EP4348255A1 - Criblage peptidique - Google Patents

Criblage peptidique

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Publication number
EP4348255A1
EP4348255A1 EP22729763.7A EP22729763A EP4348255A1 EP 4348255 A1 EP4348255 A1 EP 4348255A1 EP 22729763 A EP22729763 A EP 22729763A EP 4348255 A1 EP4348255 A1 EP 4348255A1
Authority
EP
European Patent Office
Prior art keywords
peptide
hla
interest
cell
complex
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22729763.7A
Other languages
German (de)
English (en)
Inventor
Andrew Mcmichael
Geraldine GILLESPIE
Lucy WALTERS
Max QUASTEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxford University Innovation Ltd
Original Assignee
Oxford University Innovation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB2107821.7A external-priority patent/GB202107821D0/en
Priority claimed from GBGB2119143.2A external-priority patent/GB202119143D0/en
Application filed by Oxford University Innovation Ltd filed Critical Oxford University Innovation Ltd
Publication of EP4348255A1 publication Critical patent/EP4348255A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • G01N33/56977—HLA or MHC typing
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • 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
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present invention relates to methods of identifying a peptide which is capable of forming an HLA-E:peptide complex and being recognised by a T-cell and or B-cell, and methods of identifying one or more T-cell or B-cell which recognises an HLA-E:peptide complex.
  • HLA-E is a non-polymorphic HLA class I molecule. There are two major alleles in the population differing only in one amino acid at position 107 which is outside the peptide binding groove (Strong et al., Correlating differential expression, peptide affinities, crystal structures, and thermal stabilities. J Biol Chem. 2003;278(7):5082-90).
  • the primary function of HLA-E is to bind a peptide usually termed ‘VL9’ which is derived from the signal peptide of classical HLA class I A, B, C molecules and HLA-G, but not HLA-E.
  • the peptide has the sequence VMAPRTLVL, VMAPRTVLL, VMAPRTLLL, VMAPRTLIL, or VMAPRTLFL.
  • the HLA-VL9 complex in turn binds to the NKG2A- CD94 inhibitory or NKG2C-CD94 activating receptors on natural killer cells and a subset of T cells (Braud et al., HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature. 1998 ;391 (6669) : 795 -9) .
  • HLA-E HLA-E
  • its functionally conserved murine and rhesus orthologues, Qa-1 and Mamu-E is to regulate NK-cell activity via MHC class I signal peptide presentation
  • specific contexts appear to permit diversified peptide binding repertoires and MHC-E-restricted CD8+ T-cell priming in vivo (Wu et al., 2018) (Vance et al., 1998).
  • HLA-E-restricted pathogen peptides include a number of physiologically relevant epitopes such as the immunodominant SIV derived supertope, RL9SIV (RMYNPTNIL)(SEQ ID NO: 1).
  • HLA-E is non-polymorphic and HLA-E restricted responses to pathogens have thus far been poorly characterised, further identification of T cell responses to peptide antigen bound to HLA-E may be useful for immunotherapies which could be applicable universally in the population due to a lack of HLA-E genetic polymorphism; the HLA-E locus encodes two non-synonymous functional allelic variants HLA-E*01:01 and HLA-E*01:03 that differ by a single Arg or Gly amino acid residue, respectively, at position 107, resulting in largely overlapping peptide binding repertoires (O’Callaghan et al., 1998) (Strong et al., 2003).
  • Minimal allelic polymorphism and shared peptide binding repertoires position HLA-E as a particularly attractive restriction element for T cell-targeted vaccination strategies which could potentially offer universal, MHC class I allotype-unrestricted protection.
  • TCRs and/or monoclonal antibodies specific for HLA-E in complex with a peptide antigen can be generated and used therapeutically as cytotoxic reagents, or such antibodies and TCRs could manipulated as receptors, including chimeric receptors, which are transfected or transduced into effector cells to induce immune responses against the peptide antigen. Therefore, the generation of antibodies or T cells and B- cells which recognise HLA-E bound to peptide antigens derived from a cancer, pathogen or even autoantigens has considerable therapeutic potential.
  • a multistep process is necessary to identify peptides that bind to HLA- E, then identify whether such HLA-E:peptide complexes can be recognised by a T-cell, such as a CD8+ T-cell, and/or a B-cell, which may induce an immune response such as a CD8+ T cell response or a B lymphocyte-mediated antibody response, in vitro or in vivo in humans or in animal models.
  • a T-cell such as a CD8+ T-cell
  • B-cell which may induce an immune response such as a CD8+ T cell response or a B lymphocyte-mediated antibody response
  • a method of identifying a peptide which is capable of forming an HLA-E:peptide complex and being recognised by a T-cell and/or B-cell comprising the steps of:
  • step (b) Stratifying the peptide of interest into one of the following groups based on its relative binding determined in step (a):
  • a peptide of interest stratified into group (ii) in step (b) may be further stratified into one of the following groups based on its relative binding determined in step (a):
  • the method may further comprise performing one or more step to improve the stability and/or level of binding of the HLA-E:peptide complex comprising the peptide of interest.
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may be performed for step (c) of the method.
  • the reference peptide referred to herein may be a VL9 peptide (VMAPRT[V/L][L/V/I/F]L), such as VMAPRTLVL (SEQ ID NO: 2), VMAPRTVLL (SEQ ID NO: 3), VMAPRTLLL (SEQ ID NO: 4), VMAPRTLIL (SEQ ID NO: 5), or VMAPRTLFL (SEQ ID NO: 6).
  • VMAPRTLVL SEQ ID NO: 2
  • VMAPRTVLL SEQ ID NO: 3
  • VMAPRTLLL SEQ ID NO: 4
  • VMAPRTLIL SEQ ID NO: 5
  • VMAPRTLFL SEQ ID NO: 6
  • a HLA-E:peptide complex referred to herein refers to a complex comprising or consisting of a peptide, HLA-E heavy chain and B2 microglobulin.
  • the reference HLA- E:peptide complex referred to herein may refer to a complex comprising or consisting of a VL9 peptide, HLA-E and B2 microglobulin.
  • the HLA-E may be HLA-E*01:01 or HLA-E*01:03.
  • the HLA-E may be mutated, for example at the residues described herein.
  • the HLA-E may refer to human HLA-E or a homologue from a non-human species, such as mouse or Rhesus monkey.
  • step (a) of the method allows the level of binding of a peptide of interest to HLA-E in an HLA-E:peptide complex to be assessed, relative to that of a reference peptide such as VL9, known to bind HLA-E strongly in an HLA-E:peptide complex.
  • the peptide of interest can be given a numerical value relative to VL9 (where VL9 binding is 100%), and this allows the stratification of peptides of interest into different groups.
  • the level of binding may be defined as the percentage of HLA-E:peptide complexes formed with the peptide of interest, versus complexes formed with the reference peptide, VL9.
  • Such stratification can be used to determine whether one or more step needs to be performed to improve the stability of the HLA-E:peptide complex comprising the peptide of interest, and if so, the nature of the step or steps required for a peptide of interest stratified into a given group.
  • the inventors have shown that many immunodominant peptides which bind to HLA-E, particularly peptides derived from pathogens and cancer derived epitopes, bind surprisingly weakly in an ELISA when compared to VL9. Biological investigation of such peptides in the pursuit of therapeutics such as T-cells and antibodies targeting such pathogens and cancers, and indeed infected or malignant cells, is difficult as current techniques rely on biological functional assays which utilise stable HLA-E:peptide complexes and multimers of such complexes.
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may not need to be performed.
  • a peptide of interest which binds to HLA-E in a HLA-E:peptide complex at more than about 70% of the level of binding relative to the reference peptide will be stable enough to progress to biological functional biological assays without further stabilisation.
  • One or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may still be performed for a peptide of interest stratified into group (i), if desired.
  • step (b) If in the assay of step (b) the peptide of interest is stratified into group (ii), one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may be performed.
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may comprise one or more of:
  • HLA-E Introducing one or more crosslink between amino acids in HLA-E;
  • D Introducing one or more mutation in HLA-E, such as one mutation, two mutations or three mutations, to increase the stability of the binding of a peptide of interest in the HLA-E:peptide complex; or
  • HLA-E in any of (A)-(D) may refer to human HLA-E, which may be or be derived from HLA-E*0101/ E*0103
  • WSDSAQGSESHSL (SEQ ID NO: 8), which are the amino acid sequences of HLA- E*0101 and E*0103 respectively without an N-terminal signal sequence.
  • Mamu-E may optionally be mutated to enhance peptide binding, stability or reactivity with monoclonal antibodies such as the anti -HLA-E antibody 3D 12.
