WO2012044999A2 - Reversible protein multimers, methods for their production and use - Google Patents

Reversible protein multimers, methods for their production and use Download PDF

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Publication number
WO2012044999A2
WO2012044999A2 PCT/US2011/054335 US2011054335W WO2012044999A2 WO 2012044999 A2 WO2012044999 A2 WO 2012044999A2 US 2011054335 W US2011054335 W US 2011054335W WO 2012044999 A2 WO2012044999 A2 WO 2012044999A2
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Prior art keywords
molecule
mhc
tag
peptide
multimer
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PCT/US2011/054335
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French (fr)
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WO2012044999A3 (en
Inventor
Immanuel F. Luescher
Julien Schmidt
Philippe Guillaume
Danijel Dojcinovic
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Ludwig Institute For Cancer Research Ltd.
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Application filed by Ludwig Institute For Cancer Research Ltd. filed Critical Ludwig Institute For Cancer Research Ltd.
Priority to US13/877,200 priority Critical patent/US10023657B2/en
Publication of WO2012044999A2 publication Critical patent/WO2012044999A2/en
Publication of WO2012044999A3 publication Critical patent/WO2012044999A3/en
Priority to US16/008,430 priority patent/US20180346606A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units

Definitions

  • Fluorescent MHC-peptide multimers commonly referred to as tetramers are widely used to detect, enumerate, analyze and sort antigen- specific CD8+ T cells (1, 2).
  • Monomeric MHC-peptide complexes are produced by refolding of a MHC heavy and light chain in the presence of a peptide of interest and subsequently are biotinylated at a C-terminally added biotinylation sequence peptide (BSP, LHHILDAQKMVWNHR, SEQ ID NO: 1) the biotin- transferase BirA.
  • Fluorescent conjugates are obtained by reaction of biotinylated MHC-I- peptide monomers with phycoerythrin (PE) or allophycocyanine (APC) labeled streptavidin (2, 3).
  • PE phycoerythrin
  • APC allophycocyanine
  • Some aspects of this invention relate to reagents for reversible staining of cells.
  • Some aspects of this invention provide staining reagents that comprise a core structure, referred to herein as a carrier molecule, to which a plurality of monomeric proteins can reversibly bind to form a protein multimer.
  • the carrier molecule itself is a detectable molecule, for example, a fluorophore.
  • the proteins that reversibly bind to the carrier molecule are proteins that also specifically bind a molecular target, for example, a specific cellular ligand or receptor, allowing for staining of the molecular target with the protein multimer. Since the bond of the protein and the carrier molecule is reversible, the protein multimer complex can be dissolved, resulting in dissociation of the proteins from the carrier molecule and, thus, in re-monomerization of the bound proteins.
  • the protein multimers provided by some embodiments described herein are useful, for example, for cell staining and cell isolation procedures.
  • the multimers provided by some embodiments of this invention can be dissolved under non-toxic, physiological conditions, for example, by addition of an agent interfering with the reversible binding of protein and core structure of the multimer.
  • one advantage of the instantly provided protein multimers is that, in contrast to most other staining agents, they do not expose stained cells to the undesirable effects of permanent staining.
  • Dissolution of the multimer structure used for staining live cells avoids undesired biological effects of continued association of cells with a dye or a multimeric protein structure, for example, continued activation of T-cell receptor signaling in MHC-stained T-cells.
  • Staining procedures using the instantly described protein multimers thus, allow for "minimally invasive" staining of cells, which is a requirement for the isolation of native cells that are sensitive to staining procedures, including, for example, certain T-cell populations.
  • Some aspects of this invention provide methods and reagents for the generation of peptide-loaded MHC molecules, for example, of peptide-loaded MHC class II molecules. In some embodiments, methods for the efficient production of molecularly defined,
  • MHC class II molecules are loaded with an antigenic peptide that is conjugated to a tag, for example, a tag allowing for peptide or protein isolation and/or purification by
  • tagged MHC class II binding peptides are provided.
  • the tag is an acidic tag, for example, a tag comprising a plurality of acidic amino acid residues, or an acidic fluorophore, for example, an acidic cyanine dye.
  • the tag for example, the acidic tag, is reversibly conjugated to the antigenic peptide of interest, for example via a cleavable linker.
  • the cleavable linker is a photocleavable linker.
  • the cleavable linker comprises a cleavage site for an enzyme, for example, a protease, or a chemical.
  • Some aspects of this invention provide protein multimers that include (a) a
  • the multivalent carrier molecule and (b) a plurality of proteins bound to the carrier molecule.
  • at least one of the proteins is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant ⁇ > K D ⁇ lOfM.
  • at least one of the peptides or proteins is conjugated to the carrier molecule via a chelate complex bond.
  • Some aspects of this invention provide multivalent chelants that comprise a water- soluble carrier molecule and a plurality of chelant moieties conjugated to the carrier molecule.
  • Some aspects of this invention provide multivalent chelants that comprise a carrier molecule or structure, and a plurality of chelant moieties conjugated to the carrier molecule or structure, wherein the carrier molecule or structure has a diameter of less than O.lnm, less than 0.2nm, less than 0.25nm, less than 0.3nm, less than 0.4nm, less than 0.5nm, less than 0.6nm, less than 0.7nm, less than 0.75nm, less than 0.8nm, less than 0.9nm, less than lnm, less than l.lnm, less than 1.2nm, less than 1.3nm, less than 1.4nm, less than 1.5nm, less than 1.6nm, less than 1.7nm, less than 1.8nm, less than 1.9nm, less than 2nm, less than 2.5nm, less than 3nm, less than 4nm, less than 5nm, less than 6nm, less than 7nm, less than 8nm, less
  • the diameter of the carrier molecule is less than 20nm, less than 30nm, less than 40nm, less than 50nm, less than 60nm, less than 70nm, less than 80nm, less than 90nm, less than lOOnm, less than 200nm, less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm, or less than ⁇ .
  • Some aspects of this invention provide methods for the production of protein multimers that comprise a step of contacting a monomeric chelant moiety-conjugated MHC molecule with a carrier molecule, for example, a water-soluble carrier molecule or a carrier molecule or structure described herein, that is conjugated to a plurality of chelant moieties under conditions suitable for formation of a chelate complex between the chelant moieties conjugated to the MHC molecule and the chelant moieties conjugated to the carrier molecule.
  • a carrier molecule for example, a water-soluble carrier molecule or a carrier molecule or structure described herein
  • Some aspects of this invention provide methods for the production of MHC molecules that are conjugated to a ligand via a chelate complex bond, comprising a step of contacting an MHC molecule conjugated to a first chelant with a ligand molecule conjugated to a second chelant under conditions suitable for formation of a chelate complex between the first and the second chelant.
  • the resulting MHC molecule is then contacted with a multivalent binding molecule binding the ligand to produce reversible MHC multimers.
  • Some aspects of this invention provide methods to generate peptide-loaded MHC molecules that are conjugated to a chelant using MHC binding peptides conjugated to a tag, the methods comprising the steps of providing an MHC molecule bound to an antigenic MHC molecule-binding peptide that is conjugated to a tag via a cleavable linker, removing the tag from the antigenic peptide, and conjugating a chelant moiety to a heavy chain of the MHC molecule.
  • Some aspects of this invention provide methods for staining, detecting, and isolating cells, the methods comprising a step of contacting a population of cells with a protein multimer as described herein, for example, with a multimer comprising a chelate bond and a detectable label, and performing an assay to detect a cell binding the multimer.
  • Some aspects of this invention provide method for the isolation of cells that bind a multimer as described herein, the methods comprising the steps of (a) contacting a population of cells with a protein multimer as provided herein, for example, with a multimer comprising a chelate bond and a detectable label, (b) optionally, detecting a cell binding the multimer, and (c) isolating the cell binding the multimer.
  • Some aspects of this invention provide methods for the manipulation of T-cell populations with protein multimers, the methods comprising a step of contacting a population of cells expressing a T-cell receptor with an MHC multimer as described herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC class I molecule interaction to activate a T-cell expressing the T-cell receptor and binding the MHC multimer.
  • Some aspects of this invention provide cells or cell populations that are contacted with a protein multimer as provided herein, for example, with an MHC multimer. Some aspects of this invention provide kits comprising pep tide-loaded or empty MHC molecules as provided herein.
  • Some aspects of this invention provide isolated peptide-loaded MHC molecules that comprise an MHC heavy chain, and an antigenic peptide.
  • the peptide is conjugated to a tag, for example, a tag for ion exchange chromatography.
  • Some aspects of this invention provide methods for the generation of peptide-loaded MHC molecules, for example, MHC class II molecules, comprising a step of contacting an empty MHC molecule with an antigenic peptide conjugated to a tag under conditions suitable for the antigenic peptide to bind the MHC molecule.
  • the tag is a tag for ion exchange chromatography and the method, in some embodiments, includes a step of isolating peptide-loaded MHC molecules by performing a chromatography procedure.
  • Some aspects of this invention provide methods for the generation of MHC class II molecules, empty or peptide-loaded, the method comprising a step of contacting an MHC class II type alpha heavy chain with an MHC class II type beta heavy chain under conditions suitable for the alpha and the beta chain to form a heterodimeric MHC class II molecule.
  • at least one of the MHC class II heavy chains is conjugated to a tag.
  • a step of isolating the MHC class II molecule is performed, wherein the isolating comprises a step of chromatography, for example, affinity chromatography.
  • a protein multimer comprising (a) a multivalent carrier molecule, and (b) a plurality of proteins bound to the carrier molecule.
  • at least one of the plurality of proteins is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant ⁇ > KD > lOfM.
  • the dissociation constant is KD > O. lpM.
  • the dissociation constant is KD > lpM.
  • the dissociation constant is KD > lOpM.
  • the dissociation constant is KD > lOOpM.
  • the dissociation constant is KD > InM. In some embodiments, the dissociation constant is KD > lOnM. In some embodiments, the dissociation constant is KD ⁇ lOOnM. In some embodiments, the dissociation constant is KD ⁇ lOnM. In some embodiments, the dissociation constant is KD ⁇ InM. In some embodiments, the dissociation constant is KD ⁇ lOOpM. In some embodiments, the dissociation constant is KD ⁇ lOpM.
  • the chelate complex bond is a bond with a dissociation constant 5 ⁇ > KD lfM.
  • the protein is an MHC molecule and the protein multimer is an MHC multimer.
  • the chelate complex bond comprises a chelant conjugated to the MHC molecule, and a chelant conjugated to the carrier molecule.
  • the chelant conjugated to the carrier molecule is of a different structure than the chelant conjugated to the MHC molecule.
  • the MHC molecule comprises an MHC a chain.
  • the MHC molecule further comprises an MHC a chain or a ⁇ 2 microglobulin chain.
  • the MHC molecule is an MHC class I molecule.
  • the MHC molecule is an MHC class II molecule.
  • the chelant conjugated to the MHC molecule is C- terminally conjugated to the MHC ⁇ chain.
  • the chelant conjugated to the MHC molecule is C-terminally conjugated to the MHC ⁇ chain or the ⁇ 2 microglobulin chain.
  • the chelant conjugated to the MHC molecule is a peptide comprising a chelant moiety.
  • the peptide comprising a chelant moiety is fused to a polypeptide chain comprised by the MHC molecule.
  • the peptide comprising a chelant moiety comprises a poly-Histidine sequence.
  • the poly-Histidine sequence comprises 3-24 His residues.
  • the chelant conjugated to the MHC molecule comprises a His6 tag, a His 12 tag, or a 2x His6 tag.
  • the chelant conjugated to the carrier molecule comprises an NTA moiety.
  • the NTA moiety is bound to the carrier molecule in mono-NTA configuration.
  • the NTA moiety is bound to the carrier molecule in poly-NTA configuration.
  • the NTA moiety is bound to the carrier molecule in di-NTA, or tetra-NTA configuration.
  • the NTA moiety is bound to a linker.
  • the linker comprises a maleimide moiety or derivative.
  • the linker comprises an oxime moiety or derivative. In some embodiments, the linker is between about 9A and about 23A long. In some embodiments, the linker is covalently bound to the carrier molecule. In some embodiments, the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule. In some embodiments, the ligand is biotin and the binding molecule is streptavidin. In some embodiments, the chelate complex bond further comprises a divalent cation.
  • the divalent cation is an Ni2+, Cu2+, Zn2+, Co2+, Cd2+, Sr2+ , Mn2+, Fe2+, Mg2+, Ca2+, or Ba2+ ion.
  • the carrier molecule is a fluorophore,, a phycobilin, phycoerythrin or allophycocyanine, a quantum dot (Qdot), a microsphere (e.g., a fluorescent microsphere (e.g. fluorospheres® type), a magnetic particle, or a nanoparticle.
  • the MHC molecule is an empty MHC molecule or a peptide-loaded MHC molecule.
  • the peptide-loaded MHC molecule is chosen from the MHC molecules disclosed in Table 2.
  • the MHC molecule comprises an HLA-A*0201 heavy chain.
  • the MHC molecule is loaded with an antigenic peptide.
  • the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL (SEQ ID NO: 2).
  • the multimer is a tetramer.
  • the antigenic peptide is conjugated to a tag.
  • the tag is conjugated to the peptide via a cleavable linker.
  • the linker is a photocleavable linker.
  • the linker is an ⁇ linker. In some embodiments, the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical. In some embodiments, the tag is an acidic peptide tag. In some embodiments, the acidic peptide tag comprises a plurality of acidic amino acid sequences. In some embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY- D9, or pY-D10 tag.
  • the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
  • the tag is a desthiobiotin (DTB) tag.
  • Some aspects of this invention provide a multivalent chelant comprising a water- soluble carrier molecule, and a plurality of chelant moieties conjugated to the carrier molecule. Some aspects of this invention provide a multivalent chelant comprising a carrier molecule or structure, and a plurality of chelant moieties conjugated to the carrier molecule or structure. In some embodiments, the carrier molecule or structure has a diameter of less than O.
  • lnm less than 0.2nm, less than 0.25nm, less than 0.3nm, less than 0.4nm, less than 0.5nm, less than 0.6nm, less than 0.7nm, less than 0.75nm, less than 0.8nm, less than 0.9nm, less than lnm, less than l.lnm, less than 1.2nm, less than 1.3nm, less than 1.4nm, less than 1.5nm, less than 1.6nm, less than 1.7nm, less than 1.8nm, less than 1.9nm, less than 2nm, less than 2.5nm, less than 3nm, less than 4nm, less than 5nm, less than 6nm, less than 7nm, less than 8nm, less than 9nm, or less than lOnm.
  • the diameter of the carrier molecule is less than 20nm, less than 30nm, less than 40nm, less than 50nm, less than 60nm, less than 70nm, less than 80nm, less than 90nm, less than lOOnm, less than 200nm, less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm, or less than ⁇ .
  • the chelant moieties are nitrilotriacetic acid (NTA) moieties. In some embodiments, the NTA moieties are in mono-NTA configuration.
  • the NTA moieties are in poly-NTA configuration. In some embodiments, the NTA moieties are in di-NTA, or tetra-NTA configuration. In some embodiments, the NTA moieties are bound to a linker. In some embodiments, the linker comprises a maleimide moiety. In some embodiments, the linker comprises an oxime moiety. In some embodiments, the linker is between about 9A and about 23 A long. In some embodiments, the linker is covalently bound to the carrier molecule. In some embodiments, the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule.
  • the ligand is biotin and the binding molecule is streptavidin.
  • the carrier molecule is a fluorophore, a phycobilin, phycoerythrin or allophycocyanine, or a quantum dot (Qdot).
  • Qdot quantum dot
  • a plurality of the chelant moieties conjugated to the carrier molecule form chelate complex bonds to a plurality of chelant moiety-conjugated monomeric molecules, thus forming a multimer of the monomeric molecule.
  • the monomeric molecule is a polyprotein.
  • the monomeric molecule is a small molecule compound.
  • the monomeric molecule is a polynucleotide. In some embodiments, the monomeric molecule is a ligand of a receptor. In some embodiments, the receptor is a cell- surface receptor. In some embodiments, the receptor is a T-cell receptor. In some embodiments, the monomeric molecule is an MHC molecule. In some embodiments, the monomeric molecule is an MHC class I molecule. In some embodiments, the MHC molecule comprises an HLA-A*0201 heavy chain. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the MHC molecule is loaded with an antigenic peptide.
  • the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL (SEQ ID NO: 3).
  • the multivalent chelant is a tetravalent chelant.
  • the tag is an acidic peptide tag.
  • the acidic peptide tag comprises a plurality of acidic amino acid sequences.
  • the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, pY-D10, pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
  • the tag is a desthiobiotin (DTB) tag.
  • Some aspects of this invention provide a method comprising contacting a monomeric chelant moiety-conjugated MHC molecule with a water-soluble carrier molecule conjugated to a plurality of chelant moieties under conditions suitable for formation of a chelate complex between the chelant moieties conjugated to the MHC molecule and the chelant moieties conjugated to the carrier molecule.
  • the chelant moieties conjugated to the carrier molecule are NTA moieties.
  • the NTA moieties are in mono-NTA configuration.
  • the NTA moieties are in poly-NTA configuration.
  • the NTA moieties are in di-NTA, or tetra-NTA configuration.
  • the NTA moieties are bound to a linker.
  • the linker is between about 9A and about 23A long.
  • the linker is covalently bound to the carrier molecule.
  • the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule.
  • the ligand is biotin and the binding molecule is streptavidin.
  • the carrier molecule is a fluorophore.
  • the carrier molecule is a phycobilin.
  • the carrier molecule is phycoerythrin or allophycocyanine.
  • the carrier molecule is a quantum dot (Qdot). In some embodiments, the carrier molecule is a magnetic particle. In some embodiments, the carrier molecule is a nanoparticle. In some embodiments, the carrier molecule is PE conjugated to streptavidin. In some embodiments, the carrier molecule conjugated to a plurality of chelant moieties is generated by incubating a carrier molecule conjugated to a plurality of binding molecules with an excess of chelant-conjugated ligand under conditions suitable for the ligand to bind the binding molecule. In some embodiments, the molar ratio of carrienligand is between 1:2 and 1: 10. In some
  • the molar ratio of carrienligand is 1 :5. In some embodiments, the incubating is performed at a temperature between 2-16 °C. In some embodiments, the incubating is performed at about 4 °C. In some embodiments, the method comprises a step of incubating the carrier molecules contacted with the ligand with NiS04. In some embodiments, the method comprises a step of contacting the carrier molecule conjugated to a plurality of chelant moieties with a molar excess of the MHC molecule conjugated to a chelant. In some embodiments, the excess is 2-20 fold. In some embodiments, the excess is 10-fold. In some embodiments, the MHC molecule is an MHC class I molecule. In some embodiments, the MHC molecule comprises an HLA-A*0201 heavy chain. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the MHC molecule is loaded with an antigenic peptide.
  • Some aspects of this invention provide a method comprising contacting an MHC molecule conjugated to a first chelant with a ligand molecule conjugated to a second chelant under conditions suitable for formation of a chelate complex between the first and the second chelant.
  • the first chelant comprises a peptide.
  • the peptide is C-terminally fused to a polypeptide chain comprised by the MHC molecule.
  • the peptide comprises a poly-Histidine sequence.
  • the poly-Histidine sequence comprises between 3 and 24 His residues.
  • the chelant conjugated to the MHC molecule comprises a His6 tag, a His 12 tag, or a 2x His6 tag.
  • the second chelant is NTA.
  • the NTA is in mono-NTA configuration.
  • the NTA is in poly-NTA configuration.
  • the NTA is in di-NTA, or tetra-NTA configuration.
  • the NTA is bound to a linker.
  • the linker is covalently bound to the ligand molecule.
  • the linker is between about 9A and about 23A long.
  • the method further comprises contacting the ligand molecule with a multivalent binding molecule under conditions suitable for the ligand to bind the multivalent binding molecule.
  • the ligand is biotin and the binding molecule is streptavidin.
  • the binding molecule is conjugated to a carrier molecule.
  • the carrier molecule is a fluorophore.
  • the carrier molecule is a phycobilin.
  • the carrier molecule is phycoerythrin or allophycocyanine. In some embodiments, the carrier molecule is a quantum dot (Qdot). In some embodiments, the carrier molecule is a magnetic particle. In some embodiments, the carrier molecule is a nanoparticle. In some embodiments, the MHC molecule is an MHC class I molecule. In some embodiments, the MHC molecule comprises an HLA-A*0201 heavy chain. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the MHC molecule is loaded with an antigenic peptide.
  • the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL or a peptide comprising a sequence provided in Table 2.
  • Some aspects of this invention provide a method comprising providing an MHC molecule bound to an antigenic MHC molecule-binding peptide that is conjugated to a tag via a cleavable linker, removing the tag from the antigenic peptide, and conjugating a chelant moiety to a heavy chain of the MHC molecule.
  • the method further comprises contacting a multivalent chelant molecule with the MHC molecule under conditions suitable for the chelant moiety conjugated to the MHC molecule to form a chelate complex bind with a chelant moiety of the multivalent chelant molecule.
  • Some aspects of this invention provide a method comprising contacting a population of cells with a protein multimer according to any preceding claim, wherein the multimer comprises a chelate bond and a detectable label, and detecting a cell binding the multimer.
  • the protein multimer is an MHC multimer.
  • the detectable label is a fluorophore.
  • detecting is by fluorescent microscopy or cell sorting.
  • the detectable label is a magnetic particle.
  • detecting is by isolating the cell binding the multimer.
  • detecting comprises quantifying a number of cells binding the multimer.
  • detecting comprises quantifying a number of cells binding the multimer as a ratio to a number of cells of the population of cells that do not bind the multimer.
  • the method further comprises reversing the binding of the multimer to the cell binding the multimer by contacting the cells binding the multimer with a monomeric chelant moiety competing for the chelate complex bond comprised by the multimer.
  • the monomeric chelant moiety is an imidazole molecule.
  • Some aspects of this invention provide a method comprising contacting a population of cells with a protein multimer according to any preceding claim, wherein the multimer comprises a chelate bond and a detectable label; optionally, detecting a cell binding the multimer; and isolating the cell binding the multimer.
  • the multimer is an MHC multimer.
  • the method further comprises reversing the binding of the multimer to the cell binding the multimer by contacting the isolated cell binding the multimer with a monomeric chelant moiety competing for the chelate complex bond comprised by the multimer.
  • the monomeric chelant moiety is an imidazole molecule.
  • the isolated cell is a T-cell. In some
  • the T-cell does not undergo CD8/TCR-mediated activation during the contacting, optionally, during the detection, and during the isolation step.
  • the isolated T-cell is part of an isolated, native T-cell population.
  • Some aspects of this invention provide a method comprising, contacting a population of cells expressing a T-cell receptor with an MHC multimer provided herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC class I molecule interaction to activate a T-cell expressing the T-cell receptor and binding the MHC multimer.
  • Some aspects of this invention provide a method comprising, contacting a population of cells expressing a T-cell receptor with an MHC multimer provided herein under conditions suitable for the multimer to bind to the T-cell receptor and to render the T-cell non-responsive to a naturally occurring antigen.
  • the method further comprises contacting the population of T-cells with an agent able to release the chelate complex bond of the MHC multimer after the cells were contacted with the multimer.
  • Some aspects of this invention provide a cell or cell population comprising a cell contacted with the protein multimer of any preceding claim, wherein the multimer comprises a chelate complex bond.
  • the multimer is an MHC multimer.
  • the cell is or has been contacted with an excess of monomeric chelant moieties competing for the chelate complex bond comprised by the multimer.
  • kits comprising an MHC multimer as provided in any preceding claim.
  • the MHC multimer is loaded with an antigenic peptide.
  • the MHC multimer is an empty MHC multimer.
  • the kit further comprises at least one antigenic peptide that can be loaded onto the empty MHC multimer.
  • the kit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different antigenic peptides.
  • the antigenic peptides are antigenic peptides of tumor antigens.
  • the peptide is conjugated to a tag.
  • the tag is an affinity tag.
  • the tag is a desthiobiotin (DTB) tag.
  • the peptide is conjugated to a tag for ion exchange chromatography.
  • the MHC molecule is an MHC class II molecule.
  • the tag is an acidic tag.
  • the acidic tag is an acidic cyanine dye.
  • the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences.
  • the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag.
  • the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
  • the MHC molecule further comprises a heavy chain that is conjugated to a chelant moiety.
  • the molecule comprises a combination of a heavy chain and an antigenic peptide discloses in Table 2.
  • the tag is conjugated to the peptide via a cleavable linker.
  • the linker is a photocleavable linker.
  • the linker is an NP A linker.
  • the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
  • the MHC molecule is comprised in an MHC multimer.
  • Some aspects of this invention provide a method comprising contacting an empty MHC molecule with an antigenic peptide conjugated to a tag under conditions suitable for the antigenic peptide to bind the MHC molecule.
  • the MHC molecule is an MHC class II molecule.
  • the tag conjugated to the MHC class II binding antigenic peptide is an affinity tag that is not a polyhistidine tag.
  • the tag is a desthiobiotin (DTB) tag.
  • the tag is an acidic tag.
  • the acidic tag is an acidic cyanine dye.
  • the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences.
  • the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag.
  • the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
  • the tag is conjugated to the peptide via a cleavable linker.
  • the linker is a photocleavable linker.
  • the linker is an NP A linker.
  • the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
  • the tag is a part of a cleavable linker that remains after cleavage of the linker.
  • Some aspects of this invention provide a method comprising contacting an MHC class II type alpha heavy chain with an MHC class II type beta heavy chain under conditions suitable for the alpha and the beta chain to form a heterodimeric MHC class II molecule.
  • at least one of the MHC class II heavy chains is conjugated to a tag, and isolating the MHC class II molecule, wherein the isolating comprises a step of affinity chromatography.
  • the tag is a protein or peptide tag.
  • the tag is a poly- His tag.
  • the His tag comprises 3-12 His residues.
  • the affinity chromatography is ⁇ 2+- ⁇
  • the contacting is performed by expressing both heavy chains in a cell.
  • the cell is an insect cell.
  • the MHC class II molecule is "empty" (not loaded with an antigenic, MHC class II binding peptide).
  • the method further comprises contacting the MHC class II molecule with an MHC class II binding antigenic peptide.
  • the MHC class II binding antigenic peptide is conjugated to a tag.
  • the tag is an affinity tag.
  • the tag conjugated to the MHC class II binding antigenic peptide is an affinity tag that is not a polyhistidine tag.
  • the tag is a desthiobiotin tag.
  • the tag is an acidic tag. In some embodiments, the acidic tag is an acidic cyanine dye. In some embodiments, the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences. In some embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
  • the tag is conjugated to the peptide via a cleavable linker.
  • the tag is a desthiobiotin tag.
  • the linker is a photocleavable linker.
  • the tag is a desthiobiotin tag.
  • the linker is an ⁇ linker.
  • the tag is a
  • the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
  • the tag is a desthiobiotin tag.
  • the tag is a part of a cleavable linker that remains after cleavage of the linker.
  • FIG. 1 MHC-peptide complexes under study.
  • FIG. 1 His tagged HLA-A2 under study and their refolding efficiencies.
  • HLA-A2 heavy chains containing the different tags were refolded with ⁇ 2 ⁇ in the presence of Flu matrixs 8 _ 66 peptide. The refolding efficiencies expressed as percent with 100% being the one of HLA-A2-BSP.
  • FIG. 3 Structures of the NTA linker under studies.
  • Experimental set up consisting in first loading streptavidin coated sensor chips with biotin- mono-NTA or biotin-di-NTA, followed by saturating with NiCl 2 and washing. The changes in resonance units (RU) were measured upon injecting the different A2/Flu complexes over the NTA-Ni 2+ loaded chips.
  • Figure 6 Dissociation kinetics of different NTA 2 -biotin-steptavidin A2/Flu 5 8 66 multimers.
  • FIG. 7 Dissociation kinetics of NTA 2 -and NTA 4 biotin-streptavidin A2/Flu 58 -66 multimers.
