WO2015169853A1 - Molécule à chaîne invariante génétiquement modifiée pour chargement amélioré du complexe majeur d'histocompatibilité de classe i (cmh-i) - Google Patents

Molécule à chaîne invariante génétiquement modifiée pour chargement amélioré du complexe majeur d'histocompatibilité de classe i (cmh-i) Download PDF

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WO2015169853A1
WO2015169853A1 PCT/EP2015/059947 EP2015059947W WO2015169853A1 WO 2015169853 A1 WO2015169853 A1 WO 2015169853A1 EP 2015059947 W EP2015059947 W EP 2015059947W WO 2015169853 A1 WO2015169853 A1 WO 2015169853A1
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acid molecule
nucleic acid
antigen
sorting
cells
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PCT/EP2015/059947
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Sebastien P. WÄLCHLI
Tone Fredsvik GREGERS
Ana KUCERA
Oddmund BAKKE
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Universitetet I Oslo
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof

Definitions

  • the present invention relates to peptides presented on the cell surface of cells in the MHC class I (MHC I) context in which the invariant chain has been engineered to favor loading of specific antigens and generate CD8 + T-cell activation.
  • MHC I MHC class I
  • CD4 + T cells generally recognize exogenously derived peptides presented on the cell surface of antigen presenting cells in the MHC class II (MHC II) context.
  • MHC II MHC class II
  • the biosynthesis and transport of MHC II molecules are tightly regulated by the type II transmembrane invariant chain (Ii).
  • Ii harbors two leucine based sorting signals; Leu7/Ile8 and Metl6/Leul7 in its cytoplasmic tail. Indeed, these signals direct the Ii-MHC II complex to the endosomal pathway mainly via the cell surface, (therefore called the indirect pathway) and through binding to the adaptor proteins AP-1 and AP-2. These APs are involved in the formation of clathrin-coated vesicles at the trans Golgi and plasma membrane respectively. In addition, they play a pivotal role in cargo selection by recognizing the appropriate sorting signals of integral membrane proteins. Either one of the two Ii leucine signals is sufficient for targeting Ii to endosomal compartments.
  • Ii is sequentially degraded leaving the class II-associated Ii peptide (CLIP) bound to the MHC II groove.
  • CLIP is subsequently exchanged for antigenic peptides prior to transport to the cell surface for presentation.
  • An object of the present invention is to engineer a molecule with integrated antigens that enables loading and presentation of the antigens on the surface of cells to generate CD8 + T-cell activation.
  • An aspect of the present application relates to a nucleic acid molecule encoding a type II transmembrane invariant chain (li) which is modified by exchanging the class II-associated li peptide (CLIP) with an antigen, and wherein one or more sorting motifs is/are replaced with a AP-3 binding motif.
  • the sorting motif a leucine or a tyrosine based sorting motif.
  • cytoplasmic sorting motif selected from the group consisting of Leu7/Iles and Metie/Leui7.
  • the antigen a tumor antigen.
  • nucleic acid molecule selected from the group consisting of mRNA and DNA.
  • Another aspect of the present application relates to an amino acid molecule comprising the type II transmembrane invariant chain (Ii) according to the present invention.
  • Another aspect of the present application relates to a method of presenting a CD8 + T-cell activating antigen on a cell, comprising modifying the type II transmembrane invariant chain (Ii) by exchanging the class II-associated Ii peptide (CLIP) with an antigen, replacing one or more sorting motifs with a AP-3 binding motif, and introducing the Ii to a cell.
  • Ii type II transmembrane invariant chain
  • CLIP class II-associated Ii peptide
  • Another aspect of the present application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid molecule or the amino acid molecule according to the present invention, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the pharmaceutical composition is the pharmaceutical composition a vaccine.
  • Another aspect of the present application relates to a method for inducing a CD8 + and/or a CD4 + response, comprising administering the nucleic acid molecule or the amino acid molecule according to the present invention to an individual.
  • nucleic acid molecule or the amino acid molecule according to the present invention for use as a
  • a further aspect of the present application relates to a nucleic acid molecule or the amino acid molecule of the present invention for use in the treatment of a disease that is associated with the antigen.