  • Mamu-E comprises an almost identical peptide binding groove to HLA-E. However, Mamu-E possesses structural differences outside of the peptide binding groove which result in a more stable complex, and allowing the binding of more peptides.
  • Mamu-E may be used as an alternative to HLA-E in the Single Chain Trimer assay described herein. This enables the identification of yet further peptides which may be used as epitopes with HLA-E.
  • Mamu-E may comprise one or more of the following mutations: P57S, E79R, and/or G150A.
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may comprise:
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may comprise one or more of:
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest may comprise:
  • (A) may be performed in the presence of a molar excess of the peptide of interest, such as at IOOmM or more, to ensure stability of the complex.
  • the sensitive reference peptide may be UV-sensitive.
  • the UV-sensitive reference peptide may be a UV- sensitive VL9 peptide, such as VMAP(J*)TLVL (SEQ ID NO: 9), where J* is 3-amino- 3-(2-nitrophenyl)-propionic acid.
  • (A) may be performed in the presence or absence of UV light.
  • the crosslinking one or more amino acid of the peptide of interest to one or more amino acid of HLA-E may comprise: mutating one or more residues of HLA-E to a cysteine or lysine, and optionally substituting one or more residues of the peptide of interest, to a cysteine, lysine or synthetic amino acid, such that a crosslink is capable of being formed between the peptide of interest and HLA-E. This may involve the use of an additional small molecule to bridge the amino acids to be crosslinked. This may involve the formation of a salt bridge between oppositely charged amino acids of the peptide and HLA-E.
  • the substituting one or more residues of the peptide of interest may comprise substituting the residue in the first or second position of the peptide of interest to a cysteine, a homocysteine, or a synthetic amino acid comprising a free sulphydryl group, such that a crosslink in the form of a disulphide bond can be formed between a mutant amino acid in the HLA-E heavy chain and the amino acid at the first or second position in the peptide.
  • the crosslinking one or more amino acid of the peptide of interest to one or more amino acid of HLA-E may comprise mutating the tyrosine at position 84 of HLA-E to a cysteine, adding a glycine and cysteine to the carboxy terminus of the peptide of interest, and forming a disulphide bond between the cysteine at position 84 of HLA-E and the cysteine added to the carboxy terminus of the peptide of interest.
  • the crosslinking one or more amino acid of the peptide of interest to one or more amino acid of HLA-E may comprise mutating the methionine at position 45 of HLA-E to a cysteine, substituting the amino at position two of the peptide of interest to a cysteine, a homocysteine, or a synthetic amino acid that displays a free sulphydryl group (preferably at the end of a side chain of preferred length), and forming a disulphide bond between the cysteine at position 45 of HLA-E and the cysteine, homocysteine, or a synthetic amino acid at position two of the peptide of interest.
  • the synthetic amino acid comprising a free sulphydryl group may be a homocysteine analogue, (2S)-2-amino-5-sulfanylpentanoic acid or (2S)-2-amino-6 sulfanylhexanoic acid.
  • the one or more crosslink between amino acids in HLA-E may be introduced by mutating the tyrosine at position 84 of HLA-E to a cysteine, mutating the alanine at position 139 of HLA-E to a cysteine, and forming a disulphide bond between the two cysteine residues at position 84 and 139 of HLA-E.
  • This crosslink may improve the binding of a peptide of interest in the HLA-E:peptide complex, demonstrated by increased Tm ( Figure 8).
  • the one or more mutation in HLA-E may comprise or consist of one or more of, such as one of, two of, or all of, mutating Histidine at position 99 to Tyrosine, mutating Phenylalanine at position 116 to Tyrosine or mutating Serine at position 147 to Tryptophan.
  • These mutations close/alter different HLA-E binding pockets which are used optimally by the signal peptide VL9. This is in contrast to many low affinity pathogen or cancer-derived peptides that bind to HLA-E.
  • These mutations enhance the binding of the pathogen or cancer-derived peptides, demonstrated by increased melting temperature (Tm) of the protein ( Figures 9,11 and 12).
  • the one or more mutation in HLA-E may comprise swapping the alpha-3 domain of HLA-A3 with that of HLA-E.
  • the invention provides a mutant HLA-E heavy chain comprising one or more mutation which permits the formation of a HLA-E:peptide complex with increased stability when compared to the complex without the mutant HLA-E heavy chain.
  • the complex may further comprise b2 microglobulin.
  • the mutant HLA-E may comprise a mutation at Histidine at position 99 to Tyrosine.
  • the mutant HLA-E may comprise a mutation at Phenylalanine at position 116 to Tyrosine.
  • the mutant HLA-E may comprise a mutation at Serine at position 147 to Tryptophan.
  • the mutant HLA-E may comprise a mutation at Histidine at position 99 to Tyrosine and a mutation at Phenylalanine at position 116 to Tyrosine.
  • the mutant HLA-E may comprise a mutation at Histidine at position 99 to Tyrosine and a mutation at Serine at position 147 to Tryptophan.
  • the mutant HLA-E may comprise a mutation at Histidine at position 99 to Tyrosine, a mutation at phenylalanine at position 116 to Tyrosine, and a mutation at Serine at position 147 to Tryptophan.
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest referred to herein may also be utilised in any assay referred to herein.
  • the peptide of interest may be identified using one or more amino acid sequence prediction process.
  • a peptide of interest may be identified as comprising a HLA-E restricted sequence motif (Walters et al., 2020 EJI).
  • a peptide of interest may be identified using one or more publicly available programme such as NetMHC, or using an algorithm.
  • a peptide of interest may be identified by using one or more of the following search parameters: peptide is a nonamer; peptide comprises one or more proline between positions 3 and 7, peptide comprises a restricted set of amino acids at positions 2 and 9, such as an M, L, V A, Q, or F at position 2, and/or a L, F, I, V or M at position 9.
  • the method may further comprise, before step (a), performing an initial screening assay to determine whether a HLA-E:peptide complex comprising the peptide of interest is capable of being expressed on a cell surface, wherein if the HLA-E:peptide complex comprising the peptide of interest is determined to be capable of being expressed on a cell surface, the method proceeds to step (a).
  • the initial assay may comprise transfecting into a cell, such as HEK293T cells, a nucleic acid encoding a polypeptide comprising the peptide of interest, B2microglobulin and HLA-E heavy chain, in that order.
  • a polypeptide is also referred to herein as a
  • the nucleic acid may also encode a linker sequence between each of the peptide of interest, B2microglobulin and HLA-E heavy chain, preferably in that order.
  • the initial assay may comprise performing an experimental technique to detect the presence of the peptide of interest bound to HLA-E at the surface of the cell which expresses the SCT.
  • Many experimental techniques are known to the skilled person in the art, such as flow cytometry, and microscopy such as confocal microscopy.
  • the experimental technique may utilise a conformation-specific antibody, which recognises a correctly folded HLA-E:peptide complex, for example which can be expressed at the cell surface. Such an antibody may recognise a domain in the a3 region of HLA-E.
  • Such an initial screening assay allows the skilled person to quickly, cheaply and reliably assess whether a peptide of interest can be presented on the cell surface by HLA-E. If a peptide of interest is not expressed at the cell surface bound to HLA-E, then the method may terminate at this stage for that peptide of interest. If a peptide of interest is expressed at the cell surface bound to HLA-E, the method may proceed.
  • the one or more step to improve the stability of the HLA-E:peptide complex comprising the peptide of interest referred to herein may also be utilised in the initial screening assay.
  • the assay of step (a) may be an Enzyme-linked immunosorbent assay (ELISA).
  • ELISA Enzyme-linked immunosorbent assay
  • the ELISA may be a sandwich ELISA.
  • the sandwich ELISA assay may utilise an anti HLA-E antibody and an anti B2 microglobulin antibody.
  • the ELISA may be used to semi-quantitatively determine the level of binding of a peptide of interest in a HLA-E:peptide complex, relative to the level of binding of a reference peptide to HLA-E in a reference HLA-E complex.
  • the level of binding of a reference peptide to HLA-E in a reference HLA-E complex may be taken as 100%.
  • the reference peptide may be VL9, described above.
  • the VL9 may be VMAPRTLLL.
  • the HLA-E:peptide complex comprising the peptide of interest used in the ELISA may be formed using a peptide exchange method, in which a reference HLA-E:peptide complex comprising a UV-sensitive reference peptide is first formed, and the reference peptide is subsequently exchanged with the peptide of interest under conditions such that an HLA-E:peptide complex comprising the peptide of interest is formed.
  • the HLA-E heavy chain may be expressed in in E.coli, before being refolded by mixing with B2microglobulin and a UV-labile VL9 peptide, such as VMAP(J*)TLVL where J is 3-amino-3-(2-nitrophenyl)-propionic acid, a synthetic amino acid that is sensitive to UV light.