  • FIG. 8 Preparation of NTA 2 -PE conjugates.
  • A) Structure of PE a fluorescent protein of 240,000 Da, has a rigid structure and 24 surface exposed lysines.
  • PE was reacted with the maleimide N- hydroxy- succinimidyl ester SM(PEG) 2 (succinimidyl-maleido(PEG) 2 ), whereby maleimide groups are conjugated onto PE lysines.
  • the NTA 2 cysteine (Fig. 3E) was added, which by reacting with maleimides on PE forms stable thio-ethers.
  • Binding isotherms of NTA 2 -PE A2/Flu 58- 66 multimers Cloned 81P1 cells were incubated for 45 min at the indicated temperatures with graded concentrations of conventional BSP multimers (circles), or with multimers containing NTA 2 -PE coupled PE and A2/Flu complexes containing the 2xHis 6 tag (squares) or the His 12 tag (diamonds). After washing the cells they were analyzed by flow cytometry.
  • FIG. 10 Dissociation kinetics of NTA 2 -PE A2/Flu 5 8-66 multimers.
  • Cloned BCB 70 cells were incubated for lh at 4°C with conventional BSP multimers (circles) or multimers containing NTA 2 -PE conjugated with A2/Flu complexes containing a 2xHis 6 tag. After washing the cells they were incubated at the indicated temperatures in FACS buffer containing or not (squares) 50 mM imidazol (triangles) or 100 mM imidazol (diamonds). After the indicated periods of time the cells were analyzed by flow cytometry.
  • FIG. 11 FACS sorting with reversible NTA 2 -PE but not conventional A2/Flu multimers provides high cell viability.
  • A) Cloned BCB 70 cells were FACS sorted upon staining with the conventional A2/Flu5g_66 BSP (black bars) or NTA 2 -PE-2xHis6 multimers (dark gray bars), after 1 wash with 100 mM imidazol the cells were cultured and at the indicated intervals viable cells enumerated by trypan blue exclusion counting. As controls cells were left untreated (white bars) or washed 1 x with 100 mM imidazol (light gray bars).
  • FIG. 12 Synthesis of mono-NTA and di-NTA-biotin.
  • the Compounds shown in Fig. 3A and B were synthesized using: i) solid phase peptide synthesis on chloro-trityl resin; ii) Fmoc for transient protection and ii) coupling with TBTU (2 eq), HOBt (2 eq), DIEA (4 eq) (a) and for deprotection TFA/H20/TIPS (92.5/2.5/5) (b).
  • the intermediary thiol compounds were reacted with maleimido-NTA-lysine in 100 mM phosphate buffer, pH 7.2 (c). The distance x is about 16 A and y 23 A.
  • FIG. 13 Synthesis of an alternative di-NTA-biotin.
  • An alternative di-NTA- biotin was synthesized starting on a rink amide chloro-trityl resin to prepare the peptide back bone (a), which was reacted in solution with tri-tert. butyl-NTA lysine (b), followed by complete deprotection (c); in this compound the distance x' is about 19 A and y' 9 A.
  • FIG. 14 Synthesis of NTA_j-biotin.
  • backbone peptide was synthesized by solid phase peptide synthesis on a chloro-trityl resin ().
  • the deprotected and HPLC purified peptide was reacted in phosphate buffer with 8 equivalents of NTA maleimide (b).
  • the molecules comprises two di-NTA moieties joined by a flexible GGSGGGSGS (SEQ ID NO: 10) linker, which is the same as in the 2xHis6 tag (Fig. 2A).
  • Figure 15 Multimers containing the short di-NTA linker more avidly stain cloned CD8+ T cells than those containing the long di-NTA linker A, B) The experiment shown in Fig.
  • FIG. 17 Impact of the degree of NTA 2 conjugation of PE on NTA 2 -PE His tag multimer staining.
  • PE was conjugated with di-NTA as shown in Fig. 8 using a constant concentration of PE (10 nM) and the indicated concentrations of SM(PEG) 2 for 2h at ambient temperature, followed by alkylation NTA 2 -cysteine.
  • the resulting NTA 2 -PE conjugates were loaded with A2/Flus 8 _66 monomers containing the indicated His tags, the multimers (5 nM) were incubated at 20°C for 30 min with cloned 81P1 cells. After washing cell-associated multimers were measured by flow cytometry. For comparison conventional BSP multimers were included.
  • FIG. 18 Binding isotherms of different A2/Flu multimers on a polyclonal Flu- specific CD8+ T cell population.
  • a polyclonal population of Flu-specific CD8+ T cells was incubated at 20°C for 30 min with A2/Flu multimers containing NTA 4 -biotin and 2xHis 6 tag (light triangles, NTA 4 -biotin and His 12 (light diamonds), NTA 2 -biotin and2xHis 6 tag (dark triangles), NTA 2 -biotin and His 12 tag (dark diamonds), NTA2-PE and 2xHis 6 tag (squares) and conventional BSP multimers (circles). Cell associated fluorescence was measured by flow cytometry.
  • FIG. 21 Photochemical removal of tags from MHC II binding peptides.
  • FIG 22 Comparative staining of HA clones by conventional and immunopure DR4-HA multimers.
  • A-D The indicated HA 0 6- 3 i8-specific DR4-restricted Thl clones were incubated at 37°C for 2h with the indicated concentrations of the DR4 multimers containing: HA (conventional) (solid circles), H6-GSG-NPpA-HA 3 o6- 3 is (immunopure) (triangles), 3 ⁇ 4- GSG-NPpA-HA 0 6- 3 i8 (immunopure; after UV irradiation) (inverted triangles), HA 0 6- 3 is- NPPA-GSG-H 6 (immunopure) (diamonds), HA 30 6- 3 i8-NPpA-GSG-H 6 (immunopure, after UV irradiation) (two-colored circles). The cells were washed and the cell bound multimers (MFI
  • FIG 23 Preparation of immunopure DR4-HA306-318 monomers using Cy5.5. tagged peptides.
  • FIG. 24 Preparation of immunopure MHC II-peptide NTA multimers using the pY-D4-tag.
  • Empty MHC II molecules containing a C-terminal His tag at the acidic leucine zipper are purified by affinity chromatography on NTA columns and loaded with given peptides (e.g. HA 30 6- 3 is) containing the strongly negatively charged phospho-tyrosine-Asp 4 (pY-D 4 ) tag.
  • the peptide loading is monitored by ELISA using anti-pY mAb.
  • Immunopure monomers are obtained by anion exchange chromatography, from which the pY-D 4 tag is removed by UV irradiation and NTA multimers upon reaction with NTA-PE or NTA Qdots.
  • FIG. 25 Preparation and testing of immunopure DR4/HA 30 6- 3 is NTA multimers.
  • Figure 26 Impact of peptide loading on tetramer purity. The purity of tetramers decreases rapidly with decreasing peptide loading.
  • Figure 27 Effect of His tags on peptide binding to DR1, DR4 and DR52b.
  • FIG. 28 Neuraminidase treatment increases DR4/HA 30 6- 3 i8 multimer binding.
  • A) The indicated DR4-restricted, HA-specific T cell clones were pretreated or not with neuraminidase (30 min incubation at 37°C with 0.03 U/ml of neuraminidase) and incubated with conventional DR4/HA 30 6- 3 is multimers (20 ⁇ g/ml) at 37°C for 2h. After washing cell- associated multimers (MFI) were assessed by flow cytometry.
  • B,C) Cloned 10.5 (A) or 7.1 cells were incubated likewise with the indicated concentrations of conventional DR4/HA 3 o6- 31 8 multimers and cell associated fluorescence was assessed by flow cytometry.
  • FIG. 30 Staining isotherms on Flu stimulated PBMC with different multimers at different concentrations.
  • the x-axis provides the multimer concentration (in nM).
  • Figure 31 Exemplary NTA linkers.
  • FIG. 32 Staining performance of PE conjugates comprising A2/Fhi5 8 _66 monomers, or DR4/Flu HA 0 6- 3 i8 monomers.
  • FIG. 33 Comparison of conventional, PE-Cys-PEG2-NTA2 and PE-HNO-NTA2 multimers staining.
  • FIG. 34 Reduction of background staining by milk supplements.
  • Figure 35 Binding titration of NTAmers to determine best dilution for ex vivo staining.
  • FIG 36 Ex vivo staining with BSP and NTA multimers on fresh peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • FIG 37 Overview of an exemplary strategy for NTA-His tag multimer preparation using desthiobiotin (DTB).
  • FIG. 38 Structures of biotin and DTB.
  • FIG 40 Schematic of 2 ml of StreptActin High Capacity sepharose (IBA)
  • Figure 41 Quantification of flowthrough, washing and elution of a DTB-tagged peptide.
  • Figure 42 Purification of MHC I-DTB peptide complexes on StreptActin sepharose.
  • Figure 43 Exemplary scheme of DTB-HA peptide loading and purification.
  • Figure 44 Generation and purification of DR4-DTB-HA peptide complexes.
  • Figure 45 NTA moieties used in PE-NTA 2 and biotin-NTA 4 -SA-PE multimers.
  • Figure 46 Staining of Flu HA 306 - 3 i 8 -specific CD4 + T cells with DR4/HA 306 - 3 i 8 BSP, biotin-NTA 4 -SA-PA, or PE-NTA 2 multimers made with DTB- strep tactin purified monomers.
  • FIG. 47 Staining of HA-specific CD4 + T cell clones 9(A) or 8(B) with different concentrations of DR4/HA 306 - 3 i8 BSP, biotin-NTA 4 -SA-PA, or PE-NTA 2 multimers.
  • FIG. 50 Reversibility of multimer staining. Biotin-NTA 4 , but not BSP multimers, can be rapidly removed from stained, antigen- specific cells.
  • Figure 51 Comparative staining of a DRl/ESOu9- 143 cell line by DRl/NY-ESOu9- 143 BPS, PE-NTA 2 and biotin-NTA 4 -SA-PE multimers.
  • Some aspects of this invention relate to protein multimers in which conjugation of a plurality of monomeric proteins is based on chelate complex bonds between the monomeric proteins and a central carrier molecule. Methods for generation and the use of such protein multimers are also provided herein.
  • Some aspects of this invention provide "reversible" protein multimers that can be dissociated by releasing the chelate complex bonds between the carrier molecule and the monomeric proteins, for example, by withdrawal of a central ion from the chelate complex bond or by contacting the chelate complex with a free chelant that can displace one of the binding partners forming the chelate complex.
  • This chelate bond release results in re- monomerization of the proteins comprised in the multimer.
  • multimer assembly and/or re-monomerization are carried out under non-denaturing, physiological, and/or non-toxic conditions, rendering the respective multimers suitable for in vivo, ex vivo, and in vitro applications involving living cells and/or monomeric proteins prone to denaturation.
  • Some aspects of the invention provide, for the first time, that reversible MHC-peptide multimers can be built on Ni 2+ NTA-His tag complexes that exhibit equal or superior staining properties as compared to conventional multimers.
  • These novel staining reagents are fully reversible in that they can be rapidly dissociated into monomeric subunits upon addition of imidazol, which allows, for example, sorting of bona fide antigen-specific CD8+ T cells.
  • Some aspects of this invention provide methods and materials for the preparation of fluorescent MHC protein multimers, in which conjugation is based on chelate complex formation between nitrilotriacetic acid (NTA) and an amino acid sequence comprising a polyhistidine sequence, also referred to as a histidine tag (His tag).
  • NTA nitrilotriacetic acid
  • His tag an amino acid sequence comprising a polyhistidine sequence
  • the chelate complexes are formed in the presence of a Ni 2+ cation.
  • the His tag comprises 3-12 His residues.
  • the His tag is a hexahistidine (His 6 ) tag or a 2x His 6 tag, comprising to hexahistidine sequences separated by a short amino acid linker.
  • Some aspects of this invention are based on the recognition that reversible protein multimers based on chelate complex bonds are stable enough to be useful for various applications, for example, cell or protein staining and isolation procedures, but can be dissociated under physiological, non-toxic conditions, for example, by withdrawing a central ion that is required for the formation of the chelate complex or by contacting them with an agent competing for the chelate complex bond, and thus releasing the bond between carrier molecule and monomeric protein.
  • the reversible Ni 2+ NTA-His tag interaction- based multimers described herein are "reversible" in that they can be dissociated either by withdrawing the chelant cation (e.g.
  • Ni 2+ or Co 2+ or by adding free competing chelant, for example, free imidazol, which displaces the His tag from the chelate complex.
  • free imidazol is commercially available and is commonly used for purification of recombinant proteins on Ni 2+ NTA affinity chromatography (see e.g.
  • the interaction of one Ni 2+ NTA with a hexahistidine tag has a dissociation constant (K D ) of about 10 "6 -10 "7 M and is sufficiently stable to allow purification of His tagged recombinant proteins from culture supernatants (9, 10).
  • K D dissociation constant
  • different His tags have been examined and it has been shown that increasing the length of the His tag increases the stability of the complexes (9).
  • linking two His 6 tags via a flexible linker such as GGGSGGGSGS (SEQ ID NO: 11) provides a high increase in stability (11, 12).
  • linkers have been synthesized that contain two to four NTA groups (13-15).
  • the binding to His tags increases considerably with the number on Ni NTA entities. While, in some embodiments, the His tags are expressed tethered, or fused to a recombinant protein, the NTA compounds have to be synthesized, as only mono-NTA (NTA1) derivatives are commercially available.
  • NTA1 mono-NTA
  • reversible multimers as provided herein, can be customized to fulfill specific requirements of a wide variety of research, diagnostic, and therapeutic applications.
  • reversible MHC multimers for example, reversible MHC class I multimers
  • fluorescently-labeled soluble MHC peptide multimers are provided.
  • methods for the use of reversible MHC multimers are provided, for example, methods useful to quantitate, isolate and/or characterize antigen-specific T-cells, for example, CD8 + and CD4 + T cells.
  • methods for the use of MHC multimers are provided that are useful for phenotypic T-cell analysis or for the analysis of T-cell receptor ("TCR") repertoire in a subject.
  • MHC multimers are typically prepared by enzymatic biotinylation of monomeric MHC proteins comprising a C-terminal biotinylation sequence peptide (BSP) and subsequent conjugation with streptavidin bound to a fluorescent dye, typically phycoerythrin (PE) or allophycocyanine (APC). Due to the large size of PE and APC, conjugates with streptavidin vary in stoichiometry, accessibility, and orientation of the biotin binding sites, often resulting in variable valency and inhomogeneous populations of MHC multimers.
  • BSP C-terminal biotinylation sequence peptide
  • PLC allophycocyanine
  • Non-phycobilin-based MHC multimers have also been developed, for example Quantum dots loaded with MHC class I-peptide complexes, which allow simultaneous use of multiple MHC class I-peptide specificities in polychrome flow cytometry, Cy5-labeled dimeric, tetrameric and octameric MHC class I-peptide complexes, dextramers (Immudex, Copenhagen, Denmark) and dimeric MHC-peptide-immunoglobulin (Ig) fusion proteins.
  • exemplary reversible multimers described herein include reversible MHC protein multimers
  • the methods and reagents provided herein can be applied to generate reversible multimers of proteins other than MHC proteins.
  • the methods for the generation and use of protein multimers are universally applicable to proteins of different nature, for example, to binding proteins, such as MHC proteins, antibodies, antibody fragments, ligands, adnectins, and receptors or receptor fragments.
  • Exemplary multimers of such binding proteins namely of peptide-loaded or empty MHC molecules, are provided in the working example section.
  • binding molecule refers to a molecule that is able to bind a binding partner via non- covalent interaction.
  • a binding molecule is able to form a binding interaction with a binding partner that is strong enough to be stable under physiological conditions or under the conditions typically encountered during cell processing.
  • a binding molecule binds its binding partner with high specificity and/or high affinity.
  • binding molecules are antibodies and antibody fragments (e.g., Fab, F(ab)'2, single chain antibodies, diabodies, etc.), receptors, proteins binding a ligand, aptamers, and adnectins.
  • the term "ligand" is art-recognized and refers to a binding partner of a binding molecule.
  • Ligands can be, for example, proteins, peptides, nucleic acids, small molecules, and carbohydrates.
  • Avidins for example, streptavidin, are non-limiting examples of binding molecules that can bind a ligand, in this case, for example, biotin.
  • reversible antibody fragment for example, Fab fragment, multimers are provided, in which a plurality of Fab proteins is bound to a central carrier molecule via tone complex bonds, for example, NTA-His bonds.
  • the carrier molecule is a fluorescent microsphere, for example, a
  • Some aspects of this invention provide reversible protein multimers in which a plurality of proteins is conjugated to a central carrier molecule via a non-covalent binding interaction that can be released under physiological conditions, for example, by contacting the multimer with an agent able to displace one of the binding partners from the binding interaction.
  • protein refers to a polymer of at least three amino acid residues linked together by peptide bonds.
  • the terms are interchangeably used herein and refer to proteins, polypeptides, and peptides of any size, structure, or function.
  • a protein will be at least three amino acids long.
  • inventive proteins contain only natural amino acids, although in other embodiments non-natural amino acids (e.g., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, US Patent 7045337, which describes incorporation of non-natural amino acids into proteins) and/or amino acid analogs as are known in the art may alternatively be employed.
  • one or more of the amino acids in an inventive protein are modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation,
  • a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation
  • a protein may also be a single molecule or may be a multi-molecular complex.
  • a protein may be just a fragment of a naturally occurring protein or peptide.
  • a protein may be naturally occurring, recombinant, or synthetic, or any combination of these.
  • the protein is an MHC molecule.
  • conjugation refers to an entity, molecule, or moiety that is stably associated with another molecule or moiety via a covalent or non-covalent bond.
  • the conjugation is via a covalent bond, for example, in the case of a peptide tag conjugated to an MHC protein via fusion of the peptide to a heavy chain of the MHC protein.
  • the conjugation is via a non-covalent interactions, for example, via hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, or electrostatic interactions.
  • carrier molecule refers to a molecule that binds or is conjugated to a plurality of monomeric molecules or entities, or a plurality of binding molecules or moieties that can bind such monomeric molecules or entities.
  • the carrier molecule is a monomeric molecule, for example, a single molecule of a fluorescent dye.
  • the carrier molecule is a multimeric molecule, for example, a polymer, or a nanocrystal.
  • the carrier molecule is a fluorophore, for example, a phycobilin, conjugated to a plurality of binding molecules, for example, streptavidin.
  • the streptavidin molecules can bind monomeric molecules, for example, biotin-conjugated MHC monomers.
  • the carrier molecule is a multivalent chelant molecule.
  • the carrier molecule is a fluorescent microsphere, for example, a
  • the carrier molecule for example, a fluorophore
  • the carrier molecule is conjugated to a plurality of chelant molecules or moieties, for example, NTA molecules that can form chelate complex bonds with histidine residues in the presence of a divalent cation.
  • chelant configuration refers to the number and spacing of chelant molecules or moieties in a given structure. For example, if chelant moieties, for example, NTA moieties, are conjugated to a carrier molecule, for example, a multivalent binding molecule or a fluorophore, via a linker, then a configuration in which a single chelant moiety is conjugated to the carrier molecule via a single linker is referred to as mono-configuration (e.g.
  • NTA mono- NTA, or NTA
  • di-configuration e.g. di-NTA, or NTA 2
  • tetra-configuration e.g. tetra-NTA, or NTA 4
  • different NTA chelant configurations and linker structures affect chelate complex bond properties, including bond strength and, thus, bond stability and reversibility.
  • the His tag is a His6 tag, comprising 6 contiguous His residues, a His 12 tag, comprising 12 contiguous His residues, or a 2xHis6 tag, comprising two sequences of 6 contiguous His residues linked by a short spacer sequence as described in more detail elsewhere herein.
  • divalent cation refers to an ion that lacks two electrons as compared to the neutral atom.
  • divalent cations useful in some embodiments of this invention are Ni2+, Cu2+, Zn2+, Co2+, Cd2+, Sr2+ , Mn2+, Fe2+, Mg2+, Ca2+, and Ba2+.
  • Other useful divalent cations will be apparent to those of skill in the art and the invention is not limited in this respect.
  • the carrier molecule is a monomeric carrier molecule. In some embodiments, the carrier molecule is a multimeric or polymeric carrier molecule. For example, in some embodiments, the carrier molecule is a tetrameric or a hexameric molecule, for example, a fluorophore. In some embodiments, the carrier molecule is a fluorophore, a phycobilin, phycoerythrin or allophycocyanine, a nanocrystal, a quantum dot (Qdot), a magnetic particle, or a nanoparticle.
  • Quantum dot and “Qdot,” as used herein, refer to fluorescent inorganic semiconductor nanocrystals in which the excitons are confined in all three spatial dimensions and which are useful as detectable agents in some
  • the Qdot comprises CdSe or CdTe. In some embodiments, the Qdot comprises InP or InGaP. In some embodiments, the Qdot comprises a core/shell structure, while in other embodiments, the Qdot is a core-only Qdot.
  • Exemplary Qdots and methods for use and production are described in Rech-Genger et al., Quantum dots versus organic dyes as fluorescent labels. Nature Methods 2008 (9):763-775, incorporated herein in its entirety by reference for disclosure of fluorescent Qdots and organic dyes, and methods of production and use of same).
  • the carrier molecule is a water-soluble molecule.
  • water-soluble is art-recognized and qualifies that an agent can be dissolved in water to a certain degree, or , in other words, that a certain amount of the agent can be dissolved in a certain volume of water.
  • a water-soluble carrier molecule is a carrier molecule that exhibits a solubility in water at 25 °C and 1 ATM of more than O.
  • lg/ml more than 0.2g/ml, more than 0.25g/ml, more than 0.3g/ml, more than 0.4g/ml, more than 0.5g/ml, more than 0.6g/ml, more than 0.7g/ml, more than 0.8g/ml, more than 0.9g/ml, more than lg/ml, more than l. lg/ml, more than 1.2g/ml, more than 1.3g/ml, more than 1.4g/ml, more than 1.5g/ml, more than 1.6g/ml, more than 1.7g/ml, more than 1.8g/ml, more than 1.9g/ml, more than 2g/ml.
  • the carrier molecule is not water soluble. In some such embodiments, the carrier molecule is highly dispersible in water and/or does not precipitate in aqeous solution under physiological conditions. In some embodiments, the diameter of the carrier molecule is less than O.
  • lnm less than 0.2nm, less than 0.25nm, less than 0.3nm, less than 0.4nm, less than 0.5nm, less than 0.6nm, less than 0.7nm, less than 0.75nm, less than 0.8nm, less than 0.9nm, less than lnm, less than l.lnm, less than 1.2nm, less than 1.3nm, less than 1.4nm, less than 1.5nm, less than 1.6nm, less than 1.7nm, less than 1.8nm, less than 1.9nm, less than 2nm, less than 2.5nm, less than 3nm, less than 4nm, less than 5nm, less than 6nm, less than 7nm, less than 8nm, less than 9nm, or less than lOnm.
  • the diameter of the carrier molecule is less than 20nm, less than 30nm, less than 40nm, less than 50nm, less than 60nm, less than 70nm, less than 80nm, less than 90nm, less than lOOnm, less than 200nm, less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm, or less than ⁇ .
  • the non-covalent interaction is a non-covalent bond with a dissociation constant K D of 5 ⁇ > K D > lfM, for example, of ⁇ > K D > lpM, or of ⁇ > K D ⁇ lOOfM.
  • K D dissociation constant
  • the term "dissociation constant,” abbreviated as K D herein, is art- recognized and refers to a specific type of equilibrium constant that measures the propensity of a complex of associated molecules to separate (dissociate) reversibly into the separate molecules.
  • the dissociation constant is the inverse of the association constant. For a general reaction A x B y ⁇ xA + yB, in which a complex A x B y breaks down into x A subunits and y B
  • the dissociation constant is defined as l j 3 ⁇ 4 j 3 ⁇ 4j , where [A], [B], and
  • [A x B y ] are the concentrations of A, B, and the complex A x B y , respectively.
  • a protein multimer is provided in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant 5 ⁇ > KD > InM.
  • a protein multimer is provided in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant 5 ⁇ > KD > lpM.
  • a protein multimer in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant of less than 2 fM, less than 5 fM, less than 10 fM, less than 20 fM, less than 50 fM, less than 100 fM, less than 250 fM, less than
  • a protein multimer in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant of more than lpM and less than ⁇ , more than ⁇ and less than ⁇ , more than ⁇ and less than 500nM, more than ⁇ and less than
  • ⁇ more than ⁇ and less than ⁇ , more than ⁇ and less than 4000nM, more than 200nM and less than 3000nM, more than 500nM and less than 2000nM, or more than 500pM and less than ⁇ .
  • the carrier molecule or the protein, or both are conjugated to a chelant moiety via a covalently bound linker.
  • linker refers to a chemical structure between two molecules or moieties or between a molecule and a moiety, thus linking the two.
  • the linker is covalently bound to both linked elements.
  • the linker is covalently bound to one, but not the other linked element.
  • the linker is non-covalently bound to one or both elements.
  • a linker is covalently bound to a carrier molecule and a chelant moiety, while in other embodiments, a linker is covalently bound to a chelant moiety and non-covalently bound to a carrier molecule.
  • the linker is about 2A, about 3A, about 4A, about 5A, about 6A, about 7 A, about 8A, about 9 A, about 10A, about 11 A, about 12A, about 13A, about 14A, about 15A, about 16 A, about 17A, about 18 A, about 19 A, about 20A, about 21A, about 22A, about 23A, about 24A, about 25A, about 26A, about 27A, about 28A, about 29A, about 30A, about 35A, about 40A, about 45A, or about 50A long. In some embodiments, the linker is longer than about 50A.
  • NTA moieties are conjugated with a carrier molecule, for example, a PE molecule, using maleimide alkylation chemistry.
  • a carrier molecule for example, a PE molecule
  • maleimide alkylation chemistry for example, in some embodiments, PE is first reacted with an NHS-maleimide and the maleimido-PE
  • chelate moieties for example, NTA moieties
  • a carrier molecule for example, a PE molecule
  • oxime chemistry has several advantages: i) efficient conjugation in the pH range 5-7; ii) good stability under physiological conditions; and iii) efficient conjugation at low protein concentrations.
  • An exemplary oxime chemistry for attachment of NTA moieties to a carrier is described in the following scheme:
  • chemistry strategies provided herein for example, the maleimide and oxime chemistry strategies described, are universally applicable to generate MHC multimers, for example, multimers of MHC class I or class II as described herein. Additional maleimide and oxime chemistry strategies will be apparent to those of skill in the art and it will be appreciated that the disclosure is not limited in this respect.
  • oxime chemistry also referred to herein as oxime ligation
  • Oxime formation benefits from the exclusive specificity and reactivity between the aminooxy function and the carbonyl group, since the nitrogen atom behaves as a weak base and as an excellent nucleophile.
  • aldehydes are
  • the oxime ligation reaction can occur in mild acidic conditions (pH 5) within 1 h but also at physiological conditions (pH 7) in 10 h.
  • the resulting imine bond is covalent and stable under
  • aminooxy-containing peptides are obtained by inserting the classical fmoc-Dpr(Aoa)-OH residue at the last stage of solid phase peptide synthesis. Incorporation of this residue is efficient (quantitative yields) and does not require specialized conditions. Final TFA deprotection of the peptide results in a free aminooxy function.
  • the incorporation of the aldehyde moiety on the biomolecule to be derivatized is achieved, in some embodiments, by employing a commercially available sulfo-SFB molecule, that reacts on lysines via succinimidyl ester reaction.
  • oxime chemistry over using a thiol-maleimide chemistry strategy is that oxime formation is not subjected to a hydrolysis or degradation reaction.
  • oxime chemistry strategies lead to better incorporation yields.
  • Another benefit is that the functionalized entities can be prepared and stored for several months before being mixed together.