  • Ii constructs used. Here showing the amino acid sequences of the Ii cytoplasmic tails.
  • the Ii wt, sorting signal (QRD)L7I was replaced by an (RRP) L7I motif resulting in the trafficking mutant Ii RRP/L17A.
  • RRP sorting signal
  • Ii L7A/L17A both L signals were removed. Indicated are also; the trans membrane (TM) region, CLIP, the two N-glycosylation sites (113 and 119) and the trimerization domain (TRI).
  • TM trans membrane
  • CLIP the two N-glycosylation sites
  • TRI trimerization domain
  • Hek cells were transfected as indicated. After 24 hours, whole cell lysates (WCL) were subjected to 4-20% SDS-PAGE Tris-HEPES-SDS gels, transferred to PVDF membranes and probed with anti Ii antibody, M-B741. The samples were either boiled or non-boiled before gel loading. Ii trimers and monomers are shown. Detection of actin in the WCL was used as loading control using anti-actin antibodies.
  • Hek cells were transfected as indicated. The cells were pulsed with
  • Transfected Hek cells were subjected to a metabolic pulse with
  • M l cells were seeded on coverslips and transiently transfected with li wt and mutants as ind icated in the figure.
  • Green channel ; li luminal domain, red channel ; li cytoplasmic tail, blue channel ; Lamp- 1.
  • 25 L17A/RRP mutant increased presentation of antigenic peptide on MHC-I.
  • J76 cells constitutively expressing DMF5 were incubated for 12 hours with SupTl (for SCT) or SupTl (HLA-A2 positive) transduced with the indicated constructs.
  • control peptide (B) SupTl (for SCT) or SupTl (HLA-A2 positive) transduced with the indicated construct were incubated with sTcR ( 10 nM final concentration) for 15 minutes at RT and the bound complex was detected using anti-His-PE. This experiment was performed twice.
  • the present invention relates to the effect on MHC I antigen presentation with Ii trafficking mutants which were designed to bind AP-3 rather than AP-1 and -2.
  • nucleic acid molecule selected from the group consisting of mRNA and DNA.
  • nucleic acid molecule mRNA in another embodiment of the present invention is the nucleic acid molecule mRNA.
  • Another aspect of the present application relates to an amino acid molecule comprising the type II transmembrane invariant chain (Ii) according to the present invention.
  • the antigen also activates a CD4 + T- cell.
  • AP3 binds both leucine ([DE]xxxL[LI])and tyrosine (Yxx0) based sorting motifs through their ⁇ - and ⁇ -chain respectively (Craig HM Virology 2000),Dell'Angelica EC EMBO J 1997).
  • the sorting motif a leucine ([DE]xxxL[LI]) or a tyrosine (Yxx0) based sorting motif.
  • LIMPII is a well-known AP3 binder harboring a RAP motif in front of the LI signal.
  • An embodiment of the present invention it thus where a RAP motif is placed in front of the LI signal.
  • AP3 is required for mouse CDld (RRRSAYQDIR) mediated antigen presentation of glycosphingolipids to NKT cells (Elewaut D 2003, JEM and Lawton AP, JI 2005).
  • Human CDlb (RRRSYQNIP) is the only human CD molecule being dependent on AP3 for proper trafficking and antigen presentation (Sugita M, Immunity 2002).
  • Upstream residues are important for AP3 binding to tyrosin signals.
  • upstream residues optimized for AP-3 binding are the upstream residues optimized for AP-3 binding.
  • Lysines replace Arginines.
  • the sorting motif is a leucine or a tyrosine based sorting motif.
  • the mutant L17A and the upstream region has been optimised wherein QRD has been changed to RAP, RRP, QAP, RAD, QRP, RAD, QRP, QAD, or RRD.
  • mutant L7A and the upstream region has been optimised wherein QLP has been changed to RAP, RLP, QAP, or RRP.
  • the upstream region optimised wherein NEQLP has been replaced with DERAP is the upstream region optimised wherein NEQLP has been replaced with DERAP.
  • the one or more sorting signals is/are a cytoplasmic sorting motif selected from the group consisting of Leu7/Iles and Metie/Leui7.