  • VMAP(J*)TLVL where J is 3-amino-3-(2-nitrophenyl)-propionic acid
  • This protein complex may then be purified, for example by Fast Protein Liquid Chromatography (FPLC) size exclusion, and the protein complex of the expected mass may be collected and analysed.
  • the complex which is either fresh or previously frozen and thawed, may then be mixed with a 100-fold molar excess of the peptide, and optionally exposed to UV light.
  • the ELISA may use the methodology described in Walters et al., EJI, 2020.
  • the assay of step (a) may comprise or consist of performing Thermal melt (Tm) analysis by Differential Scanning Fluorometry (DSF), or Small Angle X-ray Scattering of the material after flowing through the size exclusion column (SEC-SAXS), which purifies the refolded HLA-E after the peptide exchange.
  • Tm Thermal melt
  • SEC-SAXS Small Angle X-ray Scattering of the material after flowing through the size exclusion column
  • Such steps may be performed on fresh or previously frozen material that has been thawed.
  • the inventors have determined that the level of binding of a peptide of interest relative to VL9 binding to HLA-E in the ELISA assay has a strong correlation with the thermal stability of the HLA-E: peptide complex and its overall structural conformation in solution ( Figure 4).
  • binding in the ELISA assay and/or thermal melt determination is predictive of structural stability.
  • the method may further comprise performing one or more further assay after step (b) to determine the biophysical and/or biochemical characteristics of the HLA-E:peptide complex comprising the peptide of interest.
  • the one or more further assays may comprise Native PAGE, Thermal melt (Tm) analysis, and/or) Size Exclusion Chromatography- Small Angle X-ray Scatter (SEC-SAXS) .
  • SAXS small angle X ray scatter
  • one or more biological assay may be performed.
  • the one or more biological assay may use of multimers of the HLA-E:peptide complex comprising the peptide of interest.
  • the multimer may be a tetramer or a pentamer.
  • a multimer described herein may comprise streptavidin or a microbead and may comprise a label such as a fluorochrome.
  • the one or more biological assay may be a FACS assay.
  • the one or more biological assay may comprise introducing the peptide of interest into a co-culture of T-cells and monocytes, in the presence of IL-2 and IL-15.
  • the peptide of interest may then bind to HLA-E on the monocytes and be recognised by one or more T-cell.
  • T-cell and/or B-cell (or the corresponding TCR/BCR) identified in such a biological assay may then be selected, isolated, sequenced and/or cloned.
  • the selected/isolated T-cell and/or B-cell may be expanded.
  • one or more identified T cell may be sorted using a multimer, and seeded at less than one cell per well into multiple wells of a tissue culture plate, and cultured with irradiated allogeneic peripheral blood mononuclear cells (PBMC) as feeders together with IL-2 and phytohemagglutinin (PHA).
  • PBMC peripheral blood mononuclear cells
  • PHA phytohemagglutinin
  • step (c) of the assay it may be also determined whether an HLA-E:peptide complex comprising the peptide of interest is capable of stimulating an immune response in one or more T-cell and/or B-cell.
  • one or more biological assay may be performed.
  • the one or more biological assay may identify whether an HLA-E:peptide complex comprising the peptide of interest is capable of inducing an increase in IFN-g, TNF-a, CD107a/b and/or CD137 expression in T-cells.
  • numerous other activation markers can be used to determine whether an immune response is stimulated in a T-cell.
  • the one or more biological assay may comprise the use of multimers of the HLA- E:peptide complex comprising the peptide of interest to determine whether an immune response in a T-cell and/or B-cell can be stimulated.
  • the multimer may be a tetramer or a pentamer or a dextramer.
  • a multimer described herein may comprise streptavidin or a microbead and may comprise a label such as a fluorochrome.
  • the one or more biological assay may comprise introducing the peptide of interest into a co-culture of T-cells and monocytes, in the presence of IL-2 and IL-15.
  • the peptide of interest may then bind to HLA-E on the monocytes and stimulate an immune response in one or more T-cell.
  • One more T-cell identified as being stimulated in such biological assays may then be selected, isolated, sequenced and/or cloned.
  • the selected/isolated T-cell may be expanded.
  • one or more identified T cell may be sorted using a multimer, and seeded at less than one cell per well into multiple wells of a tissue culture plate, and cultured with irradiated allogeneic peripheral blood mononuclear cells (PBMC) as feeders together with IL-2 and phytohemagglutinin (PHA).
  • PBMC peripheral blood mononuclear cells
  • PHA phytohemagglutinin
  • one or more biological assay may be performed.
  • the one or more biological assay may identify whether an HLA-E:peptide complex comprising the peptide of interest is capable of being bound by a BCR, and/or whether this stimulates the secretion of antibody.
  • the HLA-E:peptide complex comprising the peptide of interest, or a multimer thereof, is capable of stimulating an immune response in one or more T- cell and or B-cell, this is indicative of the T-cell and/or B-cell recognising the HLA- E:peptide complex comprising the peptide of interest.
  • a method of identifying one or more T-cell or B- cell which recognises an HLA-E:peptide complex comprising a peptide of interest comprising:
  • An immune response may be stimulated in a T-cell and/or B-cell upon recognition of the HLA-E:peptide complex comprising the peptide of interest.
  • the assay to identify a T-cell or B-cell which recognises the HLA-E:peptide complex comprising the peptide of interest may comprise isolating T-cells and/or B-cells from a sample of blood or tissue/biological fluid from the site of infection or a tumour, of a healthy subject or from a subject who has been diagnosed with or is suspected of having cancer and/or an infection.
  • One or more T-cell or B-cell which recognise the HLA- E:peptide complex comprising the peptide of interest may then be selected.
  • the subject may be a mammal.
  • the subject may be a human.
  • the method may further comprise performing an assay to determine whether a T-cell or B-cell which is identified by any of the methods described above, is capable of recognising and/or being activated by an abnormal cell.
  • An abnormal cell may be a cell which is infected with a pathogen, a tumour cell, or a cell which is undergoing a stress response, and which comprises the peptide of interest.
  • the stress response may be to one or more of: metabolic stress, physical stress, apoptotic stress, pH stress, osmotic stress, DNA Damage stress, heat stress, hypoxic stress or toxin stress.
  • an identified T-cell or T-cell clone may be exposed to an abnormal cell and an assay performed to determine whether the T-cell is activated.
  • Presence or level of activation markers such as one or more of CD137, CD107a/b, TNF alpha, or interferon-gamma may be used to determine whether the T-cell is activated by contact with the abnormal cell.
  • This step confirms that the peptide of interest is a biologically relevant epitope which is presented on an abnormal cell by HLA-E.
  • This step also confirms that a multimer used for the T cell or B-cell selection displays the same antigen/epitope as is present on the abnormal cell; that is, where one or more step to improve the stability of the HLA- E:peptide complex comprising the peptide of interest has been performed, a resulting T-cell or B-cell identified is also capable of recognising and/or being stimulated by a corresponding natural HLA-E:peptide complex, in which no step to improve the stability has been performed.
  • This step therefore excludes T cells and B-cells that recognise and/or respond to altered forms of the HLA-E (or indeed Mamu-E) multimers which result from on chemical, biochemical or mutational steps used in the one or more step to improve the stability described herein.
  • T cells or B cells, or indeed their TCR or BCR respectively may then be developed as a therapeutic reagent.
  • a resulting TCR and/or BCR may be sequenced and cloned.
  • the nucleic acid encoding a TCR can then be used to generate soluble TCR or can be transfected or transduced into live cells such that they then express the receptor.
  • nucleic acid such as DNA, cDNA or RNA
  • TCRs or antibodies See for example Robbins et al., (2008) J Immunol. 180: 6116-6131.
  • Any method described herein may further comprise generating one or more T-cell or TCR, or antibody such as a monoclonal antibody, which recognises the HLA-E:peptide complex comprising the peptide of interest.
  • the skilled person will know of various methods to generate such an antibody, including immunising an animal such as a mouse such that antibodies which recognise the HLA-E:peptide complex comprising the peptide of interest are generated, or by using the Milstein method.
  • Figure 1 - shows Blue native (BN) gels signatures for pathogen peptide versus canonical leader peptide-loaded HLA-E complexes.
  • 10pg of pre-refolded HLA-E- 2m-UV-sensitive VL9 (VMAP(J*)TLVL) material was incubated with 12M excess of test peptides (VL9, Mtb44, IL9, RL9H, RL9S and BLZF1) or the no added peptide buffer control (Eno pep) for 3 hours on ice prior to Blue Native-PAGETM Novex 4-16% Bis-Tris gel evaluation.