  • Oxime chemistry reactions, reagents, and reaction conditions are well known to those of skill in the art. Some reactions, reagents, and reaction conditions are described herein. Additional suitable reactions, reagents, and reaction conditions will be apparent to those of skill in the art and it will be appreciated that this disclosure is not limited in this respect.
  • a protein multimer in which a plurality of proteins is conjugated to a multivalent carrier molecule and wherein at least one of the proteins is conjugated to the carrier molecule via a chelate complex bond.
  • chelate complex refers to a chemical structure that comprises two or more separate, non-covalent binding interactions between a polydentate (multiple bonded) molecule or moiety, also referred to as "chelant", and a single central atom, for example, a divalent cation.
  • chelate complex bond accordingly, refers to a non-covalent bond between two or more chelants that form a chelate complex. In some embodiments, all chelants of a chelate complex are of the same structure. In other
  • a chelate complex is formed by chelants of different structures, for example, by a chelant comprising a histidine residue and a chelant comprising an NTA residue.
  • the central atom is a divalent cation, for example, an Ni 2+ cation.
  • Chelants, chelant moieties, and suitable central atoms are well known to those of skill in the art and the invention is not limited in this respect.
  • a protein multimer in which a plurality of MHC molecules is conjugated to a carrier molecule by a non-covalent bond as described herein, for example, a chelate complex bond or a bond of a K D value as provided elsewhere herein.
  • MHC molecule refers to a protein encoded by the major histocompatibility complex, and includes MHC class I and MHC class II molecules.
  • the MHC molecule is an MHC class I molecule and the MHC multimer is an MHC class I multimer.
  • the MHC molecule is an MHC class II molecule and the MHC multimer is an MHC class II multimer.
  • the MHC molecule is a human MHC molecule. In humans,
  • MHC molecules are also referred to as HLA molecules.
  • the MHC molecule is an MHC molecule of a non-human mammal, for example, of a mouse, a rat, a rabbit, a non-human primate, a cat, a dog, a goat, a cow, a sheep, a horse, or a pig.
  • MHC class-I molecules comprise one heavy chain type a that comprises three domains (al, a2, and a3). In naturally occurring MHC class I molecules, these domains are exposed to the extracellular space, and are linked to the cellular membrane through a transmembrane region. The a chain of MHC class I molecules is associated with a molecule of ⁇ 2 microglobulin, which is not encoded by an MHC gene, but also included within the scope of the term "MHC molecule".
  • MHC class II molecules comprise two heavy chains, one type a and one type ⁇ , each of which comprises two domains: al and ⁇ 2, ⁇ and ⁇ 2, respectively. In naturally occurring MHC class II molecules, these domains are exposed to the extracellular space and are linked to the cellular membrane through a transmembrane region on each of the two chains.
  • an MHC multimer comprises a genetically engineered MHC molecule.
  • an MHC molecule as provided herein comprises an extracellular domain of a naturally occurring MHC molecule, or a genetically engineered derivative thereof, but is devoid of all or part of the transmembrane domain or domains.
  • MHC class II molecules are provided that comprise a leucine zipper in place of the transmembrane domain in order to achieve dimerization of a and ⁇ chains.
  • MHC proteins for example, MHC molecules lacking transmembrane domains, MHC molecules comprising leucine zippers, single chain MHC molecules or MHC molecules fused to an antigenic peptide, are also included in the scope of the term "MHC molecule".
  • MHC molecule refers to a complete molecule, for example, an MHC heavy chain type a (genetically engineered or not) that is associated with a molecule of ⁇ 2 microglobulin in the case of an MHC class I molecule, or an MHC heavy chain type a (genetically engineered or not) that is associated with an MHC heavy chain type ⁇ (genetically engineered or not), for example, via leucine zipper interaction.
  • MHC molecule refers to a single component of an MHC molecule, for example, to an MHC heavy chain (e.g. type a or type ⁇ , genetically engineered or not), or to a ⁇ 2 microglobulin.
  • MHC molecules can bind antigenic peptides in a groove formed by the l and a2 domains of MHC class I molecules or by the al and ⁇ domains of MHC class II molecules.
  • An MHC molecule that has bound an antigenic peptide is referred to herein as a "peptide - loaded” MHC molecule, whereas an MHC molecule that has not bound an antigenic peptide is referred to herein as an "empty" MHC molecule.
  • Non-limiting examples of antigenic peptides are a peptide that is recognized by a B or T-cell, e.g. via binding to a T-cell receptor, or a peptide that binds to an antibody or antibody fragment, or a peptide that stimulates an immune response in a subject.
  • MHC molecule refers to a single MHC molecule, for example, to a single MHC heavy chain, a single MHC heavy chain associated with a ⁇ 2 microglobulin, or a heterodimer of an MHC type a heavy chain and an MHC type ⁇ heavy chain.
  • MHC multimer refers to a plurality of MHC molecules associated with each other, for example, via non-covalent interaction with a carrier molecule.
  • the term “multimer” excludes dimers, but includes trimers, and multimers of four monomers (tetramers), or of more than four monomers (pentamers, hexamers, septamers, octamers, nonamers, decamers, etc.). In some embodiments, the term “multimer” excludes dimers and trimers, but includes multimers of four and more monomers.
  • Some aspects of this invention provide a multivalent chelant, that comprises a plurality of chelant moieties conjugated to a carrier molecule.
  • Such multivalent chelants are useful for the generation of reversible multimers, for example, of reversible protein multimers (e.g. MHC multimers), as described elsewhere herein.
  • multivalent chelant molecule refers to a carrier molecule comprising or conjugated to a plurality of chelant moieties able to form chelate complex bonds with a plurality of chelant-moiety comprising molecules.
  • a tetravalent chelant molecule is a carrier molecule that is able to form chelate complex bonds to four molecules, for example, four MHC proteins comprising a compatible chelant moiety, thus forming a tetramer of the four molecules held together by chelate complex bonds.
  • a compatible chelant moiety is a chelant moiety able to form a chelate complex bond with the chelant moiety of the carrier molecule.
  • an NTA moiety and a Histidine moiety are compatible chelant moieties, since they can form a chelant complex bond in the presence of a divalent cation.
  • the multivalent chelant molecule can form a chelate complex bond with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, or 150 molecules comprising a compatible chelant moiety.
  • the multivalent chelant molecule can form a chelate complex bond with more than 150
  • the carrier molecule is a water-soluble molecule as described in more detail elsewhere herein. In some embodiments, the carrier molecule is a non-water soluble molecule as described in more detail elsewhere herein.
  • the chelant moieties are tridentate or tetradentate chelant moieties. In some embodiments, the chelant moieties are nitrilotriacetic acid (NTA) moieties. In some embodiments, the chelant moieties are histidine residues, for example, in the form of histidine tags. In some embodiments, the chelate moieties are iminodiacetic acid (IDA) moieties.
  • NTA nitrilotriacetic acid
  • the chelant moieties are histidine residues, for example, in the form of histidine tags.
  • the chelate moieties are iminodiacetic acid (IDA) moieties.
  • the chelant moieties are covalently bound to the carrier molecule via a linker, for example, via a linker described herein.
  • each linker is bound to a single chelant moiety (e.g., mono-NTA configuration).
  • two chelant moieties are bound to a single linker (e.g., di-NTA configuration).
  • four chelant moieties are bound to a single linker (e.g., tetra-NTA configuration).
  • the invention provides methods for the generation of reversible protein multimers in which a plurality of proteins is conjugated to a carrier molecule via a chelate complex bond.
  • the carrier molecule and the protein are conjugated to the respective chelant moieties via covalent bond, for example, via a covalently bound linker.
  • the only non-covalent bond between carrier molecule and protein monomer is the chelate complex bond.
  • an additional non- covalent bond is introduced between the carrier molecule and the monomeric protein, for example, a binding molecule/ligand bond, such as a streptavidin/biotin interaction.
  • the method includes a step of contacting a monomeric protein, for example, an MHC molecule, that is conjugated to a chelant moiety with a carrier molecule as provided herein, that is conjugated to a plurality of chelant moieties under conditions suitable for formation of a chelate complex between the chelant moieties conjugated to the MHC molecule and the chelant moieties conjugated to the carrier molecule.
  • a method for the generation of protein multimers for example, MHC protein multimers
  • a protein molecule for example, an MHC molecule
  • a first chelant for example, a His tag
  • a ligand molecule for example, biotin
  • conjugated to a second chelant under conditions suitable for formation of a chelate complex between the first and the second chelant.
  • the resulting product is a monomeric protein conjugated to a ligand via a non-covalent chelate complex bond.
  • Such monomers can be assembled to reversible multimers by contacting them with a carrier molecule conjugated to a plurality of ligand-binding molecules or moieties, for example, streptavidin molecules.
  • a multivalent carrier molecule is generated by contacting a carrier molecule conjugated to a plurality of chelant moieties via non-covalent interaction, for example, via biotin/streptavidin interaction.
  • a carrier molecule comprising a plurality of streptavidin moieties is contacted with a plurality of biotin molecules that are conjugated to chelant moieties, for example, NTA moieties, via a covalently bound linker as described herein.
  • methods for the production of peptide-loaded MHC multimers are provided. While stable, peptide-loaded MHC class I proteins can be obtained by refolding of MHC class I heavy chains with peptides of interest, recombinant MHC class II proteins are more difficult to obtain.
  • recombinant MHC class II molecules are produced in soluble form by insect expression systems, such as Drosophila S2 cells or baculovirus and sf9 cells. With very few exceptions, deletion of the transmembrane (TM) domains of the a and ⁇ chains in MHC class II molecules results in the dissociation of the two subunit chains. In some embodiments, chain pairing is re-established by addition of leucine zippers.
  • empty MHC class II molecules are first isolated and then loaded with an antigenic peptide of interest.
  • Such peptide-loaded MHC class II molecules can then be isolated and used in the production of MHC protein multimers.
  • peptides can be fused to the N-terminus of the ⁇ chain via a flexible linker. Such fusions of MHC class II chains and antigenic peptides, resulting in the production of recombinant, peptide-loaded MHC molecules, are well known to those of skill in the art.
  • a chelant moiety for example, a His tag
  • a chelant moiety is added at the C- terminus of an MHC chain by recombinant addition of a fusion peptide comprising a chelant moiety, for example, in the form of a His tag as described herein.
  • a chelant moiety is attached to the isolated, peptide-loaded MHC molecule after isolation. Methods for post-synthesis or post- isolation of chelants to isolated proteins are known to those of skill in the art and exemplary methods are described herein.
  • the MHC molecule is sufficiently stable without peptide cargo (e.g. HLA DRB1*0101 or DRB 1*0401) to allow the production of empty MHC molecules and MHC multimers, e.g., MHC molecules or multimers that are not loaded with an antigenic peptide.
  • the MHC monomer is loaded after isolation or purification with the peptide of interest.
  • the MHC monomer is first incorporated into a reversible MHC multimer as provided herein and subsequently loaded with a peptide of interest. The efficiency of peptide loading strongly depends on its binding strength to the respective MHC molecule. If the binding is below a critical threshold, peptide loading is inefficient and the resulting complexes are of limited stability, both physically and conformationally.
  • Some embodiments of the invention provide methods for the generation of MHC molecules and multimers that are loaded with an antigenic peptide.
  • methods and reagents for the production of peptide-loaded MHC class II molecules are provided.
  • the production of peptide-loaded MHC class II molecules is technically difficult, based on the instability of engineered MHC class II heterodimers comprising a and ⁇ heavy chains lacking a transmembrane domain, and, in many instances, the low affinity binding interactions between the MHC class II heavy chains and the antigenic peptide.
  • populations of peptide-loaded MHC class II molecules produced with conventional methods are often heterogeneous in that a significant portion of MHC class II molecules are not or not correctly peptide-loaded.
  • MHC class II multimers produced from such heterogeneous populations of MHC class II molecules often show poor staining performance, great batch-to- batch variability in staining efficiency, and some specific peptide-loaded MHC class II multimers are difficult or impossible to obtain with conventional methods.
  • Some aspects of this invention provide methods addressing these problems in the production of MHC class II molecules and multimers.
  • some aspects of this invention provide methods and reagents for the generation of peptide-loaded MHC molecule, for example, MHC class II molecules, that include the use of a tag conjugated to the antigenic peptide.
  • MHC molecules that have bound a tagged antigenic peptide are isolated and/or purified by a method that can be carried out under non-denaturing conditions, for example, by certain chromatography methods (e.g., affinity chromatography or ion exchange chromatography).
  • the tag conjugated to the antigenic peptide can be removed, for example, by cleaving a linker that connects the tag to the antigenic peptide, and methods for tag removal from tagged peptide-loaded MHC molecules, for example, MHC class II molecules, are also provided herein.
  • the antigenic peptide of interest is conjugated to a tag.
  • the tag is a peptide tag, for example, a peptide tag that is N-terminally or C-terminally fused to the antigenic peptide.
  • the tag is an affinity tag that allows for the isolation of correctly loaded MHC class II molecules by affinity chromatography.
  • Affinity tags are well known to those of skill in the art and examples of peptide tags include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags , calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags , biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags.
  • BCCP biotin carboxylase carrier protein
  • myc-tags myc-tags
  • calmodulin-tags FLAG-tags
  • HA hemagglutinin
  • the tag is a biotin tag or a biotin variant tag, for example, desthiobiotin (DTB).
  • DTB is a biotin variant that binds about lxl0 6 -fold weaker to streptavidin than biotin. DTB is readily displaced by free biotin, allowing gentle affinity purification based on the reversible DTB -streptavidin conjugation. Conjugation partners similar to streptavidin can also be employed, for example, StreptActin, a mutant of streptavidin.
  • Strep tags which are peptidic biotin analog, bind also to StreptActin.
  • Desthiobiotin, biotin, streptavidin, StreptActin, strep tags and derivatives of these reagents, as well as methods for the use of these reagents in protein and peptide purification are well known to those of skill in the art. Some methods suitable according to aspects of this invention are described herein, and additional suitable methods will be apparent to those of skill, for example, as described in Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A, Ting AY. A monovalent streptavidin with a single femtomolar biotin binding site. Nat Methods.
  • Some aspects of this invention provide tagged MHC class II binding antigenic peptides and methods of using such peptides.
  • a tag conjugated to an antigenic peptide is useful for the isolation of the tagged peptide, either alone or when bound to an MHC class II molecule.
  • Methods for isolating tagged peptides are well known to those of skill in the art and include, for example, affinity chromatography and ion exchange chromatography.
  • an MHC class II binding peptide is provided or used that is conjugated to a tag.
  • the tag is an acidic tag.
  • the acidic tag is an acidic peptide tag, for example, a peptide tag comprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, or more contiguous acidic amino acid residues.
  • the acidic amino acid residues glutamic acid (Glu, E) or aspartic acid (Asp, D) residues.
  • the antigenic peptide is conjugated to an acidic tag that allows for the isolation of correctly loaded MHC class II molecules by anion exchange chromatography.
  • the tag is a tag comprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Asp residues, for example, a pY-D4 tag
  • the acidic tag is a tag comprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Glu residues, for example, a pY-E6 (phosphor-tyrosine followed by six Glu), pY-E7, pY-E8, pY-E9, or a pY-E10 tag.
  • the acidic tag is an acidic detectable label, for example, an acidic fluorophore.
  • the acidic fluorophore is a cyanine dye tag, for example, a Cy5 tag, or a Cy5.5 tag.
  • cyanine dye tag for example, a Cy5 tag, or a Cy5.5 tag.
  • Other acidic tags suitable for peptide or protein isolation and/or purification are well known to those of skill in the art and the invention is not limited in this respect.
  • tagged MHC class II binding peptides are provided that are reversibly tagged, e.g., that are tagged with a structure that can be cleaved, resulting in a release of the conjugated tag from the peptide. Methods of using such reversibly tagged peptides are also provided.
  • the tag is conjugated to the peptide via a cleavable linker.
  • the linker is a photocleavable linker.
  • the linker is a 2-nitro-phenyl-P-Ala ( ⁇ ) linker.
  • the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
  • the tagged peptide is a peptide conjugated to a part of a cleavable linker that remains after cleavage of the linker.
  • cleavable linker is that after isolation of the peptide-loaded MHC class II molecule with a method relying on the tag, the tag can be removed. Cleavage of a cleavable linker will, in some embodiments, leave part of the linker conjugated to the MHC class II binding peptide. However, some linkers can be designed in a manner that will result in complete removal of the linker from the MHC-class II binding peptide.
  • Tag removal by linker cleavage is particularly useful in embodiments, where the tag interferes or is suspected to interfere with the binding of the peptide-loaded MHC class II molecule to its target T-cell receptor.
  • Exemplary cleavable linkers are described in more detail elsewhere herein. Additional cleavable linkers are known to those of skill in the art and the invention is not limited in this respect.
  • the cleavable linker is a photocleavable linker. Photocleavable linkers can be cleaved by irradiation with UV light. Photocleavable linkers are described herein and additional photocleavable linkers are well known in the art.
  • Photocleavable peptide-DNA conjugates synthesis and applications to DNA analysis using MALDI-MS. Nucleic Acids Res. 1999 Dec 1;27(23):4626-31; Olejnik J, Krzymanska- Olejnik E, Rothschild KJ. Photocleavable aminotag phosphoramidites for 5 '-termini DNA/RNA labeling. Nucleic Acids Res. 1998 Aug l;26(15):3572-6. Olejnik J, Krzymanska- Olejnik E, Rothschild KJ. Photocleavable affinity tags for isolation and detection of biomolecules.Met ods, Enzymol.
  • a peptide tagged via a cleavable linker is referred to as a reversibly tagged peptide.
  • an MHC class II molecule loaded with a reversibly tagged peptide is isolated, for example, by affinity or ion exchange chromatography, and the tag is removed after isolation by cleavage of the linker.
  • the MHC class II molecule loaded with a now untagged peptide is then isolated from the cleaved tag, for example, by size fractionation.
  • an MHC molecule for example, an MHC class II molecule conjugated to a chelant moiety is loaded with a tagged peptide.
  • the peptide tag is an acidic tag, as provided herein, and the chelant moiety is comprised in a peptide tag, for example, a His tag, as provided herein.
  • an MHC class II molecule is provided that comprises a heavy chain conjugated to a His tag, for example, by C-terminal fusion, and that is loaded with a tagged antigenic peptide, for example, an antigenic peptide conjugated to an acidic tag, for example, a Cy5.5 tag) via a photocleavable linker.
  • a correctly peptide-loaded MHC class II molecule is isolated by anion exchange chromatography, the tag is cleaved from the antigenic peptide, for example, by UV irradiation, and the peptide-loaded MHC class II molecule thus generated is then assembled into an MHC multimer as described herein.
  • an MHC molecule for example, an MHC molecule loaded with an antigenic peptide is conjugated with a chelant moiety or a peptide tag after production of the MHC molecule or after loading the MHC molecule with the antigenic peptide, or after cleavage of a tag, if present, from a reversibly tagged antigenic peptide bound to the MHC molecule.
  • such post-production conjugation for example, to a heavy chain of a peptide-loaded MHC class II molecule, is carried out by site- specific alkylation or a sortase-mediated transpeptidation reaction.
  • the antigenic peptide is tagged with a His tag, the tag is cleaved after peptide-loading, and a His tag is subsequently appended to a heavy chain of the MHC molecule.
  • tags can be used subsequently, or the same tag can be employed at different positions.
  • Methods for post-synthesis addition of tags to peptides and proteins are well known to those of skill in the art and include, but are not limited to, biotinylation and sortase-mediated protein labeling (for the latter see Popp et al., Site Specific Protein Labeling via Sortase -Mediated Transpeptidation Curr. Protoc. Protein Sci.
  • an MHC class II molecule comprising a sortase target sequence is loaded with a His-tagged MHC class II -binding peptide and peptide-loaded MHC class II molecules are isolated form free peptide and empty MHC class II molecules by affinity chromatography and, optionally, size fractionation. After isolation, the His-tag is cleaved off the MHC class II binding peptide and, optionally, the tag-free MHC class II molecule loaded with the peptide is separated from the cleaved-off tag.
  • the heavy chain of the MHC class II molecule is then tagged by performing a sortase-mediated transpeptidation reaction.
  • the tag appended to the MHC heavy chain is a peptide tag.
  • the sortase- appended tag is a His-tag, thus effectively yielding MHC class II molecules in which the His tag was moved from the antigenic peptide to a heavy chain.
  • a peptide-loaded MHC class II molecule is isolated by ion exchange chromatography.
  • anion exchange chromatography is used to isolate the tagged peptide or an MHC class II molecule loaded with the tagged peptide.
  • Some aspects of this invention provide methods and reagents for the generation of "empty" MHC class II molecules.
  • empty in the context of MHC class II molecules signifies that the MHC molecules are not loaded with an antigenic, MHC class II- binding peptide.
  • empty MHC class II molecules are often instable and conventional methods of high-affinity antibody-mediated isolation are typically unsuitable for the preparation of such empty MHC class II molecules because of the denaturing conditions used in such methods.
  • Some aspects of this invention provide methods that allow gentle purification of fragile "empty" (without nominal peptide cargo) His tagged MHC II molecules by affinity chromatography on Ni2+ nitrilotriacetic acid (NTA) columns.
  • methods are provided that allow for the isolation of empty MHC class II molecules that retain the correct folding and dimerization properties.
  • such methods include a step of tagging a heavy chain comprised in an MHC class II molecule.
  • the tag is a peptide tag that can be used for isolation of the tagged protein by affinity chromatography or ion exchange chromatography.
  • the tag is a His tag, for example, a tag that comprises 3-12 Histidine residues.
  • MHC class II molecules comprising a His-tag labeled heavy chain are isolated by affinity chromatography.
  • the affinity chromatography uses an ⁇ 2+- ⁇ resin. Methods for affinity chromatography for the isolation of tagged peptides and proteins are well known to those of skill in the art and it will be appreciated by those of skill that the invention is not limited in this respect.
  • a chelant moiety is conjugated to an isolated protein after the protein has been synthesized, for example, post-translationally, or after in situ synthesis, for example, after Fmoc synthesis.
  • the chelant moiety is conjugated to the protein, for example, the MHC molecule, for example, to a heavy chain of the MHC molecule by chemical or enzymatic modification.
  • tags to peptides and proteins are well known to those of skill in the art and include, but are not limited to, biotinylation and sortase-mediated protein labeling (for the latter see Popp et al., Site Specific Protein Labeling via Sortase -Mediated Transpeptidation Curr. Protoc. Protein Sci. 56: 15.3.1-15.3.9; 2009, incorporated herein by reference in its entirety for disclosure of sortase-mediated transpeptidation reactions).
  • Post-isolation addition of a chelant moiety is particularly useful in embodiments, where a chelant moiety would interfere with a synthesis or purification step, for example, in embodiments, where an MHC molecule is loaded with an antigenic peptide that comprises a chelant tag and the tag is used for isolation of peptide-loaded MHC molecules.
  • the peptide tag can be cleaved from the antigenic peptide and a chelant tag can be added subsequently to the MHC molecule, for example, to an MHC heavy chain by methods well known to those of skill in the art.
  • Some aspects of this invention provide methods for the staining, detection, and/or isolation of cells using a reversible protein multimer, for example, a reversible MHC multimer, as described herein.
  • the method comprises contacting a population of cells with a protein multimer, for example, an MHC multimer provided herein.
  • the multimer comprises a detectable label, for example, a fluorophore, either as the carrier molecule or conjugated to the multimer.
  • detectable label refers to a molecule or moiety that can be detected, for example, by performing an assay known to those of skill in the art for its detection.
  • a detectable label accordingly, may be, for example, (i) an isotopic label (e.g., a radioactive or heavy isotope, including, but not limited to, 2H, 3H, 13C, 14C, 15N, 3 IP, 32P, 35S, 67Ga, 99mTc (Tc-99m), 11 lln, 1231, 1251, 169Yb, and 186Re), (ii) an affinity label (e.g., an antibody or antibody fragment, an epitope, a ligand or a ligand-binding agent) (iii) and enzymatic label that produce detectable agents when contacted with a substrate (e.g., a horseradish peroxidase or a luciferase); (iii) a dye, (e.
  • phosphorescent, or fluorescent molecule such as a chemical compound or protein
  • Fluorophores for example, fluorescent dyes and proteins, are of particular use for embodiments of this invention that involve detection or isolation of living cells.
  • a fluorophore is a molecule or moiety that absorbs light of a specific wavelength and then re- emits light at a different specific wavelength, thus causing the molecule of moiety to be fluorescent.
  • Other detectable labels are known to those of skill in the art and the invention is not limited in this respect. It will be appreciated that a detectable label may be incorporated into any part of the multimeric structure and in any manner that does not interfere with the stability or the function, for example, the binding activity of the multimer.
  • the method includes a step of detecting the multimer bound to a cell, for example, to a surface receptor (e.g., a T-cell receptor) of a cell.
  • a surface receptor e.g., a T-cell receptor
  • the method performed to detect the multimer depends, of course, on the nature of the detectable label comprised in the multimer.
  • suitable methods for detection are fluorescence microscopy, cytometry, or FACS.
  • the method comprises a step of quantifying the number of detected cells, for example, quantifying the number of T cells expressing a specific TCR in a cell population, for example, in a cell population obtained from a subject.
  • the quantity of cells for example, of T-cells expressing a specific TCR
  • the comparison of the quantity of T-cells expressing a specific TCR in a subject to a reference quantity is used to determine an immune reaction in the subject.
  • the reference quantity is a quantity measured or expected in a healthy subject or in healthy subjects, or a quantity measured in the subject prior to a clinical intervention, for example, prior to a vaccination, and a quantity in the subject that is higher than the reference is indicative of an immune response in the subject, whereas a quantity in the subject that is lower than the reference is indicative of depletion of a specific T-cell population.
  • MHC multimers as provided herein are useful, for example, for monitoring immune responses in subjects, either in response to a clinical intervention, for example, a vaccination, or as a result of a disease or condition, for example, a
  • the clinical intervention is a vaccination against a tumor antigen.
  • the vaccination is a vaccination administered after surgical removal of a tumor expressing the tumor antigen.
  • the clinical intervention is an intervention aimed to suppress a function of the immune system, for example, by depleting a specific population of T-cells.
  • the subject is a subject having an autoimmune disease.
  • the detection method further comprises a step of releasing the chelate complex bond comprised in the multimer employed, for example, by withdrawing the cation of the complex bond, or by contacting the multimers with an agent able to displace a chelant forming the chelate complex bond.
  • T cell antigen receptors TCRs
  • MHC -peptide monomers Interactions of T cell antigen receptors (TCRs) with MHC -peptide monomers are characterized by micromolar dissociation constants (K D ) and half-lives in the range of seconds.
  • K D micromolar dissociation constants
  • the use of MHC -peptide monomers that are conjugated to reversible multimers substantially increases the overall binding avidity and decreases the dissociation rate to half- lives in the order of hours.
  • MHC multimers as provided herein allows for the efficient staining, detection, and/or isolation of T cells bearing specific TCRs, for example, by fluorescent microscopy, flow cytometry, fluorescence activated cell sorting (FACS), or magnetic- activated cell sorting (MACS).
  • FACS fluorescence activated cell sorting
  • MCS magnetic- activated cell sorting
  • MHC multimers and methods are provided that are useful for the staining of CD8 + T-cells.
  • CD8 interacts preferentially with MHC class I molecules. Accordingly, MHC multimers useful for staining of CD8 + T-cells are preferentially MHC class I multimers.
  • CD8 undergoes differentiation- and activation-dependent changes in the glycosylation and sialylation of its ⁇ chain, which can profoundly affect cognate and non- cognate MHC class I-peptide binding. Non-cognate CD8 binding to MHC class I-peptide multimers has been reported to increase non-specific multimer binding. Accordingly, in some embodiments, multimers are provided that contain the CD8 binding weakening mutation A245V in the MHC a3 domain.