  • sorting motif (QLP)l_7l is replaced with (RRP)l_7l and/or (QRD)Li7A is replaced with (RRP)Li7A.
  • the protein sequence of Iiwt (SEQ ID NO : 1) is: MDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQG RLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGN MTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRH SLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM
  • CLIP is the underlined sequence above (SEQ ID NO : 2) : MRMATPLLM
  • Iiwt-TGFbRIIp is Iiwt wherein CLIP has been changed to TGFbRIIp (SEQ ID NO: 3) :
  • Iimut-TGFbRIIp (SEQ ID NO: 5) is (SEQ ID NO: 3) three mutations have been introduced (underlined) :
  • Another aspect of the present invention relates to an isolated amino acid molecule that has an open reading frame (ORF) amino acid sequence with 80% sequence identity to the sequences of the present invention, such as 90 %, such as 95 %, such as 98 %, such as 99%.
  • identity is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length. In another embodiment the two sequences are of different length and gaps are seen as different positions.
  • Gapped BLAST may be utilised.
  • the percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted .
  • one embodiment of the present invention relates to the sequences disclosed herein, in which 50, such as 30, such as 20, such as 15, such as 10, such as 8, such as 5, such as 4, such as 3, such as 2, such as 1 amino or nucleic acid has been exchanged .
  • 50 such as 30, such as 20, such as 15, such as 10, such as 8, such as 5, such as 4, such as 3, such as 2, such as 1 amino or nucleic acid has been exchanged .
  • 50 such as 30, such as 20, such as 15, such as 10, such as 8, such as 5, such as 4, such as 3, such as 2, such as 1 amino or nucleic acid has been exchanged .
  • 50 such as 30, such as 20, such as 15, such as 10, such as 8, such as 5, such as 4, such as 3, such as 2, such as 1 amino or nucleic acid has been exchanged .
  • 50 such as 30, such as 20, such as 15, such as 10, such as 8, such as 5, such as 4, such as 3, such as 2, such as 1 amino or nucleic acid has been exchanged .
  • 50 such as 30, such as 20, such as 15,
  • DNA sequence refers such DNA sequence also to the RNA equivalent i.e. with Ts exchanged with Us as well as their complimentary sequences.
  • the nucleic acid further comprises a reporter gene, which, in one embodiment, is a gene encoding neomycin phosphotransferase, Renilla luciferase, secreted alkaline phosphatase (SEAP), Gaussia luciferase or fluorescent proteins such as green or red fluorescent protein .
  • Reporter genes can for example be beneficial in tracking intracellular movements, antigen
  • CLIP can be exchanged with all conceivable antigens.
  • MARTI p AAGIGILTV (SEQ ID NO :7) : or ALGIGILTV (SEQ ID NO :8).
  • An antigen is any substance which provokes an adaptive immune response.
  • An antigen is often foreign or toxic to the body (for example, a bacterium or virus) which, once in the body, attracts and is bound to a respective and specific antibody.
  • histocompatibility molecule Depending on the antigen presented and the type of the histocompatibility molecule, several types of immune cells can become activated.
  • Antigen was originally a structural molecule that binds specifically to the antibody, but the term now also refers to any molecule or molecular fragment that can be recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system.
  • B-cell receptor or T-cell receptor highly variable antigen receptors
  • TCR T-Cell Receptor
  • MHC major histocompatibility complex
  • the antigen a CD4 + T-cell activating antigen and/or a CD8 + T-cell activating antigen.
  • the antigen is a tumor or a viral antigen.
  • a tumor is an abnormal mass of tissue as a result of abnormal growth or division of cells. Prior to abnormal growth (known as neoplasia), cells often undergo an abnormal pattern of growth, such as metaplasia or dysplasia. However, metaplasia or dysplasia do not always progress to neoplasia.
  • neoplastic cells exceeds, and is not coordinated with, that of the normal tissues around it.
  • Neoplasms may be benign, pre- malignant (carcinoma in situ) or malignant (cancer).
  • Benign neoplasms include uterine fibroids and melanocytic nevi (skin moles). They are circumscribed and localized and do not transform into cancer.
  • Potentially malignant neoplasms include carcinoma in situ. They do not invade and destroy but, given enough time, will transform into a cancer.