  • Figure 2 - shows size exclusion chromatography-coupled small angle x-ray scattering of peptide-HLA-E complexes A.
  • Ab initio molecular envelope models displayed as small ‘dots’ represent the average conformational protein state adopted in solution and were generated from SEC-SAXS data using the DAMMIF and DAMAVER ATSAS packages in conjunction with ScAtter (Franke & Svergun, 2009) (Volkov & Svergun, 2003).
  • the top row of molecular envelope ‘dot’ models correspond to SEC- SAXS runs where HLA-E complexes were injected onto the HPLC column in the presence of 120mM excess peptide but no excess peptide was added to the HPLC elution buffer.
  • the bottom row correspond to SEC-SAXS runs where 120mM excess peptide was present in both the HPLC injection and elution buffers to ensure continual presence of excess ligand.
  • the purple-shaded molecular envelope ‘dot’ models represent canonical VL9 (VMAPRTVLL)-refolded HLA-E.
  • VMAPRTVLL canonical VL9
  • the structural coordinates of HLA-E in complex with VL9 (VMAPRTVLL) from 1MHE are shaded green and were superimposed onto SEC-SAXS-based molecular envelopes using the SUPCOMB package of ATSAS (O’Callaghan et al., 1998).
  • the neighbouring SEC- SAXS molecular envelope ‘dot’ models represent HLA-E refolded with Mtb44 (RLPAKAPLL) (SEQ ID NO: 10), onto which the previously published HLA-E- Mtb44 structural coordinates (shaded purple) were aligned (Walters et al., 2018).
  • the blue-shaded SEC-SAXS molecular envelope ‘dot’ models represents IL9 (IMYNYPAML)(SEQ ID NO: l l)-refolded HLA-E onto which the HLA-E-IL9 structural coordinates (shaded orange) were aligned.
  • the magenta-shaded SEC-SAXS molecular envelope ‘dot’ models correspond to Mtbl4 (RMAATAQVL)(SEQ ID NO: 12)-refolded HLA-E, with the HLA-E-Mtbl4 structural coordinates superimposed via SUPCOMB.
  • the neighbouring SEC-SAXS molecular envelope ‘dot’ models shaded yellow represent HLA-E refolded with RL9HIV (RMYSPTSIL) (SEQ ID NO: 13), onto which the previously published HLA-E-RL9HIV structural coordinates (shaded pink) were aligned (Walters et al., 2018).
  • Each row represents a SEC-SAXS experiment conducted in the absence or presence of 120mM excess peptide in the HPLC elution buffer.
  • the radius of gyration (Rg) and maximum dimension (dmax), both measured in A, are specified.
  • the volumes of SEC-SAXS molecular envelope ‘dot’ models (A3) displayed above in part A, are also denoted along with the % change in molecular envelope volume following the addition of excess peptide to the HPLC elution buffer.
  • C. (i), (iii), (v), (vii), (ix) & (xi) Log 10 scattering intensity plots for HLA-E SEC-SAXS experiments.
  • Figure 3 - shows size exclusion chromatography-coupled small angle x-ray scattering for peptide-HLA-A2 complexes.
  • A. (i) & (iii) Log 10 scattering intensity plots for HLA-A*02:01 SEC-SAXS experiments. Plotted on the X-axis is the scattering vector, q, measured in A-l, which for small angles is proportional to the scattering angle Q.
  • the scattered intensity, I(q) is plotted on the Y-axis with a log scale. Scattering intensity curves for HLA-A2 refolds run in the absence or presence of 120mM excess peptide in the HPLC elution buffer, are plotted together for reference.
  • Figure 4 - demonstrates that molecular envelope elongation negatively correlates with HLA-E thermal stability and peptide binding signals
  • a positive control VL9 leader peptide (VMAPRTVLL) in addition to a non binding negative control peptide derived from HIV Gag (QAISPRTLN) (SEQ ID NO: 14) are included.
  • the ‘ELISA rank’ column reflects normalised and previously published peptide binding signals which are expressed as percentages of the positive control VL9 signal (Walters et al. 2020).
  • HLA-E- 2M-peptide complex thermal stability is indicated by its melting temperature (°C) in the presence of 12M excess peptide.
  • the SEC-SAXS-obtained dmax value, measured in A, reflects the maximum dimension across the HLA-E complex in solution whereas the ‘Molecular Envelope VoT corresponds to the volume in A3 of the DAMMINF ab initio molecular envelope models presented in Figure 2.
  • the ‘no rescue’ negative control shaded in dark grey reflects a peptide exchange reaction conducted in the absence of excess test peptide.
  • VMAPRTVLL positive control VL9 leader peptide
  • the positive control VL9 leader peptide (VMAPRTVLL) corresponds to a peptide exchange reaction of 120mM excess peptide.
  • Figure 5 - shows structural characterisation of Mycobacterial peptide binding to HLA-E A.
  • Peptide residues are labelled and an electron density map contoured to 1 sigma is displayed in grey mesh overlaying the peptide (ii) Ribbon representation of the Mtbl4 (RMAATAQVL) peptide backbone shaded in hot pink from the HLA-E-Mtbl4 structure determined in this study with a superimposed VL9 (VMAPRTVLL) leader peptide backbone from the previously published HLA-E-VL9 structure, 1MHE, in violet.
  • VMAPRTVLL superimposed VL9 leader peptide backbone from the previously published HLA-E-VL9 structure, 1MHE, in violet.
  • the HLA-E binding groove is omitted and the peptide N and C termini labelled for clarity.
  • the distance (1.9 A) separating the position 7 Ca atom of the IL9 peptides from molecules 1 and 2 of the asymmetric unit is denoted (iv) Superimposed peptide backbones are depicted in ribbon-form with the HLA-E peptide binding groove omitted and N and C termini labelled for clarity.
  • the IL9 (IMYNYPAML) peptide backbones from molecules 1 and 2 of the asymmetric unit are displayed in blue.
  • the VL9 (VMAPRTVLL) leader peptide backbone from the previously published HLA-E-VL9 structure, 1MHE, is also displayed and shaded violet.
  • the associated Hepatitis C virus-derived nonamer (CINGVCWTV)(SEQ ID NO: 15) is shown in pink ribbon with the solvent-exposed position 7 side chain in stick-form projecting away from the E pocket. Visible sections of the HLA-A2 peptide binding groove including the a2 helix and b-sheet floor are shown in grey cartoon (ii) - (v) PyMol visualisation of the peptide position 7 anchor side chain-accommodating E pocket of HLA-E. Side chains of secondary E pocket-forming residues are depicted as grey sticks (Phe-116, Trp-133, Ser-147, Glu-152, Gln-156) with remaining visible regions of the peptide binding groove shown in grey cartoon.
  • VL9 (VMAPRTVLL) peptide mainchain is displayed as purple ribbon with the position 7 side chain in purple stick-form projecting downward into the secondary E pocket (PDB ID: 1MHE, O’Callaghan et al. 1998).
  • PDB ID: 1MHE, O’Callaghan et al. 1998 illustrate differential positioning at position 7 of HLA-E-bound pathogen-derived peptides relative to VL9 from 1MHE.
  • Mtb-derived IL9 IYNYPAML
  • HIV Gag- derived RL9HIV RYSPTSIL
  • Mtb-derived Mtbl4 RAATAQVL
  • peptide backbones are depicted as blue (iii), yellow (iv) and magenta (v) ribbons, respectively.
  • the position 7 side chains - Ala-7 of IL9, Ser-7 of RL9HIV and Gln-7 of Mtbl4 - are shown in blue (iii), yellow (iv) and magenta (v) stick-form, respectively.
  • VL9 peptide main chain from 1MHE is shown in purple ribbon and the distance between the aligned peptide position 7 Ca atoms is indicated by dashed lines - 2 A separates the position 7 Ca atoms of VL9 and IL9, 3.4 A separates the position 7 Ca atoms of VL9 and RL9HIV and 1.1 A separates the Ca atoms of VL9 and Mtbl4.
  • water-mediated intra- inter-chain hydrogen bonds are depicted as magenta dashed lines with the coordinated H20 molecule visualised as a cyan-shaded sphere.
  • a total of 4 hydrogen bonds are shown which indirectly link the Mtbl4 position 7 Gin side chain to the Ser-147 side chain and Ser-143 main chain of the HLA-E a2 -helix in addition to the position 8 main chain of the Mtbl4 peptide.
  • the Val-8 main chain of the Mtbl4 peptide and Ser-143 main chain of HLA-E are labelled ‘MC’ and depicted as magenta and grey sticks, respectively.