  • an MHC class I multimer as provided herein (A245V mutated or not) is used in a staining procedure at a concentration of about 5-30nM (about 2.5 to 15 ⁇ g/ml). At this concentration, non-specific staining is generally low.
  • binding of MHC class I molecules to T-cell receptors can elicit T cell activation events, such as intracellular calcium mobilization, diverse tyrosine phosphorylation and endocytosis of MHC class I-peptide engaged TCR/CD8.
  • T cell activation events such as intracellular calcium mobilization, diverse tyrosine phosphorylation and endocytosis of MHC class I-peptide engaged TCR/CD8.
  • MHC class I- peptide complex driven cell activation can induce death of effector cytotoxic T-cells (CTLs) via FasL-dependent apoptosis or severe mitochondrial damage. This can lead to changes in T-cell populations that are contacted with MHC multimers, for example, for cell staining, detection, or isolation, for example, by selective depletion of stained T-cells.
  • CTLs effector cytotoxic T-cells
  • TCR- activation-mediated cell depletion can render isolation of a non- activated T-cell population impossible.
  • Some aspects of this invention provide reversible MHC multimers and methods for their use that avoid this problem by minimizing the time of high-avidity MHC/TCR interaction by re-monomerizing the multimers, thus minimizing undesired TCR activation-mediated effects on stained cells.
  • the reversible MHC multimers provided herein can also be used in methods that do not include a chelate complex bond release step, thus employing the reversible multimers in the manner conventional multimers would be employed.
  • methods are provided that exploit MHC multimer TCR activation to deplete specific T-cell populations by MHC-mediated TCR activation.
  • reversible MHC multimers as provided herein are used to eradicate antigen- specific CD8+ CTLs.
  • MHC class II multimer binding to CD4+ T-cells can also lead to T-cell activation and death, for example, of CD4+ effector cells. Accordingly, reversible MHC class II multimers are useful in the staining of CD4+ T-cells and in the isolation of minimally manipulated or activated CD4+ T-cells.
  • Some aspects of this invention provide reversible MHC multimers and methods for the use of such multimers to analyze the state of activation or differentiation of T-cells, for example, CD8+ and/or CD4+ T-cells.
  • homogenous populations of MHC multimers of defined structure are preferable over heterogeneous MHC multimer populations.
  • homogenous populations of MHC multimers e.g. of dimers, trimers, tetramers, pentamers, hexamers, or decamers, are provided for use in such methods.
  • the MHC multimers comprise linkers of defined length and flexibility.
  • the MHC multimers comprise chelant groups in defined configurations, for example, in mono- di- or tetra-chelant configuration.
  • binding studies with defined, homogenous populations of multimers can reveal differentiation- and activation-dependent differences, for example, differentiation- and activation-dependent changes in glycosylation and sialylation of T cell surface molecules involved in antigen recognition of the cells under study which can affect, for example, CD8 participation in MHC class I molecule binding and/or aggregation of TCR and CD8.
  • MHC multimers provided herein can be employed alone or in combination with other binding and/or staining agents.
  • MHC multimers provided herein are used to stain T-cells in combination with staining the cells for an additional antigen, for example, with a staining for intracellular cytokine staining.
  • reversible MHC class I-peptide multimers are provided that comprise a mutation in the a3 domain.
  • the mutation is a mutation that ablate CD8 binding, e.g. a D227K, T228A in human MHC and D227K, Q226A in mouse MHC molecules.
  • methods are provided that use such CD8 binding- deficient MHC multimers to stain, detect, and/or isolate CD 8 -independent T cells, which typically express high affinity TCRs.
  • CD8 binding-deficient MHC multimers are provided for the staining, detection, and/or isolation of CD8+ T cells expressing high-affinity TCRs specific for tumor antigens, for example, for MELAN-A/Mart-1, gplOO, or tyrosinase. It is known to those of skill in the art that such tumor-antigen specific T-cell tend to express low affinity TCRs and that infrequent CD8+ T cells specific for tumor antigens expressing high affinity TCRs efficiently kill tumor cells.
  • the use of a reversible MHC class I multimer as provided herein enables efficient identification and isolation of such rare cells with no or only minimal TCR activation, thus allowing for the isolation of native T- cell populations that cannot be isolated with conventional MHC class I multimers.
  • CD8 binding-deficient multimers are used to selectively induce FasL (CD95L) expression, resulting in apoptosis of antigen- specific CTLs.
  • Staining with reversible MHC multimers can be performed through a wide range of temperatures. In some embodiments, staining is performed at a temperature between 0-37 °C.
  • MHC multimer staining is performed at 37 °C. While staining at 37°C is rapid, and efficient staining of CD4+ T cells with reversible MHC class II multimers is often observed upon incubations at 37°C for extended periods of time, reversible MHC class I multimers efficiently effect TCR activation at this temperature.
  • staining is performed at 0-4°C. It will be appreciated by those of skill, that MHC multimer binding at low temperatures (e.g., 0-4°C) tends to be slow, necessitating extended periods of time for staining as compared to staining at higher temperatures.
  • staining with MHC multimers is performed at ambient temperature, e.g. at 20-30°C, preferably at 22-25°C.
  • MHC staining is performed in the presence of EDTA (e.g., 5mM) and/or sodium azide (e.g., 0.02%) to inhibit cell activation. Under these conditions multimer binding is rapid.
  • staining is performed for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 20-45 minutes. In some embodiments, for example, in some embodiments using reversible MHC class II multimers, staining is performed for 30- 120min. Under these conditions, cognate MHC class II complexes binding to TCR (and CD4) are internalized and accumulate over time.
  • Multimer concentration is an important factor in achieving maximum staining efficiency, and, while exemplary MHC multimer concentrations are provided herein, it will be appreciated by those of skill that it is preferable to test a range of concentrations, for example, in the range of about 5-50nM (about 2.5-25 ⁇ g/ml), or, in the case of low affinity binding, in higher concentration ranges, for example, in the range of about 5-100nM (about 2.5-5( ⁇ g/ml).
  • a cell is contacted with an MHC multimer, for example, with an MHC class II multimer as provided herein
  • binding of the MHC multimer to the cell is facilitated by desialylation of the cell.
  • Desialylation is a process by which sialyl groups on the cell surface are removed or modified. Methods and reagents for desialylating a cell are described in detail elsewhere herein and additional methods are well known to those of skill in the art.
  • a cell is contacted with a desialylating agent in order to achieve desialylation.
  • Desialylating agents are, in some embodiments, enzymes, while, in other embodiments, chemicals are used to effect desialylation.
  • Enzymes known to desialylate cell surfaces are, for example, neuraminidases. Methods and conditions suitable for desialylation of cells by contacting them with a neuraminidase are well known to those of skill in the art.
  • the cells are pre-treated with neuraminidase under conditions suitable to achieve desialylation of the cells (e.g., treatment with 0.03 ⁇ / ⁇ 1 for 30 min at 37°C).
  • staining is increased by inhibiting TCR down modulation with the protein kinase inhibitor dasatinhib.
  • scarce antigen- specific cells can be enriched by combination of fluorescence -based methods as described herein with a non-fluorescent-based isolation method, for example, with MACS using magnetic beads coated with an antibody against an epitope of the carrier molecule.
  • useful multimers for isolation methods comprise a detectable label and the methods include a step of staining the target cell population as described in more detail elsewhere herein.
  • a method of cell separation is employed that allows for the enrichment or the isolation of homogenous populations of cells based on the cells binding the employed multimer, for example, the employed MHC multimer.
  • Such methods are well known in the art and include, for example, FACS and MACS.
  • the method of isolating cells with a reversible multimer further includes a step of releasing the chelate bond comprised in the multimer.
  • this step includes withdrawal of the central ion, for example, the central divalent cation, from the chelate complex.
  • Method for ion withdrawal are well known in the art and include, in some embodiments, washing the cells with a solution that does not contain a significant amount of the divalent cation, or with a solution that comprises an agent that sequesters the divalent cation.
  • the step of chelate complex bond release includes contacting the chelate complex bond with an agent that displaces a chelant from the chelate complex bond.
  • the chelate complex bond can be released by contacting it with an imidazole residue, for example, with free imidazole.
  • Imidazole is able to displace a chelant, in this case, the histidine chelant from the complex bond, thus releasing the chelate complex bond of the multimer.
  • the result of this release is the re-monomerization of the proteins comprised in the multimer, for example, of MHC proteins in an MHC multimer.
  • re-monomerization of the multimer after staining and/or isolation avoids detrimental effects on the cells and, in embodiments, where the cells are rare and/or sensitive to detrimental effects of staining, allows for efficient isolation of such cells that is cumbersome or impossible with conventional strategies.
  • this invention provides methods for the manipulation of T-cells using reversible monomers.
  • the method includes a step of contacting a population of cells expressing a T-cell receptor with an MHC multimer as described herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC class I interaction to effect TCR-mediated T-cell activation.
  • the contacting is performed in vitro.
  • the contacting is performed ex vivo.
  • the contacting is performed in vivo.
  • the cells are contacted with an MHC multimer for a time long enough to activate high-affinity TCR expressing T-cells, but not to activate low affinity TCR expressing T-cells.
  • the cells are cells from a subject having an autoimmune disease.
  • the cells are contacted with an MHC multimer that is loaded with an antigenic peptide recognized by T-cells that mediate an autoimmune disease.
  • the method further comprises measuring the quantity of the T-cells targeted by the MHC multimer, for example, by methods for the identification or detection of T-cells provided herein or otherwise known in the art.
  • Some embodiments of this invention provide isolated cells or cell populations, for example, isolated native, or non-activated T-cell populations obtained by using a reversible multimer or a method as provided herein.
  • an isolated cell is provided that has been contacted with a reversible multimer provided herein and isolated from a cell population based on the cell binding the multimer, for example, by a method for detection and/or isolation described in more detail elsewhere herein.
  • the cell is a T-cell.
  • the T-cell is a native T-cell, or a T-cell that has not undergone TCR-mediated cell activation.
  • the cell has been contacted with an agent releasing the chelate complex bond of the reversible multimer subsequent to its isolation.
  • the cell is a T-cell recognizing a tumor antigen.
  • the cell is a T-cell expressing a TCR that binds a tumor antigen with high affinity.
  • the cell is a therapeutically valuable cell.
  • the cell is expanded in vitro after isolation, and used in a therapeutic method.
  • the therapeutic method includes a step of administering the cell to a subject in need thereof, for example, a subject having a tumor or having an elevated risk of developing a tumor expressing a tumor antigen.
  • a subject at risk of developing a tumor expressing a tumor antigen is a subject which was diagnosed to have such a tumor and has undergone surgical removal of the tumor.
  • Soluble MHC-peptide complexes induce rapid death of CD8+ CTL.
  • Nucleophosmin leukaemic mutants contain C- terminus peptides that bind HLA class I molecules. Leukemia 2008; 22: 424-426.; and Matsuki N, Ogasawara K, Takami K, Namba K, Takahashi A, Fukui Y, Sasazuki T, Iwabuchi K, Good RA, Onoe K.Prevention of infection of influenza virus in DQ6 mice, a human model, by a peptide vaccine prepared according to the cassette theory. Vaccine 1999; 17: 1161-1168. All references are incorporated herein in their entirety by reference for disclosure of methods and materials useful for the generation, isolation, and or purification of MHC multimers and for staining, detection, and/or isolation of cells using MHC multimers.
  • Some aspects of this invention provide a novel type of MHC multimers in which MHC class I-peptide monomers are conjugated to phycobilins (PE or APC) or quantum dots (Qdots) via chelate complexes of histidine (His) tags and Ni 2+ -nitrilotriacetic acid (NTA). Hexa (His 6 ), dodeca (His 12 ) or tandem hexa (2xHis 6 ) histidine tags were fused to HLA- A*0201 (A2) heavy chain and A2-peptide monomers obtained in good yields by refolding.
  • PE phycobilins
  • Qdots quantum dots
  • NTA-His tag A2/Flus 8 _ 66 multimers indicated that: i) multimers containing 2xHis 6 tagged complexes and short di-or tetra NTA moieties were equal or superior compared to conventional multimers; ii) di-NTA or tetra- NTA can be directly coupled to the phycobilin proteins or Qdots, which circumvents the use of biotin and streptavidin, and renders synthesis simpler and cheaper; iii) these reagents are molecularly better defined than conventional multimers and hence allow better analysis of binding data; iv) NTA-His tag built multimers dissociated rapidly in the presence of 100 mM imidazol (t 1/2 ⁇ 1 min), which allows sorting of bona fide antigen-specific CD8+ T cells without inducing activation dependent cell death.
  • APC allophycocyanine
  • ⁇ 2 ⁇ beta-2- microglobulin
  • BSP biotinylation sequence peptide
  • DIEA di-isopropyl-ethyl-amine
  • EDIC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • GFC gel filtration chromatography
  • Flu influenza matrix
  • HOBt 1-hydroxy-benzo-triazole
  • NHS N-hydroxysuccinimide
  • NTA nitrilotriacetic acid
  • PE phycoerythrin
  • Qdot quantum dot
  • SPR surface plasmon resonance
  • TBTU 0-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate.
  • Protected amino acids and 2-chlorotrityl resin were obtained from Reactolab (Servion, Switzerland), TBTU and HOBt were from Multisynthec (Witten, Germany), maleimide-NTA from Dojindo Laboratories (Kumamoto, Japan).
  • RP-HPLC analyses were performed on Waters HPLC station consisting of two 515 pumps and a Waters 996 photodiode array detector.
  • the purified peptides were characterized for correct Mr using matrix- assisted laser desorption ionization time-of-flight mass spectrometer (Micromass QTOF Ultima) (Waters Ltd, En Yvelines Cedex, France).
  • the reaction vessel was purged with N2 and then continuously stirred overnight at 55°C. The volatiles were evaporated in vacuo at 60°C. Cyclohexane/ethylacetate (3: 1, 15 ml) solution was added to the partially solidified reaction mixture. The resulting slurry was filtered over sintered glass funnel and the precipitate washed three times with the same solvent (3x10 ml). The filtrate was
  • the linear peptide H-K(aminocaproyl-biotin)-PEG- A-E*-A-E*-OH (*: Fmoc-E-OtBu) was synthesized on an ABI433 peptide synthesizer.
  • Double coupling of each Fmoc-protected amino acid were performed using DIPC and HOBt as coupling reagent.
  • Fmoc was removed by 3x5 min treatment with piperidine 20% in DMF.
  • Each cycle was followed by an acetylation (N-capping) to prevent the synthesis of truncated peptides.
  • Final cleavage was performed in TFA/TIPS/H20 (92.5/5/2.5) for 2 h.
  • the peptide was precipitated with cold ether, dissolved in water and purified by semi-preparative HPLC.
  • the synthesis of SH-NTA2 was performed assembling the linear C(Acm)-PEG- C(Trt)-G-C(Trt) on a chlorotrityl resin.
  • TFA treatment the linear peptide C(Acm)- PEG-C(SH)-G-C(SH) was coupled to maleimide-NTA as described previously.
  • 1 ⁇ of lyophilized peptide was dissolved in 100 ⁇ of AcOH 20%, the pH was adjusted to 4 with aqueous ammonia. 3 ⁇ of mercury(II) acetate were added and the mixture stirred for 1 h. 5 ⁇ of DTT were then added and the mixture stirred for 1 additional hour.
  • the desired product was directly purified by semi-preparative HPLC and analyzed by mass spectrometry.
  • a RU (resonance unit) of 100 was used in all experiment. Loading of NTA with Ni 2+ was performed by injecting NiC12 solution (500 mM in eluent buffer) and regeneration of the chip with imidazole (500mM in water) followed by a regeneration solution (lOmM HEPES, 150mM NaCl, 0.005%
  • the different heavy chains were refolded in the presence of 1 ⁇ 2 ⁇ and the influenza matrix peptide 5g_66 (GILGFVFTL, SEQ ID NO: 12) and purified on a Superdex S75 column as described (3).
  • streptavidin PE multimers were prepared as described (3).
  • NTA- streptavidin PE multimers were prepared in two steps. First, streptavidin PE conjugate (Invitrogen) was incubated with NTA-biotin peptides (five- fold molar excess) at 4°C for lh followed by incubation for 30 min with NiS04 (10 mM). Excess of reagents were removed by means of min- spin columns (ZebaTM Spin Desalting Columns (Thermo scientific).
  • NTA 2 -PE conjugates were prepared by first reacting PE (Sigma) (50 nM) in 0.1 M
  • HLA-A*0201 -restricted, influenza matrix peptides 8 _66- specific CD8+ T cell clones were obtained by limiting dilution cloning from bulk cultures.
  • CD8+ PBMC from healthy donors were prepared by negative selection and were stimulated with Fhi58_66 peptides as described (4).
  • the clones were re- stimulated in 24- well plates every 15 d in RPMI 1640 medium supplemented with 8 % human serum, rIL-2 (150 U/ml) (Hoffmann-la Roche Ltd, Basel, Switzerland) with PHA (1 ⁇ g/ml; Sodiag SA, Losone, Switzerland) and 1 x 10 6 /ml irradiated alio geneic PBMC (3000 rad) as feeder cells.
  • Bulk cultures were prepared by one or two peptide stimulations of CD8+ T cells obtained from PBMC from a
  • FACS buffer OptiMEM (Invitrogen AG, Basel, Switzerland) supplemented with 0.5% BSA (Sigma- Aldrich), 15 mM HEPES, 5 mM EDTA, and 5 mM NaN 3 ).
  • BSA Sigma- Aldrich
  • cells were incubated an additional 20 min at 4°C with anti-CD8-FITC (Immuno Tools). After 30-fold dilution in FACS buffer, cell-associated fluorescence was measured on a LSRII flow cytometer (BD Biosciences).
  • NTA-His tag interactions depend on the spatial configuration of the NTA moiety.
  • tri-NTA compounds most avidly bind to His6 tags when they contain minimal spacers (15). To better understand this relationship, we are currently testing additional di and tetra NTA molecules.
  • Qdots are larger than PE, contain more streptavidin on their surface and therefore more NTA 2 groups; as discussed below, the density of NTA groups on a surface is another factor determining the stability of complexes with His tagged proteins.
  • NTA-His tag built multimers are reversible
  • NTA multimers with multimers containing NTA 2 -PE and His 12 or 2xHis 6 tagged monomers. At all temperatures tested, both NTA 2 multimers exhibited a stable binding plateau above 4 nM multimer concentration. By contrast the binding of the BSP multimer increased over the whole concentration range tested (Fig. 9). This is explained by that NTA multimers are molecularly better defined than BSP multimers. While the NTA multimers consist of one PE conjugated with variable numbers of A2/Flu monomers, conventional multimers contain multiple complexes of different sizes and stoichiometries.
  • NTA-biotin- strep tavidin PE multimers are equally heterogeneous, as reflected by their binding isotherms (Fig. 5, Fig. 18).
  • NTA multimers are better defined, they allow more conclusive binding analysis (e.g. Scatchard analysis) than conventional BSP multimers, i.e. can provide more information on given antigen- specific T cells.
  • the 20°C binding isotherms on this polyclonal population exhibited essentially the same picture as the one obtained on clones, namely: i) the binding of NTA 4 multimer binding was higher than of NTA 2 multimer; ii) multimers containing the 2xHis 6 tag bound better than those containing the His 12 tag in the case of NTA 2 , but not NTA 4 reagents; iii) the NTA 2 -PE multimer exhibited clear saturation, whereas all streptavidin-PE containing ones did not; and iv) the NTA 4 and NTA 2 -PE multimers exhibited the highest avidity, i.e. lowest concentrations for half maximal binding.
  • Reversible MHC class I-peptide complexes allow sorting of antigen-specific CD8+ T cells without inducing activation dependent cell death.
  • cloned BCB 70 cells were stained with this or conventional BSP multimers. FACS sorted cells were washed once with 100 mM imidazol and viable cells enumerated 1, 2 or 3 d afterwards. As shown in Fig. 11 A the percentage of viable cells of NTA 2 -PE multimer sorted cells was slightly lower compared to untreated cells or cells washed once with cold imidazol. By contrast cells sorted with conventional multimers exhibited only 42% viable cells after Id and merely 20% 3d after sorting.
  • Novel soluble HLA-A2/MELAN-A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in patients with melanoma. Int J Cancer 127:910-23.
  • NTA nitrilotriacetic acid
  • the methods and materials described herein are universally applicable to generate reversible MHC multimers by conjugating a plurality of MHC molecules to a multivalent carrier molecule via a chelate complex bond. While the invention is not limited to specific MHC molecules, Tables 1 and 2 provide exemplary MHC molecules and exemplary antigenic peptides that can be used to produce empty or peptide-loaded reversible MHC molecules using the concepts, methods, and materials provided by aspects of this invention and described in more detail elsewhere herein. Further MHC molecules and antigenic peptides are known in the art and described, for example, in the Tetramer Collection of the Ludwig Institute for Cancer Research (see Nicolas P, Dojcinovic D, Luescher IF. LICR tetramer collection: Soluble tetrameric MHC/peptide complexes to identify and monitor tumor antigen-specific T cells. Cancer Immun 2009; URL:
  • Tyrosinase 206-214 AFLPWHRLF 148
  • MHC class II molecules and antigenic peptides useful for the generation of reversible multimers include, but are not limited to MHC molecules comprising a mutant HLA-A*0201 chain, e.g. a chain comprising a D227K, a T228A, a D227K, a T228A, a T233A, and/or A245V mutation.
  • Additional MHC molecules useful according to aspects of this invention further include, but are not limited to HLA-A*1101, HLA-A*3001, HLA-A*3004, HLA-B*0801, HLA-B*2705, HLA-B*5101, HLA-Cw*0303, HLA-Cw*0401, HLA-Cw*0602, HLA-Cw*1402, H-2IAd, and H-2IEd molecules.
  • chimeric MHC class I multimers are provided, for example, multimers in which the comprised heavy chains are in part human and in part murine.
  • peptides comprising modified amino acid residues are provided, for example, ABA, 4-azidobenzoic acid, or Dap, diamino-propionic acid.
  • peptides are provided that comprise or are conjugated to low molecular weight fluorescent dyes (see, e.g. www.attotech.com/), for example, for flow cytometry analysis.
  • a method for isolation of MHC II molecules that have stably bound a peptide of interest conjugated to a tag which, in some embodiments, can subsequently be removed, for example, by cleavage of a linker connecting the tag to the peptide.
  • a method is provided that allows gentle purification of fragile "empty" (without nominal peptide cargo) His tagged MHC II molecules by affinity chromatography on Ni 2+ nitrilotriacetic acid (NTA) columns. After isolation of correctly peptide loaded MHC Il-peptide complexes these can be directly converted to multimers by reaction with NTA n conjugated phycobilins (e.g. phycoerythrin) or quantum dots (Qdots).
  • NTA n conjugated phycobilins
  • Qdots quantum dots
  • Abbreviations used in this example include BSP (biotinylation sequence peptide), DR1 (DRB1*0101); DR4 (DRB1*0401); ESO (NY-ESO-1); GFC (gel filtration chromatography); HA (influenza hemagglutinin); LZ (leucine zipper); ⁇ (3-(2-nitrophenyl)-P-alanine); NTA (nitrilotriacetic acid); PE (phycoerythrin); and pY (phospho-tyrosine).
  • MHC I-peptide complexes can be obtained by peptide driven refolding in good yields and high purity, soluble recombinant MHC class II proteins cannot and are typically produced by insect expression systems, e.g. Drosophila S2 cells or baculovirus and sf9 cells (1). Deletion of the transmembrane (TM) domains of the a and ⁇ chains results in their dissociation, which is re-established by addition of leucine zippers.
  • TM transmembrane
  • a biotinylation sequence peptide (BSP) sequence is added after the leucine zipper (e.g., after the acidic zipper) and enzymatic biotinylation and tetramerization is performed as for MHC I-peptide multimers (1-5).
  • BSP biotinylation sequence peptide
  • "empty" (without nominal peptide cargo) MHC II molecules are isolated from culture supernatants by immunoaffinity chromatography and subsequently loaded with a peptide of interest. The efficiency of peptide loading depends on its binding strength to the MHC II molecule; if it is below a threshold, peptide loading is inefficient and the resulting complexes of limited stability.
  • peptides can be tethered to the N-terminus of the ⁇ chain via a flexible linker (6). This strategy works for some, but not all, complexes. Also, although the peptide is part of the molecule, in the case of weak binding peptides there is no knowing whether or not it is correctly bound in the peptide binding groove.
  • the staining of antigen-specific CD4+ T cells often is weak and the frequency of stained cells ex vivo very low, usually necessitating prior in vitro peptide stimulation to permit conclusive detection.
  • multimer staining of CD8+ and CD4+ T cells such as 1) the staining with MHC II multimers is usually higher at 37°C than at lower temperatures, which is not the case for MHC I multimers (2, 3, 7); 2) Efficient CD4+ T cell staining requires longer incubation periods, which is explained, at least in part, by accumulation of MHC II multimers by endocytosis; therefore agents that affect cell vitality and cyto skeleton function inhibit CD4+ T cell staining (7, 8).
  • MHC II multimer staining therefore typically requires higher concentrations (up to 100 nM, i.e. about 50 ⁇ ) (2-5). While MHC I-peptide complexes obtained by refolding are highly pure and conformationally uniform, MHC II-peptide complexes obtained by peptide loading of "empty" MHC II proteins or containing tethered-on peptides often are not, which can seriously impair MHC II multimer staining.
  • MHC II-peptide complexes can be isolated by anion exchange chromatography when adding an acidic tag on the peptide of interest.
  • the coupling reagents TBTU and HOBt were purchased from Multisynthec (Witten, Germany), Cy5.5-maleimide from GE Healthcare and Fmoc-3-amino-(2- nitrophenyl) -propionic acid from Peptech Corporation (USA).
  • Reverse phase HPLC analyses were performed on a Waters system consisting of two Waters 515 pumps and a Waters 996 photodiode array detector. The purity of all peptides was assessed by analytical HPLC.
  • Peptide syntheses were performed using 2 equiv of N-CC-Fmoc-protected amino acid relative to the resin loading, activated in situ with 2 equiv of TBTU, 2 equiv of HOBt and 4 equiv of DIEA in DMF (10 ml/g resin) for 1 h. Coupling completion was verified by the Kaiser test. N-CC-Fmoc protecting groups were removed by treatment with a piperidine/DMF solution (1:4) (10 ml/g resin) for 5 min. Crude peptides were obtained by treating the resin with a solution of TFA/H20/TIPS (92.5/2.5/5) for 3 h at ambient temperature.
  • Extracellular coding parts of DR alpha and beta chains were PCR amplified using ctttagatctcgaccacgtttcttggagc (SEQ ID NO: 297) as the 5' primer and
  • ctttgaattccttgctctgtgcagattcag (SEQ ID NO: 298) as the 3' primer from cDNA preparations (Qiagen) of total RNA extracted from human PBMCs, digested with appropriate restriction enzymes and cloned in pMT A BiP/V5/His vector-derived cassette (Invitrogen) containing sequences for appropriate leucine zippers and the AviTag (Avidity).
  • TTAPSAQLKKKLQALKKKNAQLKWKLQALKKKLAQ SEQ ID NO: 301
  • GGGSGGS flexible linker
  • Oligos for introduction of a single His6 CTTGATATCTCAATGATGGTGATGATGGTGGCCGGTGCGCTGAGCCAGTTCCTT TTCC, SEQ ID NO: 303
  • a double His6 CTTGATATCTCAATGATGGTGATGATGGTGGCCGGTGCGCTGAGCCAGTTCCTT TTCC, SEQ ID NO: 303
  • DRA Drosophila melanogaster D.
  • Mel-2 cells a serum-free medium adapted variant of S2 cells grown in Sf900 II serum-free medium (Invitrogen) at room temperature (22-26 °C).
  • Cells were simultaneously transfected with three plasmids (the plasmid for DRA, DRB and pBS-PURO, a plasmid conferring puromycin resistance (a gift from K. Karjalainen, Nanyang Technological University, School of
  • DR1 and DR4 a population of transfected cells was used, whereas for DR52b high-yielding clones were obtained by limiting dilution.
  • the cells were grown in roller bottles (BD Falcon) rotating at 6 rev/min at room temperature to 5-10*10 6 /ml and protein production was induced by addition of 1 mM CuS0 4 for 3-5 days. The yields of purified protein were 2-5 mg per liter of medium.
  • peptide loading soluble "empty" molecules were brought to pH 5.5 (DR1 and DR4) or pH 6.0 (DR52b) by addition of 100 mM citric acid and incubated with a peptide of interest (1-5 ⁇ final concentration) for 24 h at 28 °C (DR52b) or 37 °C (DR1, DR4) in the presence of 0.2% octyl ⁇ -D-glucopyranoside (Sigma), protease and phosphatase inhibitor cocktails (Roche).
  • peptide loaded DR molecules were biotinylated with recombinant BirA biotin transferase (Avidity) according to the supplier' s
  • MHC II-peptide complexes containing His tagged peptides e.g. DR4 peptide-NPpA-SGSGHHHHHH, SEQ ID NO: 306
  • samples were passes through a HisTrap HP column (GE Healthcare Life Sciences), which after washing was eluted with 200 mM imidazole, which subsequently was removed by GFC in PBS on a Superdex S200 column (GE Healthcare Life Sciences) or by ultrafiltration.
  • biotinylation and purity were assessed by the avidin shift assay and was routinely >90%. Briefly, two different amounts of biotinylated concentrated complexes (typically 2 and 5 ⁇ g) are mixed or not with 10 ⁇ g of avidin (Pierce) and run on 12% SDS-PAGE (non-boiling, non-reducing). After staining in Gelcode blue (Pierce), gels are scanned and quantified using the ImageQuant TL software (GE Healthcare Life Sciences).
  • BSP antibody (a gift from Dr. Gennaro DiLibero, Basel University Hospital) and a secondary antibody conjugated with streptavidin-alkaline phosphatase (Sigma).
  • the plates were developed with p-nitrophenly-phosphate (pNPP) SigmaFAST substrate (Sigma) and absorbance read at 405 nm on an ELISA plate reader.
  • the concentration of His 6 tag was assessed likewise, i.e. biotinylated MHC II-peptide complexes were trapped on streptavidin- coated plates (Maxisorp, Nunc) and His tag detected by means of Ni-NTA alkaline phosphatase (His-Detector, KPL).
  • HA 30 6-3 is- specific CD4+ T cell clones were obtained by limiting dilution cloning of a DR4 HA306-318 multimer-sorted population of a CD4+ T cell line generated from PBMC of a healthy donor (HD137) that was propagated by stimulation with 2 ⁇ HA 30 6-3i8 peptide and ⁇ -irradiated CD4 " PBMC in RPMI-1640, 10% human serum AB, supplemented with 100 U/ml of hIL-2.
  • the clones were propagated by phyto-hemagglutinin (PHA) (Oxoid) stimulations every 2-3 weeks.
  • PHA phyto-hemagglutinin
  • ESO-specific CD4+ T cell lines were derived and maintained as described previously (3).
  • DIFCO complete Freund's adjuvant
  • Biotinylated DR-peptide complexes were multimerized by mixing with small aliquots of streptavidin-PE (Invitrogen) up to the calculated 4: 1 stoichiometric equivalent.
  • Cells were stained in 50 ⁇ of FACS buffer (PBS, 0.5% BSA, 2 mM EDTA, 0.05% sodium azide) for 1 hour at 37°C.
  • FACS buffer PBS, 0.5% BSA, 2 mM EDTA, 0.05% sodium azide
  • neuraminidase treatment cells were incubated with 0.03 U/ml of neuraminidase from V. cholerae (Roche) in complete medium and washed twice prior to tetramer staining.
  • fluorescent antibodies e.g. anti-CD4 were added after multimer staining and incubated at 0-4°C for 15 min. After 2 washes the cells were suspended in 300 ⁇ buffer and analyzed by flow cytometry on a FACSCalibur (
  • Propidium iodide (Invitrogen) was added just before acquisition for exclusion of dead cells. Human cells were fixed in 2% formaldehyde (Polysciences) in FACS buffer analysis. Data analysis was performed with FlowJo 7.6 software (TreeStar).
  • This tag (H 6 -GSG-NPpA) can be added N- or C- terminally to the peptide of interest (Fig. 21).
  • Fig. 21 Upon UV irradiation at 365 nm the ⁇ residue is cleft, resulting in the removal of the His tag after its use for affinity purification of MHC II-peptide complexes (Fig. 21 A).
  • ⁇ has been used for the preparation of conditional MHC I-peptide ligands and its usefulness is well documented (11, 12).
  • Cy5.5 (and Cy5) is a blue fluorescent low molecular dye that can be coupled to peptides via amides or thioethers (Fig. 23A, www.gelifesciences.com).
  • DR4 Cy5.5 labelled HA306-318 peptide (HA 30 6-3i8-GSGC-Cy5.5, SEQ ID NO: 307) and analyzed the reaction mixture by GCF on Superdex S200 column recording the OD of the eluent at 675 nm (Cy5.5) and 280 nm
  • Cy5.5 As for His 6 tagged peptides, the content of Cy5.5 (or Cy5) labelled peptide in MC II- peptide complexes can also be detected by ELISA, using Cy dye specific antibodies (Fig. 23C). Moreover and importantly Cy5.5 contains four negatively charged sulfonyl groups (S03 ), i.e. is strongly acidic. This allows quantitative separation of the Cy5.5-peptide loaded DR4 molecules from other DR4 molecules by anion exchange chromatography (Fig. 23D).
  • a negatively charged tag allows purification of correctly loaded MHC complexes
  • a related strategy namely for a negatively charged tag that can be readily synthesized and then be removed by photolysis.
  • new tag we used phospho-tyrosine-Asp 4 (pY-D 4 ), which can be detected by anti-pY antibodies in ELISA and having six negative charges is expected to allow separation of loaded MHC II molecules by anion exchange chromatography (Fig. 24).
  • the His tag can be used again to directly form fluorescent staining reagents by reaction with NTA n -PE or NTA n -Qdots (as described for MHC I-peptide complexes in the first technical report).
  • phospho-tyrosine a priori is susceptible to enzymatic de-phosphorylation (although with complete phosphates inhibitors, e.g. from Roche, this can be prevented) we also tested para-sulfate-tyrosine (e.g. Y(S0 4 )-D 4 ) as tag, but observed lesser shifts of the acidic peptide containing DR4 complexes (data not shown).
  • neuraminidase increases multimer binding by 2-5-fold. This was observed on a range of DR4-restricted, HA-specific CD4+ T cell clones (Fig. 28) and on populations of HA peptide stimulated PBMC from healthy donors. Because MHC II multimer staining tends to be weak, this observation suggests pre-treatment of the cells with neuraminidase, an enzyme that removes sialic acid residues on cellular surface proteins (18) increases MHC class II multimer staining efficiency.
  • PE-NTA-A2/peptide multimers - a new type of staining reagent based on oxime ligation.
  • PE-NTA multimers 1 nmol Phycoerythrine (PE) was first reacted in phosphate buffer, pH 7.4, with 20 mM of sulfo-SFB (sulfo-succinimidyl-formylbenzoate) for 4 h at room temperature.
  • the resulting activated-PE was subsequently dialyzed in 2 L PBS over 2 days and then coupled in phosphate buffer (pH 7.2) with a large excess of H 2 N-0- NTA 2 or H 2 N-0-NTA 4 aminooxy-containing peptide. After overnight reaction at 4 °C, bioconjugates were loaded with Ni and dialyzed in 2 L PBS over 2 days.
  • PE-NO-NTA 2 or PE-NO-NTA 4 obtained were conjugated with A2/peptide-2xHis6 complexes to generate biotin SA free PE-NTA 2 or PE-NTA 4 multimers. Staining is a function ofPE substitution degree.
  • Figure 29A illustrates a scheme of PE activation with sulfo-SFB and conjugation with H 2 N-0-NTA 2 via oxime ligation.
  • Figure 29B PE (1 nM) was activated with the indicated concentrations of sulfo-SFB and conjugated with an excess of NTA peptide.
  • 0.5 ⁇ g of PE-NO-NTA 2 were mixed with 5 ⁇ g of A2/Flu58-66-2xHis6 monomers.
  • Flu matrix 58-66-specific BC74cells were stained with 8 nM of the different conjugates at room temperature for 30 min and analyzed by flow cytometry.
  • SFB succinimidyl-p- formylbenzoate.
  • Figure 30 shows staining isotherms on Flu stimulated PBMC.
  • Figure 30A PE activated with 1, 10 or 20 mM sulfo-SFB was coupled to NTA 2 peptide via oxime ligation and subsequently mixed with A2/Flu-2xHis6 A2/Flu58-66 monomers. Flu matrix peptide stimulated PBMC were stained with different concentrations of the conjugates and analyzed by flow cytometry. The numbers indicate MFI of specific (above the bar in each graph) and non-specific (below the bar) staining. The right hand numbers indicate the input amounts of monomers (upper number) and PE-NTA2 (lower number).
  • Figure 30B Data plotted using GraphPad Prism software.
  • FIG 31 shows exemplary NTA linkers that were synthesized and tested.
  • the bioNTA4 and oxNTA2 linkers were determined to be of particular interest for the generation of MHC I and MHC II multimers and were used for further testing as described below.
  • the bioNTA 2 and bioNTA 4 compounds contain biotin- NT A2 or4, all others contain N terminal Cys(SH)-PEG 2 on NTA 2 or NTA 4 to be used for conjugation with maleimido-PE.
  • the non- biotin compounds contain N-terminal imines and NTA 2 linked via a PEG 2 Spacer, a PEG 4 spacer, or a dipeptide linker (bottom).
  • PE conjugates with these linkers were obtained and the performance of these conjugates was tested.
  • the conjugates comprised either A2/Flu58-66 monomers, which were tested in staining of Flu peptide stimulated PBMC, or comprised DR4/Flu HA306-318 monomers, which were tested in staining of Flu-Specific CD4+ T cells (Figure 32). From these initial tests, the biotin-NTA4 and SA-PE multimers appear to perform similar to conventional BSP multimers, but result in better stainings of both CD8 + and CD4 + T cells. In addition, the multimers described herein are fully reversible, as explained in more detail elsewhere herein, which allows sorting of antigen-specific T cells without causing activation dependent T cell death.
  • the data presented here indicates that the PEG2-NTA2-comprising PE-NTAmers coupled to PE via oxime bond formation exhibit an improved staining performance as compared to multimers obtained by conjugation via maleimides. This may in par be due to an increased degree of conjugation associated with the use of oxime-chemistry. Comparison of conventional, PE-Cys-PEG2-NTA2 and PE-HNO-NTA2 multimers staining.
  • Figure 33A shows the results of an experiment in which cloned, Flu matrix 58-66- specific CD8 + 81P1 cells were incubated at 20°C for 30 min with graded concentration of conventional multimers (circles), PE-Cys-PEG 2 -NTA 2 NTAmers (squares ), or PE-HNO- NTA 2 NTAmers (triangles). Cell-associated PE fluorescence was then assessed by flow cytometry.
  • the binding data of Figure 33A were subjected to Scatchard analysis (Figure 33 B), and K D and B max were calculated from the results of the Scatchard Analysis (Figure 33C).
  • Binding titration of NTAmers to determine best dilution for ex vivo staining Binding titration of NTAmers to determine best dilution for ex vivo staining.
  • Figure 35B were mixed with fresh PBMC and incubated at 20°C for 30 min with graded dilution of PE-NTA2 NTAmer (row 2), SA-PE multimers containing SA-PE biotin-NTA 2 (row 3) or SA-PE biotin-NTA 4 (row 4).
  • Figure 36 shows ex vivo flow cytometric analysis of PBMC stained with A2/peptide multimers containing BSP (conventional), PE-oxime NTA 2 , or SA-PE biotin-NTA 4 .
  • PBMCs were obtained from EBV+ (BCL 7) individuals (Figure 36A), CMV+ (BCL 8) individuals ( Figure 36B), and from two melanoma patients: LAU 616 ( Figure 36C) and LAU 1164 ( Figure 36D).
  • multimeric MHC II staining reagents described herein are generated by loading monomeric MHC molecules with a peptide of interest and purifying correctly peptide-loaded monomers for further processing and assembly to multimers.
  • a hexahistidine (His6) tag is added N-terminally at the peptide and the complexes are purified on Ni 2+ NTA columns.
  • an N- terminal polyacidic tag is used, which allows purification of complexes by anion exchange chromatography.
  • a desthiobiotin (DTB) tag is N-terminally added to the peptide using conventional solid phase peptide synthesis (SPPS) and the target complexes are purified on streptactin columns using elution with free DTB.
  • SPPS solid phase peptide synthesis
  • the column can be completely regenerated by washing with 2-(4'-hydroxyazobenzene) benzoic acid (HABA) and the Tris, pH 9.0.
  • HABA 2-(4'-hydroxyazobenzene) benzoic acid
  • the recovery yields are close to 100%, and pMHC II complexes fully active.
  • DTB is less complex than using a poly-acidic tag and anion exchange chromatography, and has the advantage that a His tag can be used on the MHC II protein. In some embodiments, this allows i) gentle and universal purification of MHC II proteins from supernatants and ii) diverse conjugate formation based on the His tag- NTA chelate complex conjugation strategies described herein.
  • An overview of an exemplary strategy for NTA-His tag multimer preparation using DTB is outlined in Figure 37. Protein expression in Drosophila cells
  • Serum-free adapted Drosophila melanogaster cell line (D.mel-2) were transfected concomitantly with two pMT-derived plasmids containing cloned extracellular parts of the a and beta chain of the MHC class II allele, followed by leucine zipper sequences and terminating with a Avi-Tag (beta chain) or a tandem His-tag (a chain).
  • a pBS-derived plasmid containing the puromycin resistance gene is co-transfected to allow for antibiotic selection. After selection in 10 ⁇ g/ml puromycin (Sigma), cells were cloned by limiting dilution and clones screened for high expression. Expression levels in supernatants of DR1, DR4 and DP4 proteins with C-terminal tandem His-tags range from 1 to 5 mg/ml as determined by ELISA.
  • DTB-containing complexes were eluted with 50 mM desthiobiotin in PBS (pH corrected to 7.4) while the pY-E8 complexes were eluted with a NaCl gradient (0-1 M NaCl in 20 CV) at 400 mM NaCl.
  • Flow rates were 1 ml/min for the StrepTrap column and 2 ml/min for the MonoQ column (both columns from GE Healthcare Life Sciences).
  • the StrepTrap column is regenerated with 1 mM HABA (Sigma) in PBS and equilibrated in 30 CV of PBS.
  • the eluted proteins were diluted with an equal volume of PBS (DTB-tagged peptide) or 50 mM Tris pH 9.0 (pY-E8-tagged peptide) before being concentrated to 0.5-1 mg/ml in an Amicon- 5 filter concentrator (Millipore). Aliquots of 10-25 ⁇ g were snap frozen in liquid nitrogen and stored at -80 C. Optional tag removal by UV. In case the tag is linked to the peptide with a photocleavable amino-acid derivative ( ⁇ ), it can be removed by irradiation with a 30 W UV lamp (365 nm) by irradiating for 10 min in V-bottom 96-well plates (1 ⁇ g MHC -peptide complex in 100 ul PBS).
  • Purified MHC class II-peptide complexes with C-terminal tandem His-tags are added to NTA-derivatized PE and SA-PE conjugates in a well of a V-bottom 96-well plate, vigorously mixed and incubated at least 2 hours at 4°C before use in staining a cell sample.
  • FIG. 39 A DTB tagged model peptide was generated and purification performance was evaluated (Figure 39).
  • the structure of a low molecular weight colored peptide used for quantification of the purification efficiency is described in Figure 39A.
  • Figure 39B shows a compilations of the efficiencies of retention, elution and regeneration for 3 different columns.
  • Figure 39C shows purification yields on StreptActin superflow high capacity column for three consecutive rounds of purification/regeneration.
  • Regeneration buffer HABA 0.1 M followed by Tris 0.2 M, pH 9.
  • BindingAVashing buffer Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8.
  • Elution Buffer Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8, 50 mM DTB.
  • Regeneration Buffer Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8, 1 mM 2-(4'-hydroxyazobenzene) benzoic acid (HABA).
  • HABA 2-(4'-hydroxyazobenzene) benzoic acid
  • FIG. 42A shows a chromatogram of a DTB-tagged protein purified on a StreptActin sepharose column.
  • Figure 42B shows the purification parameters for DTB tagged MHC class I-peptide complexes.
  • FIG 43 An exemplary scheme of DTB-HA peptide loading and purification is described in Figure 43.
  • An exemplary structure of DTB-HA and the corresponding DR42xHis DTB-HA complexes are depicted in Figure 43A.
  • a schematic of the purification and regeneration cycle of DR42xHis DTB-HA on a StreptActin sepharose column is illustrated in Figure 43B.
  • DR4-DTB-HA peptide complexes were generated and purified on a streptactin column. After peptide-loading, DR4-2xHis DTB-HA306-318 complexes were passed over a StreptActin column, washed and eluted with 50 nM DTB as indicated by arrows ( Figure 44A), and the OD of the effluent at 280 nm was monitored. Pure DR4-HA complexes eluted in a sharp peak (indicated by a box in Figure 44 A). DR4 2xHis DTB-HA306-318 complexes were analyzed by SDS-PAGE (12%) before (lane 1) and after (lane 2) purification on
  • StreptActin sepharose ( Figure 44B). SDS-PAGE was performed under non-reducing, non- boiling (NR, NB, left gel image) or reducing and boiling conditions (R, B, right gel image). Each lane was loaded with 10 ⁇ g protein and the gel was stained with Coomassie blue. DTB- tag based purification resulted in highly pure peptide-loaded MHC preparations. Surprisingly, the purity of DTB-tagged peptide-loaded MHC molecules obtained by StreptActin column purification was observed to be superior to that of purification of polyanionic peptide tagged MHC molecules, which yield highly pure peptide-loaded MHC molecules. Staining of CD4+ DR4-restricted Flu-specific T cells with DTB immunopure mult inters.
  • PE-NTA 2 Staining performance of PE-NTA 2 , biotin-NTA 4 -SA-PE, and conventional BSP multimers was compared. As described in more detail elsewhere herein, PE-NTA 2 and biotin-NTA 4 -SA-PE multimers contain the NTA moieties shown in Figure 45.
  • HA-specific CD4 + T cell clones 9(A) or 8(B) were incubated for 1 hour at 37°C with graded concentrations of DR4/HA 3 o6- 3 is multimer (as in previous experiments), washed and analyzed by flow cytometry ( Figure 47).
  • a DR4-restricted Flu matrix specific clone was used to evaluate nonspecific binding, which was subtracted. Scatchard analysis was performed and the K D (dissociation constant) and Bmax (maximal binding) values are described in the tables on the right.
  • the PE-NTA 2 multimers exhibited efficient binding/staining, particularly on clones that exhibited poor BSP multimer staining. Similarly, the biotin-NTA 4 -SA-PE multimers stained with higher efficiency than BSP multimers. It is important to note, however, that significant clonal variations were observed.
  • DR4/HA 0 6- 3 i8 SA-PE NTA 4 multimers exhibited less background staining than PE NTA 2 multimers ( Figure 48).
  • HA peptide- stimulated PBMC were incubated at room Temperature (A, B) or 4°C, (C).
  • Optimal multimer concentrations 14 ⁇ g/ml for PE NTA 2 , 28 ⁇ g/ml for SA-PE NTA 4 ) were used in (A) and (B).
  • (C) three different concentrations of the PE NTA 2 reagent were compared.
  • Non- stimulated PBMCs were used as a control.
  • the PE-NTA 2 multimers tended to exhibit increased background staining on control PBMCs. Background staining was efficiently suppressed by addition of 0.5% milk powder (as used, e.g., in Western blotting).
  • SA-PE NTA4 DR4/HA 3 o6-3 i8 multimers were able to detect more antigen-specific cells than BSP multimers over a wide range of concentrations (Figure 49).
  • Stimulated PBMCs were incubated for 1 h at 37°C (A) or room temperature (C) with 10 ⁇ g/ml of the indicated HA peptide-loaded multimers and analyzed after washing by flow cytometry.
  • Nonspecific background (control) was determined on TT 6 3 4 -653 peptide stimulated PBMCs. Specific multimer staining was assessed in cells prepared and stained as in (A) over at different concentrations of multimers (B). For comparison the frequencies of IFNy + T cells was assessed by ICS and flow cytometry with (+) or without (-) peptide stimulation.
  • Biotin-NTA 4 but not BSP multimers, can be rapidly removed from stained, antigen- specific cells (Figure 50). Peptide- stimulated PBMCs were stained with optimal
  • Biotin-NTA 4 -SA- PE multimers were observed to be stable in the absence and rapidly reversible in the presence of imidazole. This prevents multimer staining-induced death of antigen- specific T-cells, for example, CD4 + T cells or CD8 + T cells. As described elsewhere herein.
  • the staining reagents were prepared as described above from DTB/StreptActin purified DRl/NY-ESOn 9 - 14 3 monomers.
  • CD4 + cells were incubated with three different multimer dilutions (1:2, 1:4 and 1:8, corresponding to 14 ⁇ g/ml, 7 ⁇ g/ml, and 3.5 ⁇ g/ml for the SA-PE NTA 4 and the BSP multimers, and to 7 ⁇ g/ml, 3.5 ⁇ g/ml and 1.75 ⁇ g/ml for the PE NTA 2 multimers (Figure 51 white histograms).
  • Mouse splenocytes (C57BL/6J) were used as a negative control (black histograms).
  • the DRl/ESOu 9 - 14 3 cell line was derived from an HLA-DR1 transgenic mouse immunized with NY-ESOu 9 -i 4 3 peptide, and was maintained in IMDM-10, 50 ⁇ beta-mercaptoethanol, pen/strep, with 50 U/ml of rmIL-2.
  • the gradient shown as a light line rises from 0 to 1 M NaCl.
  • Figure 52 B shows an ELISA of DR4 (light line) and pY (phospho-tyrosine)(dark line).
  • Additional acidic tags that were successfully employed for the isolation of correctly peptide loaded MHC II monomers includes YP-D4-SGSG-*-HA, YS-D6-SGSG-*-HA, YP- E8-GSG-HA, YP-E8-GSG-HA, YP-DDGGDDGGDD-SGS-HA, P - P0 4 2" ,-S - SO 3" .
  • the pY-E8 tag provided the best results. While anionic tags are suitable for the isolation of peptide-loaded MHC monomers, the disadvantages of using such tags include: i) difficulties in peptide synthesis, ii) the high NaCl concentration needed to elute the complexes may partially denature them; iii) an additional desalting step is need to remove the high NaCl concentrations. While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention.
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B" can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

Some aspects of this invention are based on the recognition that reversible protein multimers in which monomeric proteins are conjugated to a carrier molecule via chelation complex bonds are stable under physiological conditions and can be dissociated in a controlled manner under physiological, nontoxic conditions. Accordingly, such protein multimers are useful for a variety of in vitro, ex vivo, and in vivo application for research, diagnostics, and therapy. Some aspect of this invention provide reversible MHC protein multimers, and methods of using such multimers in the detection and/or isolation of specific T-cells or T-cell populations. Because reversible MHC multimers can efficiently be dissociated, the time of MHC binding to T-cell receptors, and, thus, T-cell receptor- mediated T-cell activation can be minimized. The use of reversible MHC multimers as provided herein, accordingly, allows for the detection and isolation of bona fide antigen- specific CD8+ T cells without inducing activation dependent cell death, including rare, therapeutically valuable T- cells expressing T-cell receptors binding tumor antigens with high affinity. Methods for the production and use of reversible multimers are also provided.

Description

REVERSIBLE PROTEIN MULTIMERS, METHODS FOR THEIR
PRODUCTION AND USE
RELATED APPLICATION
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application serial number 61/389,092, filed on October 1, 2010, the entire disclosure of which is incorporated herein by reference.
BACKGROUND
Fluorescent MHC-peptide multimers, commonly referred to as tetramers are widely used to detect, enumerate, analyze and sort antigen- specific CD8+ T cells (1, 2). Monomeric MHC-peptide complexes are produced by refolding of a MHC heavy and light chain in the presence of a peptide of interest and subsequently are biotinylated at a C-terminally added biotinylation sequence peptide (BSP, LHHILDAQKMVWNHR, SEQ ID NO: 1) the biotin- transferase BirA. Fluorescent conjugates are obtained by reaction of biotinylated MHC-I- peptide monomers with phycoerythrin (PE) or allophycocyanine (APC) labeled streptavidin (2, 3). Although such reagents generally perform well, they have shortcomings: 1) they are heterogeneous in terms of stoichiometry and configuration, which compromises stringent binding analysis (4). 2) The enzymatic biotinylation is tedious, expensive and in the case of unstable MHC-peptide monomers causes degradation during the enzymatic reaction, which is performed at elevated temperatures (5). 3) For some studies antigen- specific CD8+ T cells are isolated by cell sorting (e.g. FACS or MACS). Conventional multimers stably bind to cells and induce strong T cell activation, which can induces death and result in loss of CD8+ T cells, questioning that the surviving cells are representative for the original populations (5- 8). To overcome this, reversible multimer have been introduced, which contain low affinity biotin analogues and hence dissociate upon addition of free biotin. With these reagents significantly improved sorting and cloning efficiencies are obtained; however, they are costly and tend to be less stable (5, 8). SUMMARY
Some aspects of this invention relate to reagents for reversible staining of cells. Some aspects of this invention provide staining reagents that comprise a core structure, referred to herein as a carrier molecule, to which a plurality of monomeric proteins can reversibly bind to form a protein multimer. In some embodiments, the carrier molecule itself is a detectable molecule, for example, a fluorophore. In some embodiments, the proteins that reversibly bind to the carrier molecule are proteins that also specifically bind a molecular target, for example, a specific cellular ligand or receptor, allowing for staining of the molecular target with the protein multimer. Since the bond of the protein and the carrier molecule is reversible, the protein multimer complex can be dissolved, resulting in dissociation of the proteins from the carrier molecule and, thus, in re-monomerization of the bound proteins.
The protein multimers provided by some embodiments described herein are useful, for example, for cell staining and cell isolation procedures. The multimers provided by some embodiments of this invention can be dissolved under non-toxic, physiological conditions, for example, by addition of an agent interfering with the reversible binding of protein and core structure of the multimer. Accordingly, one advantage of the instantly provided protein multimers is that, in contrast to most other staining agents, they do not expose stained cells to the undesirable effects of permanent staining. Dissolution of the multimer structure used for staining live cells avoids undesired biological effects of continued association of cells with a dye or a multimeric protein structure, for example, continued activation of T-cell receptor signaling in MHC-stained T-cells. Staining procedures using the instantly described protein multimers, thus, allow for "minimally invasive" staining of cells, which is a requirement for the isolation of native cells that are sensitive to staining procedures, including, for example, certain T-cell populations.
Some aspects of this invention provide methods and reagents for the generation of peptide-loaded MHC molecules, for example, of peptide-loaded MHC class II molecules. In some embodiments, methods for the efficient production of molecularly defined,
homogeneously peptide-loaded MHC class II molecules are provided. In some embodiments, MHC class II molecules are loaded with an antigenic peptide that is conjugated to a tag, for example, a tag allowing for peptide or protein isolation and/or purification by
chromatography, for example, by affinity or ion exchange chromatography. In some embodiments, tagged MHC class II binding peptides are provided. In some embodiments, the tag is an acidic tag, for example, a tag comprising a plurality of acidic amino acid residues, or an acidic fluorophore, for example, an acidic cyanine dye. In some embodiments, the tag, for example, the acidic tag, is reversibly conjugated to the antigenic peptide of interest, for example via a cleavable linker. In some embodiments, the cleavable linker is a photocleavable linker. In some embodiments, the cleavable linker comprises a cleavage site for an enzyme, for example, a protease, or a chemical.
Some aspects of the invention, including aspects and embodiments not mentioned in this summary are described in more detail elsewhere in the specification and in the claims.
Some aspects of this invention provide protein multimers that include (a) a
multivalent carrier molecule, and (b) a plurality of proteins bound to the carrier molecule. In some embodiments, at least one of the proteins is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant ΙμΜ > KD≥ lOfM. In some embodiments, at least one of the peptides or proteins is conjugated to the carrier molecule via a chelate complex bond.
Some aspects of this invention provide multivalent chelants that comprise a water- soluble carrier molecule and a plurality of chelant moieties conjugated to the carrier molecule.
Some aspects of this invention provide multivalent chelants that comprise a carrier molecule or structure, and a plurality of chelant moieties conjugated to the carrier molecule or structure, wherein the carrier molecule or structure has a diameter of less than O.lnm, less than 0.2nm, less than 0.25nm, less than 0.3nm, less than 0.4nm, less than 0.5nm, less than 0.6nm, less than 0.7nm, less than 0.75nm, less than 0.8nm, less than 0.9nm, less than lnm, less than l.lnm, less than 1.2nm, less than 1.3nm, less than 1.4nm, less than 1.5nm, less than 1.6nm, less than 1.7nm, less than 1.8nm, less than 1.9nm, less than 2nm, less than 2.5nm, less than 3nm, less than 4nm, less than 5nm, less than 6nm, less than 7nm, less than 8nm, less than 9nm, or less than lOnm. In some embodiments, the diameter of the carrier molecule is less than 20nm, less than 30nm, less than 40nm, less than 50nm, less than 60nm, less than 70nm, less than 80nm, less than 90nm, less than lOOnm, less than 200nm, less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm, or less than Ιμιη.
Some aspects of this invention provide methods for the production of protein multimers that comprise a step of contacting a monomeric chelant moiety-conjugated MHC molecule with a carrier molecule, for example, a water-soluble carrier molecule or a carrier molecule or structure described herein, that is conjugated to a plurality of chelant moieties under conditions suitable for formation of a chelate complex between the chelant moieties conjugated to the MHC molecule and the chelant moieties conjugated to the carrier molecule.
Some aspects of this invention provide methods for the production of MHC molecules that are conjugated to a ligand via a chelate complex bond, comprising a step of contacting an MHC molecule conjugated to a first chelant with a ligand molecule conjugated to a second chelant under conditions suitable for formation of a chelate complex between the first and the second chelant. In some embodiments, the resulting MHC molecule is then contacted with a multivalent binding molecule binding the ligand to produce reversible MHC multimers.
Some aspects of this invention provide methods to generate peptide-loaded MHC molecules that are conjugated to a chelant using MHC binding peptides conjugated to a tag, the methods comprising the steps of providing an MHC molecule bound to an antigenic MHC molecule-binding peptide that is conjugated to a tag via a cleavable linker, removing the tag from the antigenic peptide, and conjugating a chelant moiety to a heavy chain of the MHC molecule.
Some aspects of this invention provide methods for staining, detecting, and isolating cells, the methods comprising a step of contacting a population of cells with a protein multimer as described herein, for example, with a multimer comprising a chelate bond and a detectable label, and performing an assay to detect a cell binding the multimer.
Some aspects of this invention provide method for the isolation of cells that bind a multimer as described herein, the methods comprising the steps of (a) contacting a population of cells with a protein multimer as provided herein, for example, with a multimer comprising a chelate bond and a detectable label, (b) optionally, detecting a cell binding the multimer, and (c) isolating the cell binding the multimer.
Some aspects of this invention provide methods for the manipulation of T-cell populations with protein multimers, the methods comprising a step of contacting a population of cells expressing a T-cell receptor with an MHC multimer as described herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC class I molecule interaction to activate a T-cell expressing the T-cell receptor and binding the MHC multimer.
Some aspects of this invention provide cells or cell populations that are contacted with a protein multimer as provided herein, for example, with an MHC multimer. Some aspects of this invention provide kits comprising pep tide-loaded or empty MHC molecules as provided herein.
Some aspects of this invention provide isolated peptide-loaded MHC molecules that comprise an MHC heavy chain, and an antigenic peptide. In some embodiments, the peptide is conjugated to a tag, for example, a tag for ion exchange chromatography.
Some aspects of this invention provide methods for the generation of peptide-loaded MHC molecules, for example, MHC class II molecules, comprising a step of contacting an empty MHC molecule with an antigenic peptide conjugated to a tag under conditions suitable for the antigenic peptide to bind the MHC molecule. In some embodiments, the tag is a tag for ion exchange chromatography and the method, in some embodiments, includes a step of isolating peptide-loaded MHC molecules by performing a chromatography procedure.
Some aspects of this invention provide methods for the generation of MHC class II molecules, empty or peptide-loaded, the method comprising a step of contacting an MHC class II type alpha heavy chain with an MHC class II type beta heavy chain under conditions suitable for the alpha and the beta chain to form a heterodimeric MHC class II molecule. In some embodiments, at least one of the MHC class II heavy chains is conjugated to a tag. In some embodiments, a step of isolating the MHC class II molecule is performed, wherein the isolating comprises a step of chromatography, for example, affinity chromatography. Some aspects of this invention provide a protein multimer comprising (a) a multivalent carrier molecule, and (b) a plurality of proteins bound to the carrier molecule. In some embodiments, at least one of the plurality of proteins is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant ΙμΜ > KD > lOfM. In some embodiments,the dissociation constant is KD > O. lpM. In some embodiments, the dissociation constant is KD > lpM. In some embodiments, the dissociation constant is KD > lOpM. In some embodiments, the dissociation constant is KD > lOOpM. In some
embodiments, the dissociation constant is KD > InM. In some embodiments, the dissociation constant is KD > lOnM. In some embodiments, the dissociation constant is KD < lOOnM. In some embodiments, the dissociation constant is KD < lOnM. In some embodiments, the dissociation constant is KD < InM. In some embodiments, the dissociation constant is KD < lOOpM. In some embodiments, the dissociation constant is KD < lOpM. Some aspects of this invention provide a protein multimer comprising (a) a multivalent carrier molecule, and (b) a plurality of proteins bound to the carrier molecule. In some embodiments, at least one of the plurality of proteins is conjugated to the carrier molecule via a chelate complex bond. In some embodiments, the chelate complex bond is a bond with a dissociation constant 5μΜ > KD lfM. In some embodiments, the protein is an MHC molecule and the protein multimer is an MHC multimer. In some embodiments, the chelate complex bond comprises a chelant conjugated to the MHC molecule, and a chelant conjugated to the carrier molecule. In some embodiments, the chelant conjugated to the carrier molecule is of a different structure than the chelant conjugated to the MHC molecule. In some embodiments, the MHC molecule comprises an MHC a chain. In some embodiments, the MHC molecule further comprises an MHC a chain or a β2 microglobulin chain. In some embodiments, the MHC molecule is an MHC class I molecule. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the chelant conjugated to the MHC molecule is C- terminally conjugated to the MHC β chain. In some embodiments, the chelant conjugated to the MHC molecule is C-terminally conjugated to the MHC β chain or the β2 microglobulin chain. In some embodiments, the chelant conjugated to the MHC molecule is a peptide comprising a chelant moiety. In some embodiments, the peptide comprising a chelant moiety is fused to a polypeptide chain comprised by the MHC molecule. In some embodiments, the peptide comprising a chelant moiety comprises a poly-Histidine sequence. In some embodiments, the poly-Histidine sequence comprises 3-24 His residues. In some
embodiments, the chelant conjugated to the MHC molecule comprises a His6 tag, a His 12 tag, or a 2x His6 tag. In some embodiments, the chelant conjugated to the carrier molecule comprises an NTA moiety. In some embodiments, the NTA moiety is bound to the carrier molecule in mono-NTA configuration. In some embodiments, the NTA moiety is bound to the carrier molecule in poly-NTA configuration. In some embodiments, the NTA moiety is bound to the carrier molecule in di-NTA, or tetra-NTA configuration. In some embodiments, the NTA moiety is bound to a linker. In some embodiments, the linker comprises a maleimide moiety or derivative. In some embodiments, the linker comprises an oxime moiety or derivative. In some embodiments, the linker is between about 9A and about 23A long. In some embodiments, the linker is covalently bound to the carrier molecule. In some embodiments, the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule. In some embodiments, the ligand is biotin and the binding molecule is streptavidin. In some embodiments, the chelate complex bond further comprises a divalent cation. In some embodiments, the divalent cation is an Ni2+, Cu2+, Zn2+, Co2+, Cd2+, Sr2+ , Mn2+, Fe2+, Mg2+, Ca2+, or Ba2+ ion. In some embodiments, the carrier molecule is a fluorophore,, a phycobilin, phycoerythrin or allophycocyanine, a quantum dot (Qdot), a microsphere (e.g., a fluorescent microsphere (e.g. fluorospheres® type), a magnetic particle, or a nanoparticle. In some embodiments, the MHC molecule is an empty MHC molecule or a peptide-loaded MHC molecule. In some embodiments, the peptide-loaded MHC molecule is chosen from the MHC molecules disclosed in Table 2. In some embodiments, the MHC molecule comprises an HLA-A*0201 heavy chain. In some embodiments, the MHC molecule is loaded with an antigenic peptide. In some embodiments, the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL (SEQ ID NO: 2). In some embodiments, the multimer is a tetramer. In some embodiments, the antigenic peptide is conjugated to a tag. In some embodiments, the tag is conjugated to the peptide via a cleavable linker. In some embodiments, the linker is a photocleavable linker. In some embodiments, the linker is an ΝΡβΑ linker. In some embodiments, the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical. In some embodiments, the tag is an acidic peptide tag. In some embodiments, the acidic peptide tag comprises a plurality of acidic amino acid sequences. In some embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY- D9, or pY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the tag is a desthiobiotin (DTB) tag.
Some aspects of this invention provide a multivalent chelant comprising a water- soluble carrier molecule, and a plurality of chelant moieties conjugated to the carrier molecule. Some aspects of this invention provide a multivalent chelant comprising a carrier molecule or structure, and a plurality of chelant moieties conjugated to the carrier molecule or structure. In some embodiments, the carrier molecule or structure has a diameter of less than O. lnm, less than 0.2nm, less than 0.25nm, less than 0.3nm, less than 0.4nm, less than 0.5nm, less than 0.6nm, less than 0.7nm, less than 0.75nm, less than 0.8nm, less than 0.9nm, less than lnm, less than l.lnm, less than 1.2nm, less than 1.3nm, less than 1.4nm, less than 1.5nm, less than 1.6nm, less than 1.7nm, less than 1.8nm, less than 1.9nm, less than 2nm, less than 2.5nm, less than 3nm, less than 4nm, less than 5nm, less than 6nm, less than 7nm, less than 8nm, less than 9nm, or less than lOnm. In some embodiments, the diameter of the carrier molecule is less than 20nm, less than 30nm, less than 40nm, less than 50nm, less than 60nm, less than 70nm, less than 80nm, less than 90nm, less than lOOnm, less than 200nm, less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm, or less than Ιμηι. In some embodiments, the chelant moieties are nitrilotriacetic acid (NTA) moieties. In some embodiments, the NTA moieties are in mono-NTA configuration. In some embodiments, the NTA moieties are in poly-NTA configuration. In some embodiments, the NTA moieties are in di-NTA, or tetra-NTA configuration. In some embodiments, the NTA moieties are bound to a linker. In some embodiments, the linker comprises a maleimide moiety. In some embodiments, the linker comprises an oxime moiety. In some embodiments, the linker is between about 9A and about 23 A long. In some embodiments, the linker is covalently bound to the carrier molecule. In some embodiments, the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule. In some embodiments, the ligand is biotin and the binding molecule is streptavidin. In some embodiments, the carrier molecule is a fluorophore, a phycobilin, phycoerythrin or allophycocyanine, or a quantum dot (Qdot). In some embodiments, a plurality of the chelant moieties conjugated to the carrier molecule form chelate complex bonds to a plurality of chelant moiety-conjugated monomeric molecules, thus forming a multimer of the monomeric molecule. In some embodiments, the monomeric molecule is a polyprotein. In some embodiments, the monomeric molecule is a small molecule compound. In some
embodiments, the monomeric molecule is a polynucleotide. In some embodiments, the monomeric molecule is a ligand of a receptor. In some embodiments, the receptor is a cell- surface receptor. In some embodiments, the receptor is a T-cell receptor. In some embodiments, the monomeric molecule is an MHC molecule. In some embodiments, the monomeric molecule is an MHC class I molecule. In some embodiments, the MHC molecule comprises an HLA-A*0201 heavy chain. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the MHC molecule is loaded with an antigenic peptide. In some embodiments, the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL (SEQ ID NO: 3). In some embodiments, the multivalent chelant is a tetravalent chelant. In some embodiments, the tag is an acidic peptide tag. In some embodiments, the acidic peptide tag comprises a plurality of acidic amino acid sequences. In some embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, pY-D10, pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the tag is a desthiobiotin (DTB) tag.
Some aspects of this invention provide a method comprising contacting a monomeric chelant moiety-conjugated MHC molecule with a water-soluble carrier molecule conjugated to a plurality of chelant moieties under conditions suitable for formation of a chelate complex between the chelant moieties conjugated to the MHC molecule and the chelant moieties conjugated to the carrier molecule. In some embodiments, the chelant moieties conjugated to the carrier molecule are NTA moieties. In some embodiments, the NTA moieties are in mono-NTA configuration. In some embodiments, the NTA moieties are in poly-NTA configuration. In some embodiments, the NTA moieties are in di-NTA, or tetra-NTA configuration. In some embodiments, the NTA moieties are bound to a linker. In some embodiments, the linker is between about 9A and about 23A long. In some embodiments, the linker is covalently bound to the carrier molecule. In some embodiments, the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule. In some embodiments, the ligand is biotin and the binding molecule is streptavidin. In some embodiments, the carrier molecule is a fluorophore. In some embodiments, the carrier molecule is a phycobilin. In some embodiments, the carrier molecule is phycoerythrin or allophycocyanine. In some embodiments, the carrier molecule is a quantum dot (Qdot). In some embodiments, the carrier molecule is a magnetic particle. In some embodiments, the carrier molecule is a nanoparticle. In some embodiments, the carrier molecule is PE conjugated to streptavidin. In some embodiments, the carrier molecule conjugated to a plurality of chelant moieties is generated by incubating a carrier molecule conjugated to a plurality of binding molecules with an excess of chelant-conjugated ligand under conditions suitable for the ligand to bind the binding molecule. In some embodiments, the molar ratio of carrienligand is between 1:2 and 1: 10. In some
embodiments, the molar ratio of carrienligand is 1 :5. In some embodiments, the incubating is performed at a temperature between 2-16 °C. In some embodiments, the incubating is performed at about 4 °C. In some embodiments, the method comprises a step of incubating the carrier molecules contacted with the ligand with NiS04. In some embodiments, the method comprises a step of contacting the carrier molecule conjugated to a plurality of chelant moieties with a molar excess of the MHC molecule conjugated to a chelant. In some embodiments, the excess is 2-20 fold. In some embodiments, the excess is 10-fold. In some embodiments, the MHC molecule is an MHC class I molecule. In some embodiments, the MHC molecule comprises an HLA-A*0201 heavy chain. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the MHC molecule is loaded with an antigenic peptide.
Some aspects of this invention provide a method comprising contacting an MHC molecule conjugated to a first chelant with a ligand molecule conjugated to a second chelant under conditions suitable for formation of a chelate complex between the first and the second chelant. In some embodiments, the first chelant comprises a peptide. In some embodiments, the peptide is C-terminally fused to a polypeptide chain comprised by the MHC molecule. In some embodiments, the peptide comprises a poly-Histidine sequence. In some embodiments, the poly-Histidine sequence comprises between 3 and 24 His residues. In some embodiments, the chelant conjugated to the MHC molecule comprises a His6 tag, a His 12 tag, or a 2x His6 tag. In some embodiments, the second chelant is NTA. In some embodiments, the NTA is in mono-NTA configuration. In some embodiments, the NTA is in poly-NTA configuration. In some embodiments, the NTA is in di-NTA, or tetra-NTA configuration. In some
embodiments, the NTA is bound to a linker. In some embodiments, the linker is covalently bound to the ligand molecule. In some embodiments, the linker is between about 9A and about 23A long. In some embodiments, the method further comprises contacting the ligand molecule with a multivalent binding molecule under conditions suitable for the ligand to bind the multivalent binding molecule. In some embodiments, the ligand is biotin and the binding molecule is streptavidin. In some embodiments, the binding molecule is conjugated to a carrier molecule. In some embodiments, the carrier molecule is a fluorophore. In some embodiments, the carrier molecule is a phycobilin. In some embodiments, the carrier molecule is phycoerythrin or allophycocyanine. In some embodiments, the carrier molecule is a quantum dot (Qdot). In some embodiments, the carrier molecule is a magnetic particle. In some embodiments, the carrier molecule is a nanoparticle. In some embodiments, the MHC molecule is an MHC class I molecule. In some embodiments, the MHC molecule comprises an HLA-A*0201 heavy chain. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the MHC molecule is loaded with an antigenic peptide. In some embodiments, the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL or a peptide comprising a sequence provided in Table 2. Some aspects of this invention provide a method comprising providing an MHC molecule bound to an antigenic MHC molecule-binding peptide that is conjugated to a tag via a cleavable linker, removing the tag from the antigenic peptide, and conjugating a chelant moiety to a heavy chain of the MHC molecule. In some embodiments, the method further comprises contacting a multivalent chelant molecule with the MHC molecule under conditions suitable for the chelant moiety conjugated to the MHC molecule to form a chelate complex bind with a chelant moiety of the multivalent chelant molecule.
Some aspects of this invention provide a method comprising contacting a population of cells with a protein multimer according to any preceding claim, wherein the multimer comprises a chelate bond and a detectable label, and detecting a cell binding the multimer. In some embodiments, the protein multimer is an MHC multimer. In some embodiments, the detectable label is a fluorophore. In some embodiments, detecting is by fluorescent microscopy or cell sorting. In some embodiments, the detectable label is a magnetic particle. In some embodiments, detecting is by isolating the cell binding the multimer. In some embodiments, detecting comprises quantifying a number of cells binding the multimer. In some embodiments, detecting comprises quantifying a number of cells binding the multimer as a ratio to a number of cells of the population of cells that do not bind the multimer. In some embodiments, the method further comprises reversing the binding of the multimer to the cell binding the multimer by contacting the cells binding the multimer with a monomeric chelant moiety competing for the chelate complex bond comprised by the multimer. In some embodiments, the monomeric chelant moiety is an imidazole molecule.
Some aspects of this invention provide a method comprising contacting a population of cells with a protein multimer according to any preceding claim, wherein the multimer comprises a chelate bond and a detectable label; optionally, detecting a cell binding the multimer; and isolating the cell binding the multimer. In some embodiments, the multimer is an MHC multimer. In some embodiments, the method further comprises reversing the binding of the multimer to the cell binding the multimer by contacting the isolated cell binding the multimer with a monomeric chelant moiety competing for the chelate complex bond comprised by the multimer. In some embodiments, the monomeric chelant moiety is an imidazole molecule. In some embodiments, the isolated cell is a T-cell. In some
embodiments, the T-cell does not undergo CD8/TCR-mediated activation during the contacting, optionally, during the detection, and during the isolation step. In some embodiments, the isolated T-cell is part of an isolated, native T-cell population.
Some aspects of this invention provide a method comprising, contacting a population of cells expressing a T-cell receptor with an MHC multimer provided herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC class I molecule interaction to activate a T-cell expressing the T-cell receptor and binding the MHC multimer. Some aspects of this invention provide a method comprising, contacting a population of cells expressing a T-cell receptor with an MHC multimer provided herein under conditions suitable for the multimer to bind to the T-cell receptor and to render the T-cell non-responsive to a naturally occurring antigen. In some embodiments, the method further comprises contacting the population of T-cells with an agent able to release the chelate complex bond of the MHC multimer after the cells were contacted with the multimer.
Some aspects of this invention provide a cell or cell population comprising a cell contacted with the protein multimer of any preceding claim, wherein the multimer comprises a chelate complex bond. In some embodiments, the multimer is an MHC multimer. In some embodiments, the cell is or has been contacted with an excess of monomeric chelant moieties competing for the chelate complex bond comprised by the multimer.
Some aspects of this invention provide a kit comprising an MHC multimer as provided in any preceding claim. In some embodiments, the MHC multimer is loaded with an antigenic peptide. In some embodiments, the MHC multimer is an empty MHC multimer. In some embodiments, the kit further comprises at least one antigenic peptide that can be loaded onto the empty MHC multimer. In some embodiments, the kit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different antigenic peptides. In some embodiments, the antigenic peptides are antigenic peptides of tumor antigens.
Some aspects of this invention provide an isolated peptide-loaded MHC molecule comprising an MHC heavy chain, and an antigenic peptide. In some embodiments, the peptide is conjugated to a tag. In some embodiments, the tag is an affinity tag. In some embodiments, the tag is a desthiobiotin (DTB) tag. In some embodiments, the peptide is conjugated to a tag for ion exchange chromatography. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the tag is an acidic tag. In some embodiments, the acidic tag is an acidic cyanine dye. In some embodiments, the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences. In some
embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the MHC molecule further comprises a heavy chain that is conjugated to a chelant moiety. In some embodiments, the molecule comprises a combination of a heavy chain and an antigenic peptide discloses in Table 2. In some embodiments, the tag is conjugated to the peptide via a cleavable linker. In some embodiments, the linker is a photocleavable linker. In some embodiments, the linker is an NP A linker. In some embodiments, the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical. In some embodiments, the MHC molecule is comprised in an MHC multimer.
Some aspects of this invention provide a method comprising contacting an empty MHC molecule with an antigenic peptide conjugated to a tag under conditions suitable for the antigenic peptide to bind the MHC molecule. In some embodiments, the MHC molecule is an MHC class II molecule. In some embodiments, the tag conjugated to the MHC class II binding antigenic peptide is an affinity tag that is not a polyhistidine tag. In some
embodiments, the tag is a desthiobiotin (DTB) tag. In some embodiments, the tag is an acidic tag. In some embodiments, the acidic tag is an acidic cyanine dye. In some embodiments, the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences. In some embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the tag is conjugated to the peptide via a cleavable linker. In some embodiments, the linker is a photocleavable linker. In some embodiments, the linker is an NP A linker. In some embodiments, the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical. In some embodiments, the tag is a part of a cleavable linker that remains after cleavage of the linker.
Some aspects of this invention provide a method comprising contacting an MHC class II type alpha heavy chain with an MHC class II type beta heavy chain under conditions suitable for the alpha and the beta chain to form a heterodimeric MHC class II molecule. In some embodiments, at least one of the MHC class II heavy chains is conjugated to a tag, and isolating the MHC class II molecule, wherein the isolating comprises a step of affinity chromatography. In some embodiments, the tag is a protein or peptide tag. In some embodiments, the tag is a poly- His tag. In some embodiments, the His tag comprises 3-12 His residues. In some embodiments, the affinity chromatography is ΝΪ2+-ΝΤΑ
chromatography. In some embodiments, the contacting is performed by expressing both heavy chains in a cell. In some embodiments, the cell is an insect cell. In some embodiments, the MHC class II molecule is "empty" (not loaded with an antigenic, MHC class II binding peptide). In some embodiments, the method further comprises contacting the MHC class II molecule with an MHC class II binding antigenic peptide. In some embodiments, the MHC class II binding antigenic peptide is conjugated to a tag. In some embodiments, the tag is an affinity tag. In some embodiments, the tag conjugated to the MHC class II binding antigenic peptide is an affinity tag that is not a polyhistidine tag. In some embodiments, the tag is a desthiobiotin tag. In some embodiments, the tag is an acidic tag. In some embodiments, the acidic tag is an acidic cyanine dye. In some embodiments, the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences. In some embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the tag is conjugated to the peptide via a cleavable linker. In some embodiments, the tag is a desthiobiotin tag. the linker is a photocleavable linker. In some embodiments, the tag is a desthiobiotin tag. the linker is an ΝΡβΑ linker. In some embodiments, the tag is a
desthiobiotin tag. the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical. In some embodiments, the tag is a desthiobiotin tag. the tag is a part of a cleavable linker that remains after cleavage of the linker.
The subject matter of this application may involve, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of a single system or article. Other advantages, features, and uses of the invention will become apparent from the following detailed description of non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. MHC-peptide complexes under study. A) Structure of a NTA-Ni2+-His6 complex in which Ni2+ forms a chelate on one hand with the three carboxyl moieties of NTA and with the imidazole groups of two histidines on the other. B) The conventional biotin- streptavidin PE-MHC class I-peptide multimer. C) NTA multimers in which His tagged MHC-peptide monomers are bound to NTA-PE, obtained by reacting PE streptavidin with biotin-NTA compounds. D) As C), but the NTA moieties are directly conjugated to PE. E) As D) using Qdots instead of PE.
Figure 2. His tagged HLA-A2 under study and their refolding efficiencies. A) Composition of the different C-terminal tags on HLA-A2 including the biotinylation sequence peptide BSP, linear His6, His12 or two His6 tags separated by a flexible
GGGSGGGSGS (SEQ ID NO:4) spacer. The underlined residues (274-276) mark the C- terminus of the A2 a3 domain. Sequences, from top to bottom, correspond to SEQ ID NOs 5-8. B) HLA-A2 heavy chains containing the different tags were refolded with β2ιη in the presence of Flu matrixs8_66 peptide. The refolding efficiencies expressed as percent with 100% being the one of HLA-A2-BSP.
Figure 3. Structures of the NTA linker under studies. A) Biotin-mono NTA in which a biotin (circled) is coupled to NTA-lysine via N-(5-(3-maleimidopropionylamino)-l- carboxy-pentyl)iminodiacetyl-cysteine-Na-acetyl-N8-amino-caproyl. B) biotin-di-NTA in which two NTA lysines were coupled via maleimide to two cysteines spaced by a glycine. C) biotin-tetra-NTA in which two di-NTA moieties are joined via GGGSGGGSGS (SEQ ID NO: 9) spacer. D) Amino-di-NTA, analogous to B) with a free amino group (circled). E) thiol-di-NTA, analogous to D) but with an N-terminal cysteine containing a free thiol (circled). F) Commercially available NTA-biotin (Biotium Inc, Hay ward, CA).
Figure 4. SPR binding studies for the different Ni-NTA linkers and His tags. A)
Experimental set up consisting in first loading streptavidin coated sensor chips with biotin- mono-NTA or biotin-di-NTA, followed by saturating with NiCl2 and washing. The changes in resonance units (RU) were measured upon injecting the different A2/Flu complexes over the NTA-Ni2+ loaded chips. B) Compilation of the dissociation constants (KD in nM), binding on-rate constants (kon in M" x sec" x 104) and binding off-rates (kQff in sec 1 x 10"3) recorded at room temperature for the differently His tagged A2/Flu complexes on mono-NTA (Fig. 3A) or di-NTA (Fig. 3B) loaded sensor chips. Figure 5. Bindings isotherms of different NTA-biotin-streptavidin PE A2/Flus8-66 multimers. A) Cloned, Flu-specific 81P1 cells were incubated at 20°C for 30 min with graded concentrations of PE streptavidin A2/Flus8_66 multimers containing NTA2 x 2xHis6 (dark A ), NTA2 x Hisi2 (light■), NTA x 2xHis6 (light A); NTA x His12 (dark■), NTA2 x His6 (light♦) or NTA x His6 (dark♦). For comparison BSP multimers were included (circles). After washing, cell-associated fluorescence was assessed by flow cytometry. B)
Alternatively 20°C binding isotherms were assessed likewise on cloned BCB 70 cells for multimers containing NTA2-biotin (light lines) or NTA4-biotin (dark lines) and A2/Fhi58-66 complexes with His12 (♦) or 2xHis6 tags (A ).
Figure 6. Dissociation kinetics of different NTA2-biotin-steptavidin A2/Flu58 66 multimers. A, B) Cloned Flu-specific 81P1 cells were incubated at 4°C for lh with multimers (5 nM) containing biotin NTA2 and His12 (A) or 2xHis6 (B) tagged A2/Flu complexes. After washing the cells were incubated at 4°C in HBSS in the absence (squares) or presence of 50 mM imidazole (triangles), 50 mM imidazole + 20 mM EDTA (dashes) or 100 mM imidazole (diamonds) and after the indicated periods of time cell- associated multimers were assessed by flow cytometry. For comparison conventional BSP multimers were included (circles). The inserted numbers indicate the times (ti/2) at which half maximal dissociation was observed.
Figure 7. Dissociation kinetics of NTA2-and NTA4 biotin-streptavidin A2/Flu58-66 multimers. A, B) Cloned BCB 70 cells were incubated at 4°C for lh with NTA2- biotin- streptavidin PE (A) or NTA4-PE A2/Flu multimers (B) containing the His12 (dark symbols) or 2x His6 tag (light symbols). After washing the cells were incubated at 4°C in HBSS in the absence (diamonds) or presence of 50 mM imidazole (triangles), or 100 mM imidazole (squares) and after the indicated periods of time cell-associated multimers were assessed by flow cytometry. For comparison conventional BSP multimers were included (circles). The inserted numbers indicate the times (ti/2) at which half maximal dissociation was observed.
Figure 8. Preparation of NTA2-PE conjugates. A) Structure of PE, a fluorescent protein of 240,000 Da, has a rigid structure and 24 surface exposed lysines. B) Strategy to couple NTA2-cysteine (Fig. 3E) on PE. In a first step PE was reacted with the maleimide N- hydroxy- succinimidyl ester SM(PEG)2 (succinimidyl-maleido(PEG)2), whereby maleimide groups are conjugated onto PE lysines. In a second step the NTA2 cysteine (Fig. 3E) was added, which by reacting with maleimides on PE forms stable thio-ethers. Figure 9. Binding isotherms of NTA2 -PE A2/Flu58-66 multimers. Cloned 81P1 cells were incubated for 45 min at the indicated temperatures with graded concentrations of conventional BSP multimers (circles), or with multimers containing NTA2-PE coupled PE and A2/Flu complexes containing the 2xHis6 tag (squares) or the His12 tag (diamonds). After washing the cells they were analyzed by flow cytometry.
Figure 10. Dissociation kinetics of NTA2 -PE A2/Flu58-66 multimers. Cloned BCB 70 cells were incubated for lh at 4°C with conventional BSP multimers (circles) or multimers containing NTA2-PE conjugated with A2/Flu complexes containing a 2xHis6 tag. After washing the cells they were incubated at the indicated temperatures in FACS buffer containing or not (squares) 50 mM imidazol (triangles) or 100 mM imidazol (diamonds). After the indicated periods of time the cells were analyzed by flow cytometry.
Figure 11. FACS sorting with reversible NTA2-PE but not conventional A2/Flu multimers provides high cell viability. A) Cloned BCB 70 cells were FACS sorted upon staining with the conventional A2/Flu5g_66 BSP (black bars) or NTA2-PE-2xHis6 multimers (dark gray bars), after 1 wash with 100 mM imidazol the cells were cultured and at the indicated intervals viable cells enumerated by trypan blue exclusion counting. As controls cells were left untreated (white bars) or washed 1 x with 100 mM imidazol (light gray bars).
Figure 12. Synthesis of mono-NTA and di-NTA-biotin. The Compounds shown in Fig. 3A and B were synthesized using: i) solid phase peptide synthesis on chloro-trityl resin; ii) Fmoc for transient protection and ii) coupling with TBTU (2 eq), HOBt (2 eq), DIEA (4 eq) (a) and for deprotection TFA/H20/TIPS (92.5/2.5/5) (b). The intermediary thiol compounds were reacted with maleimido-NTA-lysine in 100 mM phosphate buffer, pH 7.2 (c). The distance x is about 16 A and y 23 A.
Figure 13. Synthesis of an alternative di-NTA-biotin. An alternative di-NTA- biotin was synthesized starting on a rink amide chloro-trityl resin to prepare the peptide back bone (a), which was reacted in solution with tri-tert. butyl-NTA lysine (b), followed by complete deprotection (c); in this compound the distance x' is about 19 A and y' 9 A.
Figure 14. Synthesis of NTA_j-biotin. In a first step backbone peptide was synthesized by solid phase peptide synthesis on a chloro-trityl resin (). The deprotected and HPLC purified peptide was reacted in phosphate buffer with 8 equivalents of NTA maleimide (b). The molecules comprises two di-NTA moieties joined by a flexible GGSGGGSGS (SEQ ID NO: 10) linker, which is the same as in the 2xHis6 tag (Fig. 2A). Figure 15. Multimers containing the short di-NTA linker more avidly stain cloned CD8+ T cells than those containing the long di-NTA linker A, B) The experiment shown in Fig. 5A was repeated using multimers containing either the short di-NTA-biotin (A)(Fig. 3B) or the long one (B)(Suppl. Fig. S2) and A2/Flus8_66 monomers carrying the 2xHis6 tag (triangles), His12 tag (squares) or a His6 tag (diamonds). For comparison conventional BSP multimers were included (red circles).
Figure 16. BCB 70 CTL were incubated likewise with streptavidin coated Qdots6o5 were loaded with NTA2-biotin and conjugated with A2/Flus8_66 monomers containing the indicated tags; after washing the cells were analyzed by flow cytometry. For comparison Qdots loaded with biotinylated monomers were included (BSP).
Figure 17. Impact of the degree of NTA2 conjugation of PE on NTA2-PE His tag multimer staining. PE was conjugated with di-NTA as shown in Fig. 8 using a constant concentration of PE (10 nM) and the indicated concentrations of SM(PEG)2 for 2h at ambient temperature, followed by alkylation NTA2-cysteine. The resulting NTA2-PE conjugates were loaded with A2/Flus8_66 monomers containing the indicated His tags, the multimers (5 nM) were incubated at 20°C for 30 min with cloned 81P1 cells. After washing cell-associated multimers were measured by flow cytometry. For comparison conventional BSP multimers were included.
Figure 18. Binding isotherms of different A2/Flu multimers on a polyclonal Flu- specific CD8+ T cell population. A polyclonal population of Flu-specific CD8+ T cells was incubated at 20°C for 30 min with A2/Flu multimers containing NTA4-biotin and 2xHis6 tag (light triangles, NTA4-biotin and His12 (light diamonds), NTA2-biotin and2xHis6 tag (dark triangles), NTA2-biotin and His12 tag (dark diamonds), NTA2-PE and 2xHis6 tag (squares) and conventional BSP multimers (circles). Cell associated fluorescence was measured by flow cytometry.
Figure 19. Dissociation kinetics of A2/Flu58-66 DTB and NTA2-PE multimers. A,
B) Cloned BCB 70 cells were incubated at 4°C for lh with 10 nM of A2/Flu58_66 multimers containing DTB streptavidin PE (A) or NTA2-PE and 2xHis6 (B). After washing the cells were incubated at 4°C (diamonds), 20°C (squares) or 37°C (triangles) in FACS buffer supplemented with 2nM biotin (in A) or 100 mM imidazol (in B). Cell associated fluorescence was determined by flow cytometry after the indicated periods of incubation. Figure 20. Preparation of immunopure biotin- strep tavidin multimers. Empty soluble MHC II molecules containing C-terminal leucine zippers are purified from supernatants by immuno-affinity chromatography and loaded with a peptide conjugated with Hisg tag via a photo-cleavable linker. The complexes containing this peptide are isolated by affinity chromatography on a Ni2+-NTA, thereafter the tag is removed by photolysis. The resulting immunopure MHC II peptide monomers are enzymatically biotinylated and multimerized with PE strep tavidin.
Figure 21. Photochemical removal of tags from MHC II binding peptides. A) To allow photochemical removal of tags from MHC II-restricted peptides, these were added via 2-nitro-phenyl"P-Ala (ΝΡβΑ); upon UV irradiation this residue is cleft, such that the N- terminal fragment is an amid and the C-terminal one carries a 2-nitroso-phenacetyl-P-acetoyl group. B) HLA-DR4 loaded with H6-GSG-NPpA-HA306-3 is peptide was irradiated at 365 +/- 40 nm for the indicated periods of time and the % of complexes carrying the His tag was assessed by ELISA using a His tag-specific mAb. C) the same experiment was performed for DR4- HA3o6-3i8-NPpA-GSG-H6 complexes. The inserted numbers (ti/2) indicate the time at which half maximal photolysis occurred.
Figure 22. Comparative staining of HA clones by conventional and immunopure DR4-HA multimers. A-D) The indicated HA 06-3i8-specific DR4-restricted Thl clones were incubated at 37°C for 2h with the indicated concentrations of the DR4 multimers containing: HA (conventional) (solid circles), H6-GSG-NPpA-HA3o6-3is (immunopure) (triangles), ¾- GSG-NPpA-HA 06-3i8 (immunopure; after UV irradiation) (inverted triangles), HA 06-3is- NPPA-GSG-H6 (immunopure) (diamonds), HA306-3i8-NPpA-GSG-H6 (immunopure, after UV irradiation) (two-colored circles). The cells were washed and the cell bound multimers (MFI) assessed by flow cytometry. E) The TCR and CD4 expression of the clones were assessed by flow cytometry and the half maximal IFNy response (EC50) by ELISA.
Figure 23. Preparation of immunopure DR4-HA306-318 monomers using Cy5.5. tagged peptides. A) Empty MHC II molecules are loaded with Cy5.5 tagged peptide and subjected to GFC and anion exchange chromatography. Cy5.5 contains four negative charges, i.e. is strongly negatively charged. B) GFC on a Superdex S75 column of DR4 after loading with HA306-3i8-GSGC-Cy5.5 recording of the OD280 (black, protein) and OD675nm (gray, Cy5.5). C) Assessment of MHC II protein (black bars) and Cy5.5 (gray bars) by ELISA using mAb specific for DR4 and Cy5.5, respectively of HA306-3i8-GSGC-Cy5.5, DR4-HA; DR52b- ES0123-137-GSGC-Cy5.5 and DR52b-ES0123 137. D) Anion exchange chromatography on a Mono-Q column using the indicated NaCl gradient and recording the OD280 (protein) and OD675nm (Cy5.5) of the eluant.
Figure 24. Preparation of immunopure MHC II-peptide NTA multimers using the pY-D4-tag. Empty MHC II molecules containing a C-terminal His tag at the acidic leucine zipper are purified by affinity chromatography on NTA columns and loaded with given peptides (e.g. HA306-3is) containing the strongly negatively charged phospho-tyrosine-Asp4 (pY-D4) tag. The peptide loading is monitored by ELISA using anti-pY mAb. Immunopure monomers are obtained by anion exchange chromatography, from which the pY-D4 tag is removed by UV irradiation and NTA multimers upon reaction with NTA-PE or NTA Qdots.
Figure 25. Preparation and testing of immunopure DR4/HA306-3is NTA multimers. A) empty, His tagged DR4 molecules were purified on a Ni 2+-NTA affinity column, which was eluted with 200 mM imidazol, monitoring the OD280 nm of the eluate. B) The eluted DR4 was subjected to GFC on a Superdex S200 column again monitoring the OD280 nm of the eluate. C) The collected fractions (gray lines) were analyzed by SDS-PAGE (10% non- reducing) applying a low (2μg/ml) or high (5μg/ml) concentration. D) Empty DR4 was loaded with pY-D4-GSG-NPpA-HA306-3is peptide followed by purification by anion exchange chromatography on Mono-Q column, which was eluted with the indicated NaCl gradient (upper panel). Fractions (0.5 ml) were collected and their content of DR4 (middle panel) and pY (phospho-tyrosine) (lower panel), respectively, determined by ELSA as shown in the lower two panels.
Figure 26. Impact of peptide loading on tetramer purity. The purity of tetramers decreases rapidly with decreasing peptide loading.
Figure 27. Effect of His tags on peptide binding to DR1, DR4 and DR52b.
The binding of the indicated NY-ESO-1 peptides carrying a N-terminal His6 tag or not to DR1, DR4 and DR52b was determined in a competition as described in Materials and Methods.
Figure 28. Neuraminidase treatment increases DR4/HA306-3i8 multimer binding. A) The indicated DR4-restricted, HA-specific T cell clones were pretreated or not with neuraminidase (30 min incubation at 37°C with 0.03 U/ml of neuraminidase) and incubated with conventional DR4/HA306-3is multimers (20 μg/ml) at 37°C for 2h. After washing cell- associated multimers (MFI) were assessed by flow cytometry. B,C) Cloned 10.5 (A) or 7.1 cells were incubated likewise with the indicated concentrations of conventional DR4/HA3o6- 318 multimers and cell associated fluorescence was assessed by flow cytometry.
Figure 29. Staining is a function of PE substitution degree.
Figure 30. Staining isotherms on Flu stimulated PBMC with different multimers at different concentrations. The x-axis provides the multimer concentration (in nM).
Figure 31. Exemplary NTA linkers.
Figure 32. Staining performance of PE conjugates comprising A2/Fhi58_66 monomers, or DR4/Flu HA 06-3i8 monomers.
Figure 33. Comparison of conventional, PE-Cys-PEG2-NTA2 and PE-HNO-NTA2 multimers staining.
Figure 34. Reduction of background staining by milk supplements.
Figure 35. Binding titration of NTAmers to determine best dilution for ex vivo staining.
Figure 36. Ex vivo staining with BSP and NTA multimers on fresh peripheral blood mononuclear cells (PBMCs).
Figure 37. Overview of an exemplary strategy for NTA-His tag multimer preparation using desthiobiotin (DTB).
Figure 38. Structures of biotin and DTB.
Figure 39. Evaluation of DTB tag peptide purification efficiency.
Figure 40. Schematic of 2 ml of StreptActin High Capacity sepharose (IBA)
Figure 41. Quantification of flowthrough, washing and elution of a DTB-tagged peptide.
Figure 42. Purification of MHC I-DTB peptide complexes on StreptActin sepharose. Figure 43. Exemplary scheme of DTB-HA peptide loading and purification.
Figure 44. Generation and purification of DR4-DTB-HA peptide complexes.
Figure 45. NTA moieties used in PE-NTA2 and biotin-NTA4-SA-PE multimers. Figure 46. Staining of Flu HA306-3i8-specific CD4+ T cells with DR4/HA306-3i8 BSP, biotin-NTA4-SA-PA, or PE-NTA2 multimers made with DTB- strep tactin purified monomers.
Figure 47. Staining of HA-specific CD4+ T cell clones 9(A) or 8(B) with different concentrations of DR4/HA306-3i8 BSP, biotin-NTA4-SA-PA, or PE-NTA2 multimers.
Scatchard analysis was performed and the KD (dissociation constant) and Bmax (maximal binding) values are described in the tables on the right. Figure 48. Background staining of DR4/HA306-3 i8 SA-PE NTA4 and PE NTA multimers. Background staining was efficiently suppressed by addition of 0.5% milk powder (see Figure 34).
Figure 50. Reversibility of multimer staining. Biotin-NTA4, but not BSP multimers, can be rapidly removed from stained, antigen- specific cells.
Figure 51. Comparative staining of a DRl/ESOu9-143 cell line by DRl/NY-ESOu9-143 BPS, PE-NTA2 and biotin-NTA4-SA-PE multimers.
Figure 52. Evaluation of additional tags for purification of peptide-loaded MHC monomers.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Some aspects of this invention relate to protein multimers in which conjugation of a plurality of monomeric proteins is based on chelate complex bonds between the monomeric proteins and a central carrier molecule. Methods for generation and the use of such protein multimers are also provided herein.
Some aspects of this invention provide "reversible" protein multimers that can be dissociated by releasing the chelate complex bonds between the carrier molecule and the monomeric proteins, for example, by withdrawal of a central ion from the chelate complex bond or by contacting the chelate complex with a free chelant that can displace one of the binding partners forming the chelate complex. This chelate bond release results in re- monomerization of the proteins comprised in the multimer. In some embodiments, multimer assembly and/or re-monomerization are carried out under non-denaturing, physiological, and/or non-toxic conditions, rendering the respective multimers suitable for in vivo, ex vivo, and in vitro applications involving living cells and/or monomeric proteins prone to denaturation.
Some aspects of the invention provide, for the first time, that reversible MHC-peptide multimers can be built on Ni2+ NTA-His tag complexes that exhibit equal or superior staining properties as compared to conventional multimers. These novel staining reagents are fully reversible in that they can be rapidly dissociated into monomeric subunits upon addition of imidazol, which allows, for example, sorting of bona fide antigen-specific CD8+ T cells. Some aspects of this invention provide methods and materials for the preparation of fluorescent MHC protein multimers, in which conjugation is based on chelate complex formation between nitrilotriacetic acid (NTA) and an amino acid sequence comprising a polyhistidine sequence, also referred to as a histidine tag (His tag). In some embodiments, the chelate complexes are formed in the presence of a Ni2+ cation. In some embodiments, the His tag comprises 3-12 His residues. In some embodiments, the His tag is a hexahistidine (His6) tag or a 2x His6 tag, comprising to hexahistidine sequences separated by a short amino acid linker.
Some aspects of this invention are based on the recognition that reversible protein multimers based on chelate complex bonds are stable enough to be useful for various applications, for example, cell or protein staining and isolation procedures, but can be dissociated under physiological, non-toxic conditions, for example, by withdrawing a central ion that is required for the formation of the chelate complex or by contacting them with an agent competing for the chelate complex bond, and thus releasing the bond between carrier molecule and monomeric protein. For example, the reversible Ni2+ NTA-His tag interaction- based multimers described herein are "reversible" in that they can be dissociated either by withdrawing the chelant cation (e.g. Ni2+ or Co2+) or by adding free competing chelant, for example, free imidazol, which displaces the His tag from the chelate complex. Imidazol is commercially available and is commonly used for purification of recombinant proteins on Ni2+ NTA affinity chromatography (see e.g.
products.invitrogen.com/ivgn/product/K95001 ?ICID==Search-Product; and
QIAExpressionist Handbook, Qiagen Inc., March 2001, available from www. Qiagen.com; both incorporated herein in their entirety by reference).
For example, the interaction of one Ni2+ NTA with a hexahistidine tag (His6) has a dissociation constant (KD) of about 10"6 -10"7M and is sufficiently stable to allow purification of His tagged recombinant proteins from culture supernatants (9, 10). Previous studies have investigated ways to render this interaction more stable. For example, different His tags have been examined and it has been shown that increasing the length of the His tag increases the stability of the complexes (9). In particular it has been demonstrated that linking two His6 tags via a flexible linker, such as GGGSGGGSGS (SEQ ID NO: 11) provides a high increase in stability (11, 12). Further, linkers have been synthesized that contain two to four NTA groups (13-15). The binding to His tags, in particular longer ones, increases considerably with the number on Ni NTA entities. While, in some embodiments, the His tags are expressed tethered, or fused to a recombinant protein, the NTA compounds have to be synthesized, as only mono-NTA (NTA1) derivatives are commercially available. Some aspects of this invention provide optimized configurations of NTA bound to a linker and optimized pairings of specific NTA configurations with specific His-tags to achieve a modulation of chelate complex bond stability over multiple orders of magnitude.
Accordingly, reversible multimers, as provided herein, can be customized to fulfill specific requirements of a wide variety of research, diagnostic, and therapeutic applications.
In some embodiments, reversible MHC multimers, for example, reversible MHC class I multimers, are provided. In some embodiments, fluorescently-labeled soluble MHC peptide multimers are provided. In some embodiments, methods for the use of reversible MHC multimers are provided, for example, methods useful to quantitate, isolate and/or characterize antigen-specific T-cells, for example, CD8+ and CD4+ T cells. In some embodiments, methods for the use of MHC multimers are provided that are useful for phenotypic T-cell analysis or for the analysis of T-cell receptor ("TCR") repertoire in a subject.
Conventional MHC multimers are typically prepared by enzymatic biotinylation of monomeric MHC proteins comprising a C-terminal biotinylation sequence peptide (BSP) and subsequent conjugation with streptavidin bound to a fluorescent dye, typically phycoerythrin (PE) or allophycocyanine (APC). Due to the large size of PE and APC, conjugates with streptavidin vary in stoichiometry, accessibility, and orientation of the biotin binding sites, often resulting in variable valency and inhomogeneous populations of MHC multimers. Other types of PE- or APC-based MHC -peptide staining reagents have been described, including Streptamers, desthiobiotin (DTB) multimers, pentamers (Proimmune Inc., FL, USA). Non-phycobilin-based MHC multimers have also been developed, for example Quantum dots loaded with MHC class I-peptide complexes, which allow simultaneous use of multiple MHC class I-peptide specificities in polychrome flow cytometry, Cy5-labeled dimeric, tetrameric and octameric MHC class I-peptide complexes, dextramers (Immudex, Copenhagen, Denmark) and dimeric MHC-peptide-immunoglobulin (Ig) fusion proteins.
While exemplary reversible multimers described herein include reversible MHC protein multimers, it will be apparent to those of skill in the art that the methods and reagents provided herein can be applied to generate reversible multimers of proteins other than MHC proteins. The methods for the generation and use of protein multimers, accordingly, are universally applicable to proteins of different nature, for example, to binding proteins, such as MHC proteins, antibodies, antibody fragments, ligands, adnectins, and receptors or receptor fragments. Exemplary multimers of such binding proteins, namely of peptide-loaded or empty MHC molecules, are provided in the working example section. Further, those of skill in the art will appreciate that virtually any kind of protein can be engineered to form a reversible multimer as provided by aspects of this invention as long as the monomeric protein is amenable to conjugation to a chelant moiety, for example, to addition of a His-tag fusion.
Accordingly, those of skill in the art will appreciate that the methods for the generation of protein multimers described herein can be applied to the generation of additional chelate complex bond-based binding molecules. For example, some embodiments, provide multimers of protein or non-protein binding molecules. The term "binding molecule" as used herein, refers to a molecule that is able to bind a binding partner via non- covalent interaction. Typically, in the context of cells, cell culture, or processing of living cells (e.g. staining, FACS sorting), a binding molecule is able to form a binding interaction with a binding partner that is strong enough to be stable under physiological conditions or under the conditions typically encountered during cell processing. In some embodiments, a binding molecule binds its binding partner with high specificity and/or high affinity. Non- limiting examples of binding molecules are antibodies and antibody fragments (e.g., Fab, F(ab)'2, single chain antibodies, diabodies, etc.), receptors, proteins binding a ligand, aptamers, and adnectins. The term "ligand" is art-recognized and refers to a binding partner of a binding molecule. Ligands can be, for example, proteins, peptides, nucleic acids, small molecules, and carbohydrates. Avidins, for example, streptavidin, are non-limiting examples of binding molecules that can bind a ligand, in this case, for example, biotin.
For example, in some embodiments, reversible antibody fragment, for example, Fab fragment, multimers are provided, in which a plurality of Fab proteins is bound to a central carrier molecule via chalet complex bonds, for example, NTA-His bonds. In some embodiments, the carrier molecule is a fluorescent microsphere, for example, a
FluoSpheres® type fluorosphere (see
http ://products .invitrogen.com/ivgn/product/F8781 ?ICID=search-product) . Protein mult inters
Some aspects of this invention provide reversible protein multimers in which a plurality of proteins is conjugated to a central carrier molecule via a non-covalent binding interaction that can be released under physiological conditions, for example, by contacting the multimer with an agent able to displace one of the binding partners from the binding interaction.
The terms "protein," "polypeptide," or "peptide," as used herein, refer to a polymer of at least three amino acid residues linked together by peptide bonds. The terms are interchangeably used herein and refer to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long. In some embodiments, inventive proteins contain only natural amino acids, although in other embodiments non-natural amino acids (e.g., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, US Patent 7045337, which describes incorporation of non-natural amino acids into proteins) and/or amino acid analogs as are known in the art may alternatively be employed. In some embodiments, one or more of the amino acids in an inventive protein are modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation,
functionalization, or other modification, etc. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be just a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these. In some embodiments, the protein is an MHC molecule.
The term "conjugated," as used herein, refers to an entity, molecule, or moiety that is stably associated with another molecule or moiety via a covalent or non-covalent bond. In some embodiments, the conjugation is via a covalent bond, for example, in the case of a peptide tag conjugated to an MHC protein via fusion of the peptide to a heavy chain of the MHC protein. In other embodiments, the conjugation is via a non-covalent interactions, for example, via hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, or electrostatic interactions.
The term "carrier molecule," as used herein in the context of multimers, refers to a molecule that binds or is conjugated to a plurality of monomeric molecules or entities, or a plurality of binding molecules or moieties that can bind such monomeric molecules or entities. In some embodiments, the carrier molecule is a monomeric molecule, for example, a single molecule of a fluorescent dye. In other embodiments, the carrier molecule is a multimeric molecule, for example, a polymer, or a nanocrystal. For example, in some embodiments, the carrier molecule is a fluorophore, for example, a phycobilin, conjugated to a plurality of binding molecules, for example, streptavidin. The streptavidin molecules, in turn, can bind monomeric molecules, for example, biotin-conjugated MHC monomers. In some embodiments, the carrier molecule is a multivalent chelant molecule. In some embodiments, the carrier molecule is a fluorescent microsphere, for example, a
FluoSpheres® type fluorosphere (see
http://products.invitrogen.com/ivgn/product/F878 l ?ICID=search-product). In some embodiments, the carrier molecule, for example, a fluorophore, is conjugated to a plurality of chelant molecules or moieties, for example, NTA molecules that can form chelate complex bonds with histidine residues in the presence of a divalent cation.
Some aspect of this invention provide optimized chelate complex bond formation between NTA and His chelants by using certain chelant configurations. The term "chelant configuration," as used herein refers to the number and spacing of chelant molecules or moieties in a given structure. For example, if chelant moieties, for example, NTA moieties, are conjugated to a carrier molecule, for example, a multivalent binding molecule or a fluorophore, via a linker, then a configuration in which a single chelant moiety is conjugated to the carrier molecule via a single linker is referred to as mono-configuration (e.g. mono- NTA, or NTA , a configuration in which two chelant moieties are conjugated to the carrier molecule via a single linker is referred to as di-configuration (e.g. di-NTA, or NTA2), a configuration in which four chelant moieties are conjugated to the carrier molecule via a single linker is referred to as tetra-configuration (e.g. tetra-NTA, or NTA4), and so forth. According to some aspects of this invention, different NTA chelant configurations and linker structures affect chelate complex bond properties, including bond strength and, thus, bond stability and reversibility. Similarly, the number and configuration of Histidine residues in a His tag has been described to affect chelate complex bond properties. In some embodiments, the His tag is a His6 tag, comprising 6 contiguous His residues, a His 12 tag, comprising 12 contiguous His residues, or a 2xHis6 tag, comprising two sequences of 6 contiguous His residues linked by a short spacer sequence as described in more detail elsewhere herein. The term "divalent cation" as used herein, refers to an ion that lacks two electrons as compared to the neutral atom. Examples of divalent cations useful in some embodiments of this invention are Ni2+, Cu2+, Zn2+, Co2+, Cd2+, Sr2+ , Mn2+, Fe2+, Mg2+, Ca2+, and Ba2+. Other useful divalent cations will be apparent to those of skill in the art and the invention is not limited in this respect.
In some embodiments, the carrier molecule is a monomeric carrier molecule. In some embodiments, the carrier molecule is a multimeric or polymeric carrier molecule. For example, in some embodiments, the carrier molecule is a tetrameric or a hexameric molecule, for example, a fluorophore. In some embodiments, the carrier molecule is a fluorophore, a phycobilin, phycoerythrin or allophycocyanine, a nanocrystal, a quantum dot (Qdot), a magnetic particle, or a nanoparticle. The terms "quantum dot" and "Qdot," as used herein, refer to fluorescent inorganic semiconductor nanocrystals in which the excitons are confined in all three spatial dimensions and which are useful as detectable agents in some
embodiments of the invention. In some embodiments, the Qdot comprises CdSe or CdTe. In some embodiments, the Qdot comprises InP or InGaP. In some embodiments, the Qdot comprises a core/shell structure, while in other embodiments, the Qdot is a core-only Qdot. Exemplary Qdots and methods for use and production are described in Rech-Genger et al., Quantum dots versus organic dyes as fluorescent labels. Nature Methods 2008 (9):763-775, incorporated herein in its entirety by reference for disclosure of fluorescent Qdots and organic dyes, and methods of production and use of same).
In some embodiments, the carrier molecule is a water-soluble molecule. The term "water-soluble" is art-recognized and qualifies that an agent can be dissolved in water to a certain degree, or , in other words, that a certain amount of the agent can be dissolved in a certain volume of water. For example, in some embodiments described here, a water-soluble carrier molecule is a carrier molecule that exhibits a solubility in water at 25 °C and 1 ATM of more than O. lg/ml, more than 0.2g/ml, more than 0.25g/ml, more than 0.3g/ml, more than 0.4g/ml, more than 0.5g/ml, more than 0.6g/ml, more than 0.7g/ml, more than 0.8g/ml, more than 0.9g/ml, more than lg/ml, more than l. lg/ml, more than 1.2g/ml, more than 1.3g/ml, more than 1.4g/ml, more than 1.5g/ml, more than 1.6g/ml, more than 1.7g/ml, more than 1.8g/ml, more than 1.9g/ml, more than 2g/ml. more than 2.25g/ml, more than 2.5mg/ml, more than 3g/ml, more than 4g/ml, more than 5g/ml, more than 6g/ml, more than 7g/ml, more than 8g/ml, more than 9g/ml, more than 10 mg/ml, or more than 20 mg/ml. In some embodiments, the carrier molecule is not water soluble. In some such embodiments, the carrier molecule is highly dispersible in water and/or does not precipitate in aqeous solution under physiological conditions. In some embodiments, the diameter of the carrier molecule is less than O. lnm, less than 0.2nm, less than 0.25nm, less than 0.3nm, less than 0.4nm, less than 0.5nm, less than 0.6nm, less than 0.7nm, less than 0.75nm, less than 0.8nm, less than 0.9nm, less than lnm, less than l.lnm, less than 1.2nm, less than 1.3nm, less than 1.4nm, less than 1.5nm, less than 1.6nm, less than 1.7nm, less than 1.8nm, less than 1.9nm, less than 2nm, less than 2.5nm, less than 3nm, less than 4nm, less than 5nm, less than 6nm, less than 7nm, less than 8nm, less than 9nm, or less than lOnm. In some embodiments, the diameter of the carrier molecule is less than 20nm, less than 30nm, less than 40nm, less than 50nm, less than 60nm, less than 70nm, less than 80nm, less than 90nm, less than lOOnm, less than 200nm, less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm, or less than Ιμιη.
In some embodiments, the non-covalent interaction is a non-covalent bond with a dissociation constant KD of 5μΜ > KD > lfM, for example, of ΙΟΟηΜ > KD > lpM, or of ΙΟΟηΜ > KD≥ lOOfM. The term "dissociation constant," abbreviated as KD herein, is art- recognized and refers to a specific type of equilibrium constant that measures the propensity of a complex of associated molecules to separate (dissociate) reversibly into the separate molecules. The dissociation constant is the inverse of the association constant. For a general reaction AxBy→ xA + yB, in which a complex AxBy breaks down into x A subunits and y B
[A]' x [Bf
subunits, the dissociation constant is defined as lj ¾ j¾j , where [A], [B], and
[AxBy] are the concentrations of A, B, and the complex AxBy, respectively. In some embodiments, a protein multimer is provided in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant 5μΜ > KD > InM. In some embodiments, a protein multimer is provided in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant 5μΜ > KD > lpM. In some embodiments, a protein multimer is provided in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant of less than 2 fM, less than 5 fM, less than 10 fM, less than 20 fM, less than 50 fM, less than 100 fM, less than 250 fM, less than
500 fM, less than lpM, less than 2 pM, less than 5 pM, less than 10 pM, less than 20 pM, less than 50 pM, less than 100 pM, less than 250 pM, less than 500 pM, less than InM, less than 5 nM, less than 10 nM, less than 20 nM, less than 50 nM, less than 100 nM, less than 250 nM, less than 500 nM, less than 600 nM, less than 700 nM, less than 800 nM, less than 900 nM, less than 1000 nM, less than 1500 nM, less than 2000 nM, less than 2500 nM, less than 3000 nM, less than 3500 nM, less than 4000 nM, less than 4100 nM, less than 4200 nM, less than 4300 nM, less than 4400 nM, less than 4500 nM, less than 4600 nM, less than 4700 nM, less than 4800 nM, less than 4900 nM, less than 5000 nM, more than 1 fM, more than 5 fM, more than 10 fM, more than 20 fM, more than 25 fM, more than 50 fM, more than 100 fM, more than 200 fM, more than 500 fM, more than 1 pM, more than lpM, more than 5 pM, more than 10 pM, more than 20 pM, more than 25 pM, more than 50 pM, more than 100 pM, more than 200 pM, more than 500 pM, more than InM, more than 5 nM, more than 10 nM, more than 20 nM, more than 50 nM, more than 100 nM, more than 200 nM, more than 500 nM, more than 1000 nM, more than 2000 nM, more than 2500 nM, more than 3000 nM, more than 3500 nM, more than 4000 nM, more than 4100 nM, more than 4200 nM, more than 4300 nM, more than 4400 nM, more than 4500 nM, more than 4600 nM, more than 4700 nM, more than 4800 nM, or more than 4900 nM, or any possible combination of any of these values. For example, in some embodiments, a protein multimer is provided in which at least one of the protein monomers is conjugated to the carrier molecule via a non-covalent bond with a dissociation constant of more than lpM and less than ΙΟηΜ, more than ΙΟρΜ and less than ΙΟΟΟηΜ, more than ΙΟΟρΜ and less than 500nM, more than ΙΟηΜ and less than
ΙΟΟΟηΜ, more than ΙΟηΜ and less than ΙΟΟηΜ, more than ΙΟΟηΜ and less than 4000nM, more than 200nM and less than 3000nM, more than 500nM and less than 2000nM, or more than 500pM and less than ΙΟΟΟηΜ.
In some embodiments, the carrier molecule or the protein, or both, are conjugated to a chelant moiety via a covalently bound linker. The term "linker," as used herein, refers to a chemical structure between two molecules or moieties or between a molecule and a moiety, thus linking the two. In some embodiments, the linker is covalently bound to both linked elements. In some embodiments, the linker is covalently bound to one, but not the other linked element. In some embodiments, the linker is non-covalently bound to one or both elements. For example, in some embodiments, a linker is covalently bound to a carrier molecule and a chelant moiety, while in other embodiments, a linker is covalently bound to a chelant moiety and non-covalently bound to a carrier molecule. In some embodiments, the linker is about 2A, about 3A, about 4A, about 5A, about 6A, about 7 A, about 8A, about 9 A, about 10A, about 11 A, about 12A, about 13A, about 14A, about 15A, about 16 A, about 17A, about 18 A, about 19 A, about 20A, about 21A, about 22A, about 23A, about 24A, about 25A, about 26A, about 27A, about 28A, about 29A, about 30A, about 35A, about 40A, about 45A, or about 50A long. In some embodiments, the linker is longer than about 50A.
In some embodiments, NTA moieties are conjugated with a carrier molecule, for example, a PE molecule, using maleimide alkylation chemistry. For example, in some embodiments, PE is first reacted with an NHS-maleimide and the maleimido-PE
subsequently with NTA-Cys (SH), resulting in the formation of a stable thio-ether. It is important to note that the maleimido group readily hydrolyzes under certain conditions, which can limit the reproducibility and the degree of conjugation.
In some embodiments, chelate moieties, for example, NTA moieties, are conjugated to a carrier molecule, for example, a PE molecule, with an oxime formation chemistry
strategy. Using oxime chemistry has several advantages: i) efficient conjugation in the pH range 5-7; ii) good stability under physiological conditions; and iii) efficient conjugation at low protein concentrations. An exemplary oxime chemistry for attachment of NTA moieties to a carrier is described in the following scheme:
Figure imgf000033_0001
Aldehyde imine Oxime ion PE) (on NTAj jPE-NTA conjugate)
Exemplary oxime chemistry scheme.
The chemistry strategies provided herein, for example, the maleimide and oxime chemistry strategies described, are universally applicable to generate MHC multimers, for example, multimers of MHC class I or class II as described herein. Additional maleimide and oxime chemistry strategies will be apparent to those of skill in the art and it will be appreciated that the disclosure is not limited in this respect.
The use of oxime chemistry, also referred to herein as oxime ligation, has some advantages in the context of the preparation of large bioconjugates, for example, as its formation is compatible with other functional groups often used, namely thiol-maleimide reactions or click chemistry. Oxime formation benefits from the exclusive specificity and reactivity between the aminooxy function and the carbonyl group, since the nitrogen atom behaves as a weak base and as an excellent nucleophile. In general, aldehydes are
substantially more reactive than ketones, mainly due to steric effects. The oxime ligation reaction can occur in mild acidic conditions (pH 5) within 1 h but also at physiological conditions (pH 7) in 10 h. The resulting imine bond is covalent and stable under
physiological conditions. In some embodiments, aminooxy-containing peptides are obtained by inserting the classical fmoc-Dpr(Aoa)-OH residue at the last stage of solid phase peptide synthesis. Incorporation of this residue is efficient (quantitative yields) and does not require specialized conditions. Final TFA deprotection of the peptide results in a free aminooxy function. The incorporation of the aldehyde moiety on the biomolecule to be derivatized is achieved, in some embodiments, by employing a commercially available sulfo-SFB molecule, that reacts on lysines via succinimidyl ester reaction. One of the advantages of using oxime chemistry over using a thiol-maleimide chemistry strategy is that oxime formation is not subjected to a hydrolysis or degradation reaction. In some embodiments, oxime chemistry strategies lead to better incorporation yields. Another benefit is that the functionalized entities can be prepared and stored for several months before being mixed together.
Oxime chemistry reactions, reagents, and reaction conditions are well known to those of skill in the art. Some reactions, reagents, and reaction conditions are described herein. Additional suitable reactions, reagents, and reaction conditions will be apparent to those of skill in the art and it will be appreciated that this disclosure is not limited in this respect.
Suitable reactions, reagents, and reaction conditions are described, for example, in Mathieu Galibert, Olivier Renaudet, Pascal Dumy, and Didier Boturyn. Angew. Chem. Int. Ed. 2011, 50, 1 - 5; Youhei Sohma and Stephen B. H. Kent. J. AM. CHEM. SOC. 2009, 131, 16313- 16318 9 16313; Anouk Dirksen and Philip E. Dawson. Bioconjugate Chem. 2008, 19, 2543- 2548; Jenks, W. P. J. Am. Chem. Soc. 1959, 81, 475-481; Rose, K. J. Am. Chem. Soc. 1994, 116, 30-33; Shao, J.; Tarn, J. P. J. Am. Chem. Soc. 1995, 117, 3893-3899; Decostaire, I. P.; Lelie vre, D.; Zhang, H.; Delmas, A. F. Tetrahedron Lett. 2006, 47, 7075-7060; Garanger, E.; Boturyn, D.; Renaudet, O.; Defrancq, E.; Dumy, P. J. Org. Chem. 2006, 71, 2402-2410; and Boturyn, D.; Coll, J. L.; Garanger, E.; Favrot, M. C; Dumy, P. J. Am. Chem. Soc. 2004, 126, 5730-5739; the entire contents of each of which are incorporated herein by reference.
In some embodiments, a protein multimer is provided in which a plurality of proteins is conjugated to a multivalent carrier molecule and wherein at least one of the proteins is conjugated to the carrier molecule via a chelate complex bond. The term "chelate complex," as used herein, refers to a chemical structure that comprises two or more separate, non-covalent binding interactions between a polydentate (multiple bonded) molecule or moiety, also referred to as "chelant", and a single central atom, for example, a divalent cation. The term "chelate complex bond," accordingly, refers to a non-covalent bond between two or more chelants that form a chelate complex. In some embodiments, all chelants of a chelate complex are of the same structure. In other
embodiments, a chelate complex is formed by chelants of different structures, for example, by a chelant comprising a histidine residue and a chelant comprising an NTA residue. In some embodiments, the central atom is a divalent cation, for example, an Ni2+ cation.
Chelants, chelant moieties, and suitable central atoms are well known to those of skill in the art and the invention is not limited in this respect.
In some embodiments, a protein multimer is provided in which a plurality of MHC molecules is conjugated to a carrier molecule by a non-covalent bond as described herein, for example, a chelate complex bond or a bond of a KD value as provided elsewhere herein.
The term "MHC molecule," as used herein, refers to a protein encoded by the major histocompatibility complex, and includes MHC class I and MHC class II molecules. In some embodiments, the MHC molecule is an MHC class I molecule and the MHC multimer is an MHC class I multimer. In some embodiments, the MHC molecule is an MHC class II molecule and the MHC multimer is an MHC class II multimer.
In some embodiments, the MHC molecule is a human MHC molecule. In humans,
MHC molecules are also referred to as HLA molecules. In some embodiments, the MHC molecule is an MHC molecule of a non-human mammal, for example, of a mouse, a rat, a rabbit, a non-human primate, a cat, a dog, a goat, a cow, a sheep, a horse, or a pig.
MHC class-I molecules comprise one heavy chain type a that comprises three domains (al, a2, and a3). In naturally occurring MHC class I molecules, these domains are exposed to the extracellular space, and are linked to the cellular membrane through a transmembrane region. The a chain of MHC class I molecules is associated with a molecule of β2 microglobulin, which is not encoded by an MHC gene, but also included within the scope of the term "MHC molecule". MHC class II molecules comprise two heavy chains, one type a and one type β, each of which comprises two domains: al and α2, βΐ and β2, respectively. In naturally occurring MHC class II molecules, these domains are exposed to the extracellular space and are linked to the cellular membrane through a transmembrane region on each of the two chains.
In some embodiments, of this invention, an MHC multimer comprises a genetically engineered MHC molecule. In some embodiments, an MHC molecule as provided herein comprises an extracellular domain of a naturally occurring MHC molecule, or a genetically engineered derivative thereof, but is devoid of all or part of the transmembrane domain or domains. In some embodiments, MHC class II molecules are provided that comprise a leucine zipper in place of the transmembrane domain in order to achieve dimerization of a and β chains. Genetically engineered MHC proteins, for example, MHC molecules lacking transmembrane domains, MHC molecules comprising leucine zippers, single chain MHC molecules or MHC molecules fused to an antigenic peptide, are also included in the scope of the term "MHC molecule". In some embodiments, the term "MHC molecule" refers to a complete molecule, for example, an MHC heavy chain type a (genetically engineered or not) that is associated with a molecule of β2 microglobulin in the case of an MHC class I molecule, or an MHC heavy chain type a (genetically engineered or not) that is associated with an MHC heavy chain type β (genetically engineered or not), for example, via leucine zipper interaction. In some embodiments, the term "MHC molecule" refers to a single component of an MHC molecule, for example, to an MHC heavy chain (e.g. type a or type β, genetically engineered or not), or to a β2 microglobulin.
MHC molecules can bind antigenic peptides in a groove formed by the l and a2 domains of MHC class I molecules or by the al and βΐ domains of MHC class II molecules. An MHC molecule that has bound an antigenic peptide is referred to herein as a "peptide - loaded" MHC molecule, whereas an MHC molecule that has not bound an antigenic peptide is referred to herein as an "empty" MHC molecule. The term "antigenic peptide," as used herein, refers to a peptide comprising a structure that is recognized by the immune system of a subject. Non-limiting examples of antigenic peptides are a peptide that is recognized by a B or T-cell, e.g. via binding to a T-cell receptor, or a peptide that binds to an antibody or antibody fragment, or a peptide that stimulates an immune response in a subject.
The term "monomeric MHC molecule," "MHC monomer," and "MHC molecule monomer," as used herein, refer to a single MHC molecule, for example, to a single MHC heavy chain, a single MHC heavy chain associated with a β2 microglobulin, or a heterodimer of an MHC type a heavy chain and an MHC type β heavy chain. The term "MHC multimer," as used herein, refers to a plurality of MHC molecules associated with each other, for example, via non-covalent interaction with a carrier molecule.
In some embodiments, the term "multimer" excludes dimers, but includes trimers, and multimers of four monomers (tetramers), or of more than four monomers (pentamers, hexamers, septamers, octamers, nonamers, decamers, etc.). In some embodiments, the term "multimer" excludes dimers and trimers, but includes multimers of four and more monomers.
Core multimer structure
Some aspects of this invention provide a multivalent chelant, that comprises a plurality of chelant moieties conjugated to a carrier molecule. Such multivalent chelants are useful for the generation of reversible multimers, for example, of reversible protein multimers (e.g. MHC multimers), as described elsewhere herein.
The term "multivalent chelant molecule," as used herein, refers to a carrier molecule comprising or conjugated to a plurality of chelant moieties able to form chelate complex bonds with a plurality of chelant-moiety comprising molecules. For example, a tetravalent chelant molecule is a carrier molecule that is able to form chelate complex bonds to four molecules, for example, four MHC proteins comprising a compatible chelant moiety, thus forming a tetramer of the four molecules held together by chelate complex bonds. A compatible chelant moiety is a chelant moiety able to form a chelate complex bond with the chelant moiety of the carrier molecule. For example, an NTA moiety and a Histidine moiety are compatible chelant moieties, since they can form a chelant complex bond in the presence of a divalent cation. In some embodiments, the multivalent chelant molecule can form a chelate complex bond with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, or 150 molecules comprising a compatible chelant moiety. In some embodiments, the multivalent chelant molecule can form a chelate complex bond with more than 150
compatible molecules.
In some embodiments, the carrier molecule is a water-soluble molecule as described in more detail elsewhere herein. In some embodiments, the carrier molecule is a non-water soluble molecule as described in more detail elsewhere herein.
In some embodiments, the chelant moieties are tridentate or tetradentate chelant moieties. In some embodiments, the chelant moieties are nitrilotriacetic acid (NTA) moieties. In some embodiments, the chelant moieties are histidine residues, for example, in the form of histidine tags. In some embodiments, the chelate moieties are iminodiacetic acid (IDA) moieties.
In some embodiments, the chelant moieties are covalently bound to the carrier molecule via a linker, for example, via a linker described herein. In some embodiments, each linker is bound to a single chelant moiety (e.g., mono-NTA configuration). In some embodiments, two chelant moieties are bound to a single linker (e.g., di-NTA configuration). In some embodiments, four chelant moieties are bound to a single linker (e.g., tetra-NTA configuration).
Methods for generation of protein multimers
In some embodiments, the invention provides methods for the generation of reversible protein multimers in which a plurality of proteins is conjugated to a carrier molecule via a chelate complex bond. In some such embodiments, the carrier molecule and the protein are conjugated to the respective chelant moieties via covalent bond, for example, via a covalently bound linker. In such embodiments, the only non-covalent bond between carrier molecule and protein monomer is the chelate complex bond. In other embodiments, an additional non- covalent bond is introduced between the carrier molecule and the monomeric protein, for example, a binding molecule/ligand bond, such as a streptavidin/biotin interaction.
Some aspects of this invention provide methods for the generation of protein multimers, for example, of MHC protein multimers. In some embodiments, the method includes a step of contacting a monomeric protein, for example, an MHC molecule, that is conjugated to a chelant moiety with a carrier molecule as provided herein, that is conjugated to a plurality of chelant moieties under conditions suitable for formation of a chelate complex between the chelant moieties conjugated to the MHC molecule and the chelant moieties conjugated to the carrier molecule.
In some embodiments, a method for the generation of protein multimers, for example, MHC protein multimers is provided that includes contacting a protein molecule, for example, an MHC molecule, that is conjugated to a first chelant, for example, a His tag, with a ligand molecule, for example, biotin, conjugated to a second chelant under conditions suitable for formation of a chelate complex between the first and the second chelant. The resulting product is a monomeric protein conjugated to a ligand via a non-covalent chelate complex bond. Such monomers can be assembled to reversible multimers by contacting them with a carrier molecule conjugated to a plurality of ligand-binding molecules or moieties, for example, streptavidin molecules. In some embodiments, a multivalent carrier molecule is generated by contacting a carrier molecule conjugated to a plurality of chelant moieties via non-covalent interaction, for example, via biotin/streptavidin interaction. For example, in some embodiments, a carrier molecule comprising a plurality of streptavidin moieties is contacted with a plurality of biotin molecules that are conjugated to chelant moieties, for example, NTA moieties, via a covalently bound linker as described herein.
In some embodiments, methods for the production of peptide-loaded MHC multimers are provided. While stable, peptide-loaded MHC class I proteins can be obtained by refolding of MHC class I heavy chains with peptides of interest, recombinant MHC class II proteins are more difficult to obtain. In some embodiments, recombinant MHC class II molecules are produced in soluble form by insect expression systems, such as Drosophila S2 cells or baculovirus and sf9 cells. With very few exceptions, deletion of the transmembrane (TM) domains of the a and β chains in MHC class II molecules results in the dissociation of the two subunit chains. In some embodiments, chain pairing is re-established by addition of leucine zippers. In some embodiments, empty MHC class II molecules are first isolated and then loaded with an antigenic peptide of interest. Such peptide-loaded MHC class II molecules can then be isolated and used in the production of MHC protein multimers. In some embodiments, for example, in embodiments where the binding interaction between the peptide of interest and an MHC class II molecule is of low strength, peptides can be fused to the N-terminus of the β chain via a flexible linker. Such fusions of MHC class II chains and antigenic peptides, resulting in the production of recombinant, peptide-loaded MHC molecules, are well known to those of skill in the art.
In some embodiments, a chelant moiety, for example, a His tag, is added at the C- terminus of an MHC chain by recombinant addition of a fusion peptide comprising a chelant moiety, for example, in the form of a His tag as described herein. In some embodiments, for example, in some embodiments, in which the peptide-loaded MHC monomer is isolated via a method using a tag attached to the antigenic peptide, a chelant moiety is attached to the isolated, peptide-loaded MHC molecule after isolation. Methods for post-synthesis or post- isolation of chelants to isolated proteins are known to those of skill in the art and exemplary methods are described herein. While some MHC molecules are instable without a bound antigenic peptide, in some embodiments, the MHC molecule is sufficiently stable without peptide cargo (e.g. HLA DRB1*0101 or DRB 1*0401) to allow the production of empty MHC molecules and MHC multimers, e.g., MHC molecules or multimers that are not loaded with an antigenic peptide. In some such embodiments, the MHC monomer is loaded after isolation or purification with the peptide of interest. In some embodiments, the MHC monomer is first incorporated into a reversible MHC multimer as provided herein and subsequently loaded with a peptide of interest. The efficiency of peptide loading strongly depends on its binding strength to the respective MHC molecule. If the binding is below a critical threshold, peptide loading is inefficient and the resulting complexes are of limited stability, both physically and conformationally.
Some embodiments of the invention provide methods for the generation of MHC molecules and multimers that are loaded with an antigenic peptide. In some embodiments, methods and reagents for the production of peptide-loaded MHC class II molecules are provided. The production of peptide-loaded MHC class II molecules is technically difficult, based on the instability of engineered MHC class II heterodimers comprising a and β heavy chains lacking a transmembrane domain, and, in many instances, the low affinity binding interactions between the MHC class II heavy chains and the antigenic peptide. As a result, populations of peptide-loaded MHC class II molecules produced with conventional methods are often heterogeneous in that a significant portion of MHC class II molecules are not or not correctly peptide-loaded. MHC class II multimers produced from such heterogeneous populations of MHC class II molecules often show poor staining performance, great batch-to- batch variability in staining efficiency, and some specific peptide-loaded MHC class II multimers are difficult or impossible to obtain with conventional methods.
Some aspects of this invention provide methods addressing these problems in the production of MHC class II molecules and multimers. For example, some aspects of this invention provide methods and reagents for the generation of peptide-loaded MHC molecule, for example, MHC class II molecules, that include the use of a tag conjugated to the antigenic peptide. In some embodiments MHC molecules that have bound a tagged antigenic peptide are isolated and/or purified by a method that can be carried out under non-denaturing conditions, for example, by certain chromatography methods (e.g., affinity chromatography or ion exchange chromatography). In some embodiments, the tag conjugated to the antigenic peptide can be removed, for example, by cleaving a linker that connects the tag to the antigenic peptide, and methods for tag removal from tagged peptide-loaded MHC molecules, for example, MHC class II molecules, are also provided herein.
In some embodiments, the antigenic peptide of interest is conjugated to a tag. In some embodiment, the tag is a peptide tag, for example, a peptide tag that is N-terminally or C-terminally fused to the antigenic peptide. In some embodiments, the tag is an affinity tag that allows for the isolation of correctly loaded MHC class II molecules by affinity chromatography. Affinity tags are well known to those of skill in the art and examples of peptide tags include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags , calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags , biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. In some embodiments, the tag is a biotin tag or a biotin variant tag, for example, desthiobiotin (DTB). DTB is a biotin variant that binds about lxl06-fold weaker to streptavidin than biotin. DTB is readily displaced by free biotin, allowing gentle affinity purification based on the reversible DTB -streptavidin conjugation. Conjugation partners similar to streptavidin can also be employed, for example, StreptActin, a mutant of streptavidin. Strep tags, which are peptidic biotin analog, bind also to StreptActin.
Desthiobiotin, biotin, streptavidin, StreptActin, strep tags and derivatives of these reagents, as well as methods for the use of these reagents in protein and peptide purification are well known to those of skill in the art. Some methods suitable according to aspects of this invention are described herein, and additional suitable methods will be apparent to those of skill, for example, as described in Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A, Ting AY. A monovalent streptavidin with a single femtomolar biotin binding site. Nat Methods. 2006;3:267-73; Hirsch JD, Eslamizar L, Filanoski BJ, Malekzadeh N, Haugland RP, Beechem JM, Haugland RP. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal Biochem. 2002;308:343-57; Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S. Comparison of affinity tags for protein purification. Protein Expr Purif. 2005;41:98-105; Wu SC, Wong SL. Development of an enzymatic method for site- specific incorporation of desthiobiotin to recombinant proteins in vitro. Anal Biochem. 2004 ;331(2):340-8; Maier T, Drapal N, Thanbichler M, Bock A. Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis. Anal Biochem. 1998 ;259:68-73; Chen R, Folarin N, Ho VH, McNally D, Darling D, Farzaneh F, Slater NK. Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labeling. J Chromatogr B Analyt Technol Biomed Life Sci.
2010;878: 1939-45; Gloeckner CJ, Boldt K, Schumacher A, Roepman R, Ueffing M. A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics. 2007;7:4228-34; Cass B, Pham PL, Kamen A, Durocher Y. Purification of recombinant proteins from mammalian cell culture using a generic double - affinity chromatography scheme. Protein Expr Purif. 2005 Mar;40(l):77-85; Korndorfer IP, Skerra A. Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site. Protein Sci. 2002; 11:883-93; the entire contents of each of which are incorporated herein by reference.
Sequences of peptide tags useful in some embodiments of this invention, for example, the tags described herein, are well known to those of skill in the art, and exemplary tags are described, for example, in Kimple, M.E., and Sondek, J. Overview of affinity tags for protein purification. Curr Protoc Protein Sci. 2004 Sep; Chapter 9:Unit 9.9, incorporated in its entirety herein for disclosure of affinity tags