  • Secondary neoplasm refers to any of a class of cancerous tumor that is either a metastatic offshoot of a primary tumor, or an apparently unrelated tumor that increases in frequency following certain cancer treatments such as chemotherapy or radiotherapy.
  • the antigen can also originate from a bacteria or a virus.
  • compositions comprising the nucleic acid molecule or the amino acid molecule according to the present invention, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • a pharmaceutical composition comprising the nucleic acid molecule or the amino acid molecule according to the present invention, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the pharmaceutical composition is the pharmaceutical composition a vaccine.
  • this invention provides for compositions comprising an isolated nucleic acid, vector or cell of this invention, or an isolated nucleic acid obtained via the methods of this invention.
  • compositions of this invention can be administered by any appropriate route, for example, orally, parenterally, intravenously,
  • any applicable drug delivery system may be used with the compositions and/or agents/vectors/cells/nucleic acids of this invention, for administration to a subject, and is to be considered as part of this invention.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response. Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response.
  • BCG BacilleCalmette-Guerin
  • Corynebacteriumparvmm aluminum hydroxide +MPL, Addavax, MF59, CAFOl, CAF04, CAF05 and CAF09, and Sigma adjuvant system.
  • the adjuvant is pharmaceutically acceptable.
  • Another aspect of the present application relates to a method of presenting a CD8 + T-cell activating antigen on a cell, comprising modifying the type II transmembrane invariant chain (Ii) by exchanging the class II-associated Ii peptide (CLIP) with an antigen, replacing one or more sorting motifs with a AP-3 binding motif, and introducing the li to a cell.
  • Ii type II transmembrane invariant chain
  • CLIP class II-associated Ii peptide
  • This method will allow CD8 + and optionally CD4 + T-cell activation to the antigen inserted instead of CLIP.
  • Another aspect of the present application relates to a method for inducing a CD8 + and/or a CD4 + response, comprising administering the nucleic acid molecule or the amino acid molecule according to the present invention to an individual.
  • nucleic acid molecule or amino acid molecule administered using electroporation or transfection. Other means of administration are listed above.
  • Another aspect of the present application relates to the nucleic acid molecule or the amino acid molecule according to the present invention for use as a
  • Example 1 Sorting of an engineered Invariant chain molecule directly to endosomal pathway leads to improved MHC class I loading
  • CD4 + T cells recognize exogenously derived peptides presented on the cell surface of antigen presenting cells in the MHC class II (MHC II) context.
  • MHC II MHC class II
  • the biosynthesis and transport of MHC II molecules are tightly regulated by the type II
  • Ii transmembrane invariant chain
  • the APs are involved in the formation of clathrin-coated vesicles at the trans Golgi and plasma membrane respectively. In addition, they play a pivotal role in cargo selection by recognizing the apropriate sorting signals of integral membrane proteins. Either one of the two Ii leucine signals is sufficient for targeting Ii to endosomal compartements, but the Leu7/Ile8 is more potent in doing so.
  • Ii is sequentially degraded leaving the class II-associated Ii peptide (CLIP) bound to the MHC II groove.
  • CLIP is subsequently exchanged for antigenic peptides prior to transport to the cell surface for presentation.
  • MHC I binds mainly endogenously derived peptides generated by the proteasome in the cytosol which are targeted to the endoplasmic reticulum (ER) via TAP transporters. After MHC I peptide loading, the complex is presented to cytotoxic CD8+ T cells. MHC I is independent of Ii, however, it was recently demonstrated that Ii plays a physiological role for targeting MHC I to the endosomal pathway for loading of viral peptides and cross-presentation to CD8+ T cells. Additional evidence for an Ii-MHC I interaction came from van Luijn and colleagues who showed that CLIP efficiently binds to several MHC I molecules in leukemic cells.
  • the Ii mutants were thereafter subcloned into pcDNA3 at KpnI-BamHI, behind the T7-RNA polymerase promoter.
  • Ii constructs described above were used as templates for PCR quick change 15 mutagenesis (all reagents used were included in the kit; QuickChange® Site- Directed Mutagenesis (Stratagen, La Jolla, CA, USA)) in order to generate the AP- 3 binding motif RRP.