  • Tm values are listed for the canonical HLA-B7-derived VL9 leader peptide (VMAPRTVLL) and the Mtb-derived Mtbl4 peptide (RMAATAQVL) in addition to corresponding position 7 variant peptides in which a non-canonical polar Gin was introduced at position 7 in VL9 (VMAPRTQVL) (SEQ ID NO: 16) or a canonical hydrophobic Val was introduced at position 7 in Mtbl4 (RMAATAVVL) (SEQ ID NO: 17).
  • VMAPRTQVL VL9
  • RMAATAQVL Mtb-derived Mtbl4 peptide
  • VMAPRTVLL wild-type VL9 leader peptide
  • the Mtb-derived Mtbl4 peptide (RMAATAQVL) which also contains a non-canonical position 7 Gin was included for reference.
  • D. (i) & (iii) Log 10 scattering intensity plots for HLA-E SEC- SAXS experiments. Plotted on the X-axis is the scattering vector, q, measured in ⁇ -1, which for small angles is proportional to the scattering angle Q. The scattered intensity, I(q), is plotted on the Y-axis with a log scale.
  • Superimposed scattering intensity curves for peptide-HLA-E refolds are colour-coded according to the corresponding figure legend (ii) & (iv) Normalised Kratky plots with superimposed curves from HLA-E SEC-SAXS experiments.
  • Superimposed modulated Gaussian curves are colour-coded according to figure legends in log 10 intensity plots. Plotted on the X-axis of the normalised Kratky plot is the scattering vector multiplied by the radius of gyration. On the Y-axis, the scattering intensity, I(q), is divided by the experiment’s 1(0) and multiplied by (q*Rg)2.
  • Figure 7 - demonstrates that distinct that structural motifs emerge in the absence of HLA-E-associated VL9 leader peptide A.
  • SA short-arm of the a2 helix
  • LA long-arm labelled ‘LA’
  • residue positions denoted The a2 helix from the VL9 (VMAPRTVLL)-associated HLA-E structure, 1MHE, is shaded grey.
  • HLA-E-Mtb44 HLA-E-RL9HIV
  • RYSPTSIL HLA-E-RL9HIV
  • RAATAQVL HLA-E-Mtbl4
  • IMYNYPAML HLA-E-IL9
  • HLA- E-associated peptides Ca backbones of superimposed HLA- E-associated peptides are depicted as ribbons.
  • the canonical VL9 leader peptide (VMAPRTVLL) peptide from 1MHE is shaded grey.
  • Pathogen-derived HLA-E- associated peptides are coloured green (Mtb44 - RLPAKAPLL), yellow (RL9HIV - RMYSPTSIL), magenta (Mtbl4 - RMAATAQVL) and blue (IL9 - IMYNYPAML).
  • Mtb44 - RLPAKAPLL yellow
  • RL9HIV - RMYSPTSIL yellow
  • magenta Mtbl4 - RMAATAQVL
  • blue IL9 - IMYNYPAML
  • Position 5 Ca atoms are circled with the maximum distance separating the VL9 peptide Arg-5 Ca from pathogen-derived peptide position 5 Ca atoms, denoted. 2.3 A separates the position 5 Ca atoms of VL9 and RL9HIV, 1.9 A separates the position 5 Ca atoms of VL9 and IL9, 1.9 A separates the Ca atoms of V9 and Mtbl4 and 1.5 A separates the position 5 Ca atoms of VL9 and Mtb44. C.
  • the HLA-E a2 helix is shown in grey cartoon with superimposed pathogen-derived peptide backbones, RL9HIV (RMYSPTSIL), IL9 (IMYNYPAML) and Mtbl4 (RMAATAQVL) in yellow, blue and magenta ribbon, respectively.
  • the superimposed VL9 leader peptide (VMAPRTVLL) peptide backbone from 1MHE is shown in grey ribbon.
  • Ser-147, Glu- 152 and Gln-156 side chains of the HLA-E a2 helix are shown in stick-form and are colour-coded according the corresponding peptide.
  • the 2 salt bridges connecting Glu- 152 and the Arg-5 side chain of the VL9 peptide are shown as grey dashed lines.
  • a hydrogen bond between the Gln-156 side chain and the main chain Oxygen of VL9 Arg- 5 is also depicted as grey dashed lines (ii)
  • the HLA-E a2 helix is shown in grey cartoon with the Mtb-derived peptide Mtb44 (RLPAKAPLL) peptide backbone (green) superimposed to that of VL9 (VMAPRTVLL) from 1MHE (grey).
  • the HLA-E a2 helix is shown in grey cartoon with the superimposed pathogen-derived peptides, RL9HIV (RMYSPTSIL) and IL9 (IMYNYPAML), in yellow and blue ribbon, respectively.
  • the Tyr-3 side chain of RL9HIV and IL9 is shown in yellow and blue stick-form, respectively.
  • HLA-E a2 helix Glu-152 side chains from the HLA-E-RL9HIV and HLA-E-IL9 structures are shown as yellow and blue sticks, respectively, with corresponding hydrogen bonds depicted as yellow/blue dashed lines.
  • HLA-E-VL9 VMAPRTVLL
  • VMAPRTVLL Glu-152 side chain from the a2 helix of HLA-E-VL9
  • VMAPRTVLL VMAPRTVLL
  • E Table detailing 7 previously published non-receptor-bound HLA-E structures in addition to the 2 novel HLA-E structures presented in this study.
  • HLA-E-associated peptide IDs, organisms of origin and amino acid sequences are specified along with the corresponding HLA-E allelic variant, PDB accession code (AC) and reference.
  • Figure 8 Demonstrates thermal gain of HLA-EC84-C139 over canonical HLA-E when incubated with 100M excess peptide.
  • IOUM of pre-refolded HLA-E and HLA- EC84-C139 material was incubated with 100M excess test peptides (P1-P9) for 30 minutes at room temperature prior to thermal melt analysis using a Prometheus NT.48 Series Differential Scanning Fluorimetry instrument. Test samples were split between two Prometheus NT.48 Series nanoDSF Grade Standard Capillaries and a ramp rate of 1 °C/min from 20 °C to 95 °C was applied.
  • the ratio for fluorescence emission at 330 nm and 350 nm was used to derive the thermal melt of unfolding (Tm). Shown are the relative Tm data for canonical HLA-E (left column) and HLA-EC84-C139 (right column) datasets, where the corresponding no-peptide control Tm data for canonical HLA-E and HLA-EC84-C139 have been subtracted, respectively. The numbers plotted above the red bars denote the equivalent Tm loss/gains obtained for the HLA-EC84- C139 variant over canonical HLA-E.
  • Figure 9 Demonstrates thermal gain of HLA-EW147over canonical HLA-E when incubated with 100M excess peptide.
  • IOuM of pre-refolded HLA-E or HLA- EW147material was incubated with 100M excess test peptides (P1-P9) for 30 minutes at room temperature prior to thermal melt analysis using a Prometheus NT.48 Series Differential Scanning Fluorimetry instrument. Test samples were split between two Prometheus NT.48 Series nanoDSF Grade Standard Capillaries and a ramp rate of 1 °C/min from 20 °C to 95 °C was applied.
  • the ratio for fluorescence emission at 330 nm and 350 nm was used to derive the thermal melt of unfolding (Tm). Shown are the relative control Tm gains for canonical HLA-E (left column) and HLA-EW147 (right column) datasets, where the corresponding no-peptide control Tm data for canonical HLA-E and HLA-EW147 have been subtracted, respectively. The numbers plotted above the red bars denote the equivalent Tm loss/gains obtained for the HLA- EW147variant over canonical HLA-E.
  • Figure 10 - demonstrates that pathogen-derived epitopes drive suboptimal HLA-E complex formation, despite previously reported immunogenicity in vivo A.
  • Rg radius of gyration
  • the x-axis denotes HPLC-eluted x-ray exposed frames across the HLA-E-VL9 protein peak, whereas the Y-axis corresponds to the SAXS scattering intensity signal (ii)
  • Ab initio molecular envelope model displayed as small purple ‘dots’ representing the average conformational state of the leading peak fraction of HPLC-eluted HLA-E refolded with the canonical VL9 leader peptide.
  • This molecular envelope ‘dot’ model corresponds to a SEC-SAXS run where an HLA-E*01:03 refold was injected onto the HPLC column in the presence of 120mM excess VL9 peptide but no excess peptide was added to the HPLC elution buffer.
  • This molecular envelope ‘dot’ model corresponds to a SEC-SAXS run where an HLA-E*01:03 refold was injected onto the HPLC column in the presence of 120mM excess IL9 peptide but no excess peptide was added to the HPLC elution buffer.
  • the structural coordinates for HLA-E-IL9 are aligned to the SAXS-generated molecular envelope and displayed in orange cartoon-form (iii) Same as B. (ii), but for tailing peak fraction (iv) Log 10 scattering intensity plot for HLA-E SEC-SAXS. Plotted on the X-axis is the scattering vector, q, measured in A-l, which for small angles is proportional to the scattering angle Q.
  • the scattered intensity, I(q), is plotted on the Y-axis with a log scale.
  • Scattering intensity curves for HLA-E refolds run in the absence of 120mM excess peptide in the HPLC elution buffer are plotted for the leading and tailing peak fractions of IL9-refolded HLA-E and are colour- coded according to the corresponding figure legend (v) Normalised Kratky plot with superimposed curves corresponding to SEC-SAXS leading and tailing peak fractions for IL9-refolded HLA-E in the absence of 120mM excess peptide in the HPLC elution buffer, colour-coded according to the figure legend in B. (iv).
  • This molecular envelope ‘dot’ model corresponds to a SEC-SAXS run where an HLA-E*01:03 refold was injected onto the HPLC column in the presence of 120mM excess RL9HIV peptide but no excess peptide was added to the HPLC elution buffer.
  • the previously published structural coordinates for HLA-E-RL9HIV (PDB AC: 6GL1) are aligned to the SAXS-generated molecular envelope and displayed in pink cartoon-form (iii) Same as C. (ii), but for tailing peak fraction (iv) LoglO scattering intensity plot for HLA-E SEC-SAXS.
  • the scattered intensity, I(q), is plotted on the Y- axis with a log scale.
  • Scattering intensity curves for HLA-E refolds run in the absence of 120mM excess peptide in the HPLC elution buffer are plotted for the leading and tailing peak fractions of RL9HIV-refolded HLA-E and are colour-coded according to the corresponding figure legend (v) Normalised Kratky plot with superimposed curves corresponding to SEC-SAXS leading and tailing peak fractions for RL9HIV-refolded HLA-E in the absence of 120mM excess peptide in the HPLC elution buffer, colour- coded according to the figure legend in C. (iv). Plotted on the X-axis is the scattering vector multiplied by the radius of gyration.
  • FIG. 11 Thermal gain of HLA-E H99Y , HLA-E f116Y and HLA-E S147W over canonical HLA-E when incubated with 10M excess peptide lOuM of pre-refolded HLA-E and HLA-E H99Y (A), HLA-E F116Y (B), or HLA-E S147W (C) material was incubated with 10M excess test peptides (from panel pA to pG) for 30 minutes at room temperature prior to thermal melt analysis using a Prometheus NT.48 Series Differential Scanning Fluorimetry instrument.
  • Test samples were split between two Prometheus NT.48 Series nanoDSF Grade Standard Capillaries and a ramp rate of 1 °C/min from 20 °C to 95 °C was applied. The ratio for fluorescence emission at 330 nm and 350 nm was used to derive the thermal melt of unfolding (Tm). Shown are the relative control Tm gains for canonical HLA-E (left columns) and HLA-E H99Y , HLA- E f116Y or HLA-E S147W (right columns) datasets, where the corresponding no-peptide control Tm data for canonical HLA-E and HLA-E H99Y , HLA-E f116Y or HLA-ES147W have been subtracted, respectively. The numbers plotted above the right-hand columns denote the equivalent Tm gains obtained for the HLA-E H99Y , HLA-E f116Y orHLA-E sl47W variants over canonical HLA-E, respectively.
  • Figure 12 Thermal stability and Blue Native gel analysis of canonical HLA-E and versus single, double and triple HLA-E mutants refolded with RL9HIV peptide.
  • Example 1 Distinct blue native gel signatures for VL9- versus pathogen epitope- associated HLA-E
  • Example 2 SEC-SAXS reveals striking peptide-dependent differences in the HLA- E conformational ensemble Given that multiple distinct gel band signatures were identified in BNG analyses indicative of sample non-uniformity, size exclusion chromatography-coupled small angle x-ray scattering (SEC-SAXS) was used to further probe potential discrepancies in the protein conformational ensemble for canonical VL9 versus pathogen peptide- associated HLA-E. Individual HLA-E*01:03 protein refolds were assembled for each HLA-E-restricted pathogen-derived peptide present in the aforementioned ‘intermediate’ binding panel in addition to the positive control VL9 peptide, VMAPRTVLL.
  • SEC-SAXS size exclusion chromatography-coupled small angle x-ray scattering
  • SAXS The resolution limits of SAXS are inferior relative to those of x-ray crystallography making it challenging to decipher local, fine-tuned structural movement with SAXS data alone.
  • SAXS is capable of detecting dynamic protein folded states and large conformational adjustments of proteins in solution (Kikhney and Svergun, 2015).
  • HLA-E-restricted epitopes Hansen et al., 2016; Walters et al., EJI, 2020
  • SAXS immunodominant SIV epitope
  • EKQRESREK immunodominant SIV epitope
  • Tm DSF-generated melting temperatures
  • the negative control peptide (HIV Gag, QAISPRTLN) generated a comparable Tm (31.8°C) to the peptide-free-HLA-E background (32.0°C) whereas Mtb44 (RLPAKAPLL) generated a comparable Tm (50.6°C) to the positive control VL9-incubated HLA-E (49.4°C).
  • the remaining ‘intermediate’ binding peptides generated Tm values ranging from 35.2°C (RL9SIV, RMYNPTNIL) to 40.7°C (IL9, IMYNYPAML).
  • ELISA signals for the positive control VL9 (VMAPRTVLL) and Mtb44 (RLPAKAPLL) peptides remained significantly higher than those generated by the ‘intermediate’ pathogen-derived peptides IL9 (IMYNYPAML), Mtbl4 (RMAATAQVL), RL9HIV (RMYSPTSIL), BZLF1 (SQAPLPCVL) and RL9SIV (RMYNPTNIL), even when such ‘intermediate’ binding pathogen peptides were supplied at 1.2mM concentrations.
  • Such observations not only demonstrate the tiny selectivity of HLA-E for its canonical VL9 leader peptide but also reveal the suboptimal nature of diverse pathogen-derived epitope sampling by HLA-E.
  • Tm values and normalised ELISA signals exhibited strong negative correlations with measuremets obtained via SEC-SAXS analyses including the maximal linear dimension (as indicated by dmax values measured in A) and molecular envelope volume (as indicated by ab initio model volume measured in A3) of HLA-E complexes in solution ( Figure 4, B, i-iv).
  • Tm and ELISA signals increase, the average conformation of the protein ensemble in solution contracts and more closely aligns to previously obtained HLA-E crystallographic coordinates.
  • Tm thermal stability of peptide-HLA-E complexes
  • HLA-E*01:03 heavy chain and b2M light chain were crystallised in complex with the HLA-E-restricted Mycobacterial epitopes, IL9 (IMYNYPAML) and Mtbl4 (RMAATAQVL) (McMurtrey et al., 2017a) (Joosten et al., 2010).
  • HLA-E-IL9 (IMYNYPAML) crystals diffracted to 1.7 A whereas diffraction data was collected to 2.05 A for HLA-E-Mtbl4 (RMAATAQVL).
  • RMAATAQVL HLA-E-IL9
  • Mtbl4 (RMAATAQVL) adopted the classical conformation also adopted by canonical VL9, with buried termini and a solvent-exposed central kink at residues 4 & 5 ( Figure 5, A).
  • the less constrained central kinked region of Mtbl4 displayed the greatest movement relative to VL9 with 1.6 and 1.7 A separating Ca atoms at positions 4 and 5, respectively, resulting in the central portion of the Mtbl4 backbone leaning closer toward the HLA-E al helix.
  • the alternative IL9 peptide configuration features a position 7 Ca atom that projects 2.0 A further toward solvent relative to VL9 (VMAPRTVLL) and a position 7 secondary anchor side chain which projects away from its corresponding E pocket in the HLA-E groove.
  • HLA-E-IL9 INPYNYPAML
  • HLA- E-Mtbl4 RMAATAQVL
  • RYSPTSIL HLA-E-RL9HIV
  • the secondary E pocket of HLA-E comprises a deep hydrophobic recess with a more defined pocket-like aspect in contrast to classical MHC class la molecules which contain a large, highly conserved E pocket-occluding Trp side chain at position 147 ( Figure 6, A, i).
  • the E pocket of HLA-E classically accommodates a downward-projecting medium sized hydrophobic side chain at position 7 of the bound peptide, such as the highly conserved Val or Leu present in VL9 variants ( Figure 6, A, ii).
  • the unusual conformational dimorphism of the IL9 (IMYNYPAML) peptide primarily involves position 7 with the position 7 Ca atom projecting 2 A further toward solvent relative to canonical VL9 in one of the two observed peptide configurations in the asymmetric unit.
  • HLA-E-associated peptides including IL9 commonly participate in crystal packing interfaces, such structural polymorphism may reflect weak tethering of the small position 7 Ala side chain of IL9 to the deep secondary E pocket of HLA-E, in turn rendering it susceptible to crystal packing-induced repositioning.
  • a polar, non-canonical Gin at position 7 of Mtbl4 (RMAATAQVL) is buried within the hydrophobic E pocket and forms water- mediated hydrogen bonds to Ser-143 and Ser-147 of the HLA-E a2 helix with resulting entropic penalties ( Figure 6, A, V).
  • Combined mutagenesis and ELISA-based HLA-E peptide binding or thermal melt assays support a major contribution of the E pocket to HLA-E complex stability; the introduction of a canonical position 7 Val in place of the polar Gin in Mtbl4 resulted in a 36% increase in the ELISA-based binding signal and a 3.6°C increase in thermal stability (Figure 6, B & C).
  • VMAPRTVLL a polar Gin at position 7 of VL9
  • Example 6 Unique structural features distinguish canonical VL9 versus pathogen peptide-associated HLA-E Comparative structural analyses revealed a distinct configuration located in the a2 -helical kink region which distinguishes VL9-associated versus non-VL9-bound HLA-E ( Figure 7, A).
  • Mtb44 was the only pathogen peptide which exhibited comparable binding to VL9 in previous ELISA-based screens in addition to being the only pathogen peptide with partially preserved position 5 hydrogen bonding, perhaps eluding to the importance of centrally- positioned intermolecular bonds for HLA-E complex stability (Walters, McMichael and Gillespie, 2020).
  • HLA-E complex stability Wang, McMichael and Gillespie, 2020.
  • the loss of the hydrogen bond between Gln-156 and the VL9 position 5 main chain results in Gln-156 side chain repositioning of up to 2.2 A ( Figure 7, C, i).
  • the Glu-152 side chain in pathogen peptide-bound HLA-E structures projects up to 4 A further toward the N-terminus of the peptide binding groove relative to Glu-152 in VL9-bound HLA-E, which in turn facilitates novel inter chain hydrogen bonding with the position 3 Tyr side chains present in IL9 (IMYNYPAML) and RL9HIV (RMYSPTSIL) peptides ( Figure 7, D).
  • IL9 IL9
  • RYSPTSIL RL9HIV
  • Figure 7, D A conserved position 3 Tyr side chain orientation with clear electron density is adopted by both peptides, in which the superposed position 3 Tyr side chains project toward the HLA-E a2 -helix and form a novel hydrogen bond with the re-orientated Glu-152.
  • Glu-152 side chain orientation present in HLA-E-VL9 structures prohibits such hydrogen bond formation as it is positioned 5.5 A from the position 3 Tyr side chain present in the RL9HIV (RMYSPTSIL) or IL9 (IMYNYPAML) peptides.
  • RYSPTSIL RL9HIV
  • IL9 IL9
  • canonical VL9 peptides contain a highly conserved Ala at position 3 which projects downward into the shallow secondary D pocket, these data help redefine what can be stably accommodated at the secondary anchor position 3 of non-VL9 HLA-E binding peptides.
  • compensatory, non-pocket-based peptide- HLA-E hydrogen bonding can permit secondary anchor side chain accommodation when a lack of shape complementarity for the corresponding pocket prohibits peptide side- chain occupancy.
  • Example 7 - single mutations or a combination of H99Y, F116Y and S147W mutations dramatically improve the binding of the RL9HIV peptide to HLA-E
  • Pathogen-derived peptide HLA-E complexes including RL9SIV RMYNPTNIL, RL9HIV RMYSPTSIL, BZLF1 SQAPLPCVL, Mtbl4 RMAATAQVL and IL9 IMYNYPAML yielded conformationally heterogeneous populations and elongated molecular envelopes with average dimensions exceeding those of superimposed crystallographic coordinates.
  • crystal structures of IL9-, Mtbl4- and RL9HIV-bound HLA-E likely represent a subpopulation of the heterogeneous conformational ensemble observed in SEC-SAXS analyses which is also signified by the faint compact band observed in blue native gel analyses.
  • differential local structural features were identified and may be linked to complex instability and elongated molecular envelopes observed in solution.
  • IL9, Mtbl4 and RL9HIV peptides contain the canonical primary anchor Met at position 2 and Leu at position 9 which optimally occupy the primary B and F pockets, respectively.
  • Trp- 147 is highly conserved among classical MHC class I molecules with its bulky side chain largely occluding the E pocket whereas the smaller Ser-147 present in HLA-E and its murine and rhesus counterparts, Qa-1 and Mamu-E, results in a deep hydrophobic recess with a discrete pocket-like nature (O’Callaghan et al., 1998).
  • Another structural feature which distinguishes pathogen peptide- versus canonical VL9- associated HLA-E is a differentially positioned a2 helical kink region.
  • Glu-152 is a common TCR-interacting residue and Glu-152 to Ala mutation results in a > 10-fold reduction in CD94/NKG2A binding REF, its exclusive repositioning in structures of HLA-E lacking associated leader peptide could signal loss of VL9 to both HLA-E-restricted CD8+ T cells and NK cells, respectively, in an innate-like manner that is not wholly dictated by peptide-specific interactions (Sullivan et al., 2007). Accordingly, semi-invariant Qa-1 -restricted lymphocytes with a common Va recognised non-Qdm peptides presented by Qa-1 in a TAP-independent manner (Doorduijn et al., 2018).
  • Innate-like T cell recognition has previously been reported for other unconventionally-restricted subsets such as semi-invariant natural killer T (iNKT) cells restricted by CD Id and mucosal-associated invariant T (MAIT) cells restricted by MR1 (Cotton et al., 2018). Further, a number of structural studies have demonstrated multiple distinct modes of CD 1 -restricted TCR recognition which transcend the epitope/antigen-presenting-molecule co-recognition paradigm with minimal direct contact between the TCR and CD 1 -associated lipid antigen (Birkinshaw et al., 2015) (Wun et al., 2018).
  • canonical VL9 peptides contain a highly conserved Ala at position 3 which projects into the shallow secondary D pocket, Glu-152 repositioning and resultant hydrogen bond formation with the position 3 Tyr side chain of HLA-E-bound pathogen peptides, IL9 and RL9HIV, redefines what can be stably accommodated at the secondary anchor position 3 of non-VL9 HLA-E binding peptides - such distinct intermolecular hydrogen bonding appears to compensate for a lack of anchor side chain shape complementarity with its corresponding D pocket.
  • a VL9-prevalent MHC- E-presented ligandome in healthy cells would in turn maintain inhibitory NK cell interactions through CD94/NKG2A engagement, whereas an abundance of alternative HLA-E-restricted peptides with comparable or higher binding affinity than VL9 could conceivably disrupt this immunoregulatory interaction. Consistent with this, the inventors have demonstrated that more diverse peptide sampling by HLA-E, in settings where MHC class I trafficking pathways are disrupted - such as in Mtb infection or following RhCMV 68-1 vaccination - is largely suboptimal.
  • This non-optimal HLA-E peptide binding repertoire yields unstable, heterogeneous protein populations which at the structural level, share altered conformational adjustments relative to HLA-E-VL9 involving distinct a2-helical kink configurations that are likely to impact TCR and CD94/NKG2 class receptor recognition.
  • the inventors have demonstrated an optimised process to identify peptide epitopes presented by HLA-E on normal and abnormal cells and recognised by specific T cells and/or B-cells. Similarly, optimised methods to generate stable HLA-E-peptide complexes that can be used to make multimers to detect antigen specific T and B cells are demonstrated.
  • Methods are described to identify which peptides bind well, moderately or poorly to HLA-E, and then apply biochemical methods to enhance peptide binding to HLA-E, such as mutational methods to enhance peptide binding to HLA-E, chemical cross linking to enhance peptide binding, use of mutated Mamu-E to enhance peptide binding and to allow use of Mamu-E multimers as surrogates for HLA-E-peptide multimers. Additionally, the inventors demonstrate efficient validation of the epitopes identified by selecting, cloning and testing T cells/ B-cells specific for HLA-E-peptide complex presented on abnormal cells, thereby validating the therapeutic potential of those identified peptides, otherwise overlooked in conventional screening.
  • Peptides were purchased as lyophilised powder at >85% purity from Genscript USA prior to reconstitution in DMSO (200mM) and storage at -80 °C.
  • a UV-labile peptide based on the HLA-B leader peptide (VMAPRTLVL) with a 3-amino-3-(2-nitrophenyl)- propionic acid residue (J residue) substitution at position 5 was synthesised by Dris Elatmioui at LUMC The Netherlands, for use in peptide binding assays.
  • HLA-E*01:03 protein refolds were assembled in the traditional macro-refolding buffer for MHC class I molecules comprising 100 mM Tris pH8.0, 400mM L-arginine monohydrochloride, 2mM EDTA, 5mM reduced glutathione and 0.5mM oxidised Glutathione, prepared in MiliQ water.
  • 2-Microglobulin in Urea-Mes was initially refolded for 30 min at 4°C at a final concentration of 2 mM.
  • Test peptide was subsequently added to the refold at a concentration of 30-60 pM followed by HLA- E*01:03 heavy chain which was pulsed into the refolding buffer to reach a final concentration of 1 pM.
  • Refolds were subject to a 72 hour incubation period at 4°C prior to filtration through 1.0 pM cellular nitrate membranes to ensure the removal of aggregated material. Refolds were concentrated by a VivaFlow 50R system and VivaSpin Turbo Ultrafiltration centrifugal devices, both with 10 kDa molecular weight cut-offs. Refolded and concentrated material was used at a concentration of lOmg/mL in SEC-SAXS experiments without subsequent chromatographic separation.
  • Refolded and concentrated material intended for ELISA-based HLA-E peptide binding assays, DSF or crystallisation screening was subject to subsequent fast protein liquid chromatography (FPLC) size separation on an AKTA Start System using a Superdex S75 16/60 column.
  • FPLC fast protein liquid chromatography
  • HLA-E protein complex peaks were eluted into 20 mM Tris pH8, 100 mM NaCl and discriminated from non-associated b2M and large misfolded aggregates via elution profile visualisation by UV absorbance at 280 mAU.
  • FPLC- purified protein peaks fractions were combined and concentrated to a desired concentration for subsequent experiments using 10 kDa cut-off VivaSpin Turbo Ultrafiltration centrifugal devices - the final protein concentration was obtained by measurement of the absorbance at 280nm using a NanoDrop ND-1000 Spectrophotometer.
  • the composition of eluted protein samples was also analysed by non-reducing SDS-PAGE electrophoresis on NuPAGETM 12% Bis-Tris protein gels to demonstrate the presence of non-aggregated HLA-E heavy chain and b2M.
  • HLA-E- 2m complexes previously refolded with the UV-sensitive VL9 peptide were incubated in the presence of molar excess test peptide and evaluated via the Blue Native-PAGETM Novex Bis-Tris gel system (life technologies) (Walters et al., 2018b).
  • pre-refolded and purified HLA-E in complex with the UV-sensitive peptide was incubated at RT in the presence of 12 M excess test peptide prior to the addition of 3 pL 4x Native-PAGETM Sample Buffer per 10 pg (10 pL) of sample.
  • HLA-E- 2M-peptide refolds were assembled in the L-Arginine-Tris macro refolding buffer according to the protein refolding method detailed above and incubated for 72hrs at 4°C prior to concentration with the VivaFlow 50R system with a lOkDa molecular weight cut-off (Sartorius).
  • HPLC high-performance liquid chromatography
  • HPLC elution buffers corresponded exactly to the L- Arginine Tris pH 8 macro-refolding injection buffer minus the protein components - although 60pM or 120pM excess peptide was added to the elution buffer for certain SEC-SAXS experiments.
  • SEC-SAXS data were collected at Diamond Light Source Beamline B21 and images were taken every 3 seconds of X-ray-exposed HPLC-purified material over the course of a 32 minute elution period. Scattering data were circularly integrated prior to buffer subtraction followed by Guinier fitting and pairwise distribution function calculations, which were performed in the SAXS-dedicated software, ScAtter, developed by Robert Rambo (Franke et al., 2017).
  • Peptide exchange ELISA-based HLA-E peptide binding assays were conducted according to a previously published method that was developed and optimised by the inventors. Peptide exchange micro-reactions were assembled in the traditional macro refolding buffer for MHC class I molecules comprising 100 mM Tris pH8.0, 400 mM 1- arginine monohydrochloride, 2 mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxidized Glutathione, prepared in MiliQ water.
  • Peptide exchange reactions were subsequently interrogated by sandwich ELISA.
  • 96- well ELISA plates were coated in lOmg/mL 3D12, an anti-human HLA-E capture antibody prior to a 12 h incubation period at 4°C.
  • ELISA plate wells were blocked with 300 pL of 2% IgG-free BSA for 2 h at RT. Blocked wells were washed five times in 0.05% Tween-based ELISA wash buffer (BioLegend) followed by a single wash in PBS prior to the addition of 50 pL of peptide exchange reaction diluted 1: 100 in 2% BSA to each well.
  • ELISA plates containing peptide exchange reaction samples were incubated for 1 h at RT and subsequently washed in 0.05% Tween-based ELISA wash buffer and PBS.
  • a polyclonal anti-human b2M HRP- conjugated IgG detection antibody (ThermoFisher Scientific) was diluted 1:2500 in 2% BSA and 50pL added to each ELISA well.
  • ELISA plates were incubated in the dark for 30 min prior to wash steps in a 0.05% Tween-based ELISA wash buffer and PBS.
  • TMB 3,3',5,5'-tetramethyl benzidine
  • Pearson product-moment correlation coefficients were calculated for DSF-determined melting temperatures and normalised ELISA-based peptide binding signals to establish the congruity between these techniques. Additionally, linear correlations were identified between melting temperatures or normalised ELISA-based signals and various SEC-SAXS parameters including the maximal dimensions of the average conformation in solution (dmax) or the volume of the DAMMIF ab initio molecular envelope model (A3).
  • Crystals were cryopreserved in 25% glycerol by Dr. Karl Harlos of Oxford University and diffraction data were collected at the Diamond Light Source, beamlines i04 (HLA-E-Mtbl4 structure) and i03 (HLA-E-IL9 structure). Diffraction data were auto-indexed by Xia2 DIALS. Since the outer shell CCl/2 exceeded the minimum threshold (>0.3) for both datasets, no reflections were excluded from downstream analysis. A more conservative data truncation approach according to the Rmerge and I/sigma cut-offs has been shown to result in the elimination of useful data which would otherwise have contributed to model quality (Karplus and Diederichs, 2012).
  • thermostability of canonically refolded HEA-E-b2ih peptide complexes and C terminus extended peptides with a cysteine refolded with HLA-E containing a tyrosine to cysteine mutation was determined by heat-induced fluorescent dye incorporation, using the commercially available Protein Thermal Shift Dye KitTM (Applied Biosystems). 5 pg of test HEA-E-b2ih complexes was aliquoted into 0.1 mL MicroAmp Fast Optical 96-well plates containing pre-mixed Protein Thermal Shift Dye and Protein Thermal Shift Buffer.
  • Sample buffer (either PBS or Tris pH8, 100 mM NaCL) was added to achieve a final volume of 20 pL.
  • Control samples reconstituted with buffer were prepared to monitor background fluorescent signal. Both samples and controls were set up in quadruplicate.
  • Thermal-driven dye incorporation was measured on an Applied Biosystem Real-Time 7500 Fast PCR System. Data was collected over a temperature ramp ranging from 25 to 95 °C, with 1 °C intervals. Melt curve data were analysed using Protein thermal Shift Software vl.3, and median Derivative Tm values (°C) are reported.
  • Excitation power was pre-adjusted to obtain between 8000 and 20,000 Raw Fluorescence Units for fluorescence emission at 330 nm and 350 nm.
  • a thermal ramp ranging from 20 °C to 95 °C, at a rate of 1 °C/min, was applied.
  • Automated thermal melt data calling was generated by the analysis software within PR.ThermControl, (version 2.1.5) software.

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Abstract

L'invention concerne des méthodes d'identification d'un peptide qui est capable de former un complexe HLA-E:peptide et d'être reconnu par un lymphocyte T et/ou un lymphocyte B, et des méthodes d'amélioration de la stabilité du complexe HLA-E:peptide. En outre, l'invention concerne des méthodes d'identification d'un ou plusieurs lymphocytes T ou lymphocytes B qui reconnaissent un complexe HLA-E:peptide.
EP22729763.7A 2021-06-01 2022-05-31 Criblage peptidique Pending EP4348255A1 (fr)

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GBGB2107821.7A GB202107821D0 (en) 2021-06-01 2021-06-01 Peptide screen
GBGB2119143.2A GB202119143D0 (en) 2021-12-31 2021-12-31 Peptide screen
PCT/GB2022/051383 WO2022254200A1 (fr) 2021-06-01 2022-05-31 Criblage peptidique

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