 63x Oil objective (Zeiss).
  • a Lambda LS light source containing a 300W Xenon Arc lamp (Sutter) was used for illumination, and light intensity was reduced to optimal levels by placing a 0.3 and a 0.6 neutral density filter into the light path.
  • Excitation and emission spectra were separated using a TRITC/Dil filter set (Chroma).
  • Cells were imaged in three dimensions by z-stack acquisition (21 planes, 1 ⁇ m apart). Image acquisition and analysis was performed using Metamorph software (Universal Imaging) as described (Irvine et ah. Nature (419), p845-9, and Purbhoo et al, Nature Immunology (5), p524-30.).
  • the IFN ⁇ pre-incubation slightly increased the number of the SLLMWITQC-HLA- A*0201 complexes present on the surface of the SK-MeI -37 cells.

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Abstract

La présente invention concerne un procédé d’essai comprenant la fourniture d’une pluralité de cellules de test, qui présente un ligand du TCR donné, mis en contact avec les cellules de l’essai avec un excès de récepteurs de cellules αβ T (TCR) qui reconnaissent spécifiquement et se lient audit ligand du TCR, lesdits TCR ayant un Kd pour leur interaction avec ledit ligand du TCR de 50 nM ou moins, lesdits TCR étant marqués, ou adaptés pour être marqués, avec un signal détectable ; la séparation des TCR, non liés aux cellules des cellules, dans le cas dans lequel les TCR étaient adaptés pour être marqués plutôt que marqués, le marquage des TCR liés aux cellules avec le signal détectable, puis la détection et la quantification des signaux des marques parmi une ou une pluralité de cellules, et l’estimation à partir de la ou des quantification(s) des signaux résultants du nombre moyen dudit ligand du TCR présenté par cellule. L’information recueillie à partir de cette analyse peut fournir une information utile en termes de diagnostic et de thérapie.
PCT/GB2005/003002 2004-08-12 2005-08-01 Procédé d’essai de ligand du tcr cellulaire WO2006016113A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7569357B2 (en) 1998-01-20 2009-08-04 Board Of Trustees Of The University Of Illinois High affinity TCR proteins and methods
JP2020516594A (ja) * 2017-04-07 2020-06-11 ユーティーアイ リミテッド パートナーシップ ナノメディシンにおける受容体リガンド相互作用の効力を測定するためのアッセイ
CN113684258A (zh) * 2020-05-18 2021-11-23 上海赛比曼生物科技有限公司 用于检测鼠源tcr转基因拷贝数的试剂盒及方法

Citations (1)

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WO2004044004A2 (fr) * 2002-11-09 2004-05-27 Avidex Limited Presentation de recepteurs pour l'antigene des lymphocytes t

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WO2004044004A2 (fr) * 2002-11-09 2004-05-27 Avidex Limited Presentation de recepteurs pour l'antigene des lymphocytes t

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HOLLER P D ET AL: "In vitro evolution of a T cell receptor with high affinity for peptide/MHC.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 9 MAY 2000, vol. 97, no. 10, 9 May 2000 (2000-05-09), pages 5387 - 5392, XP002361464, ISSN: 0027-8424 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7569357B2 (en) 1998-01-20 2009-08-04 Board Of Trustees Of The University Of Illinois High affinity TCR proteins and methods
JP2020516594A (ja) * 2017-04-07 2020-06-11 ユーティーアイ リミテッド パートナーシップ ナノメディシンにおける受容体リガンド相互作用の効力を測定するためのアッセイ
CN113684258A (zh) * 2020-05-18 2021-11-23 上海赛比曼生物科技有限公司 用于检测鼠源tcr转基因拷贝数的试剂盒及方法

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