  • ELAGIGILTV was performed with IiMARTl forw/IiMARTl rev primers for IiMARTl and IiCD20 forw/IiCD20 rev for IiCD20 (table 1) . After sequence verification these constructs were recombined into a Gateway-converted pCI-pA102 (Walchli et al .
  • M l cells, HEK293 cells, human epithelial HeLa-Kyoto and Madin Darby Canine Kid ney (MDCK) cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Bio Witthaker, Walkersville, MD, USA) . All med ias were supplemented with heat- inactivated 10% fetal calf serum (FCS, HyClone, Logan, UT, USA) . J76 were a kind gift from Miriam Hemskerk (Leiden University Med ical Center, The Nederland), SupTl from Martin Pule (University College London, U K), both cell lines were grown in RPMI+ 10% fetal calf serum . PBMC from healthy donor etc.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS HyClone
  • J76 were a kind gift from Miriam Hemskerk (Leiden University Med ical Center, The Nederland), SupTl from Martin Pul
  • the Bu43 antibody was kind ly provided by D. Hard ing (Birmingham, U K) .
  • M-B741 was purchased from BD Biosciences (Franklin Lakes, NJ, USA) .
  • Pin- 1, anti-Lamp- 1 and anti-actin were all purchased from AbCam, (Cambridge, U K) .
  • the secondary antibodies anti murine IgM FITC, goat anti-rabbit alexa 647, goat anti-mouse alexa 555, sheep anti-mouse-HRP, were all aquired from
  • Metabolic labeling was done using 35 S-labeled Cystein/Methionine (Perkin Elmer, Waltham, MA, USA) .
  • Cells were seeded to 60%-70% confluence; washed three times in Cys/Met-free DMEM ; incubated in Cys/Met-free DMEM for 45 min followed by a 30 min pulse with Cys/Met-free DMEM supplemented with 50 ⁇ S35.
  • the pulse chase assay the cells were washed three times in DMEM containing 2mM L-glutamine, primocin, and 30% FCS and chased for indicated time periods.
  • Immunoprecipitations were done at at 4 °C over night with l-2[ig ml- 1 antibody in lysis buffer (50 mM Tris-HCI, pH 7,5, 150 mM NaCI, 1% TxlOO) supplemented with the protease inhibitor coctail Protease Arrest (GBiosciences, St. Louis, MO, USA) .
  • Antigen-antibody complexes were captured with Protein G- coupled Dynabeads (Invitrogen) .
  • protease inhibitors the same procedure was followed as for metabolic labeling .
  • Transfected M l cells were grown to 50-70% confluence imag ing dishes. Cells were then fixed with 3% paraformaldehyde, stained with ind icated primary and secondary antibodies in 0.1% saponine and mounted onto object-glasses with Mowiol (Sigma-Aldrich, St Louis, MO, USA) .
  • the microscope used was Olympus FV1000 confocal scanning laser upright microscope (BX61WI) with a PlanApo 60x/1.10 oil objective.
  • Three channel PMT detector unit Fluorochromes were exited with 488 nm Argon, 543 and 647 nm HeNe lasers. All image acquisition was done by seq uential line scanning to eliminate bleed-through . Images were processed with ImageJ (NIH, USA) . Results
  • the Iiwt, sorting signal (QRD)L7I which aids in AP- 1 and AP-2 binding was replaced by an (RRP) L7I motif by means of QuickChange PCR mutagenesis. This particular mutation was chosen based on previous work by Gupta et al . where it was found that RRP inserted into the Ii cytoplasmic tail bound AP-3 in vitro .
  • Transfected cells were pulsed with with 35Met/35Cys containing media, and chased for various time points, followed by an immunoprecipitation of Ii.
  • the Tl/2 of liwt is approximately 3 hours, whereas IiRRP/L17A has a half-life closer to 1 hour, which suggests a faster transport to more proteolytic late endosomal compartments.
  • Iil_7A/L17A was found to accumulate within the four hours of chase due to inhibited internalization (Simonsen et al 1993) .
  • IiRRP/L17A We designed IiRRP/L17A to follow an AP3 sorting pathway. While API and -2 are located at the TGN and plasma membrane, AP3 is involved in binding to and sorting of protein at TGN and early endosomes, such as LIMPII, bringing them to late endosomes/lysosomes, and is thus involved in endosomal maturation. We therefore performed RNAi mediated depletion of AP3 to further investigate the effect on the Ii protein levels. As many protolytic enzymes also traffic via an AP3 route to late endosomes/lysosomes one might expect that wild type invariant chain is effected. A mutant molecule which is also dependent on AP3 trafficking should then be more influenced.
  • Ii L7A/L17A was permanently located at the plasma membrane unable to internalize as the sorting signals were removed (figure 2 D). This was also expected and has previously been described for Ii constructs were the Ii N- terminal tail was fused to neuraminidase[15] . We still observed some cells with Bu43 positive endosomes most likely due to cconstitutive membrane
  • Cells expressing Ii carrying tumor-associated epitopes efficiently load HLA-A2 and specifically activate CD8+ T cells.
  • TcRs soluble TcRs like TcR are low affinity antigen-specific protein, therefore the detection of endogenous protein revealed difficult.
  • Iiwt_MART-l expressing cells 2%, MFI : 105
  • cells expressing Ii L17A/RRP_MART-1 were clearly detected (21%, MFI: 194), suggesting that the peptide loading was increased with this mutant.
  • Ii is known to depend on the adaptor proteins API and AP2 for proper trafficking from Golgi, via the cell surface to the endosomal pathway [1-5] .
  • AP3 binding motifs within the Ii tail of a TfR-Ii fusion construct Bakke and colleagues has previously shown evidence for direct sorting to late endosomal/lysosomal structures in cells [15].
  • AP3 binds both leucine ([DE]xxxL[LI])and tyrosine (Yxx0) based sorting motifs through their ⁇ - and ⁇ -chain respectively (Craig HM Virology 2000),Dell'Angelica EC EMBO J 1997).
  • the residues N terminal to the two (iso)leucines of the sorting signal determine adaptor binding (Rodionov 2002) and certain residues favor AP3 binding.
  • LIMPII is a well known AP3 binder harboring a RAP motif in front of the LI signal.
  • AP3 is required for mouse CDld (RRRSAYQDIR) mediated antigen presentation of glycosphingolipids to NKT cells ( Elewaut D 2003, JEM and Lawton AP, JI 2005).
  • Human CDlb (RRRSYQNIP) is the only human CD molecule being dependent on AP3 for proper trafficking and antigen presentation (Sugita M, Immunity 2002). These molecules harbor tyrosin based sorting motifs, however the requirement for positively charged Arginine residues seems to be similar to both leucine - and tyrosine based sorting signals. Whether upstream residues are important for AP3 binding to tyrosin signals is not investigated, yet not unlikely.

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Abstract

La présente invention concerne des peptides présentés sur la surface cellulaire de cellules dans le contexte du complexe majeur d'histocompatibilité de classe I (CMH-I) où la chaîne invariante a été génétiquement modifiée afin de favoriser le chargement d'antigènes spécifiques et de générer l'activation de lymphocytes T CD8+.
PCT/EP2015/059947 2014-05-06 2015-05-06 Molécule à chaîne invariante génétiquement modifiée pour chargement amélioré du complexe majeur d'histocompatibilité de classe i (cmh-i) WO2015169853A1 (fr)

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JP2019517790A (ja) * 2016-05-09 2019-06-27 オスロ ウニヴェルスィテーツスィーケフース ホーエフ ＴＧＦβＲＩＩのフレームシフト変異体を認識するＴ細胞受容体
CN113382747A (zh) * 2019-01-03 2021-09-10 伊沃逊生物科技股份公司 靶向肿瘤表位的疫苗

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Cited By (2)

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JP2019517790A (ja) * 2016-05-09 2019-06-27 オスロ ウニヴェルスィテーツスィーケフース ホーエフ ＴＧＦβＲＩＩのフレームシフト変異体を認識するＴ細胞受容体
CN113382747A (zh) * 2019-01-03 2021-09-10 伊沃逊生物科技股份公司 靶向肿瘤表位的疫苗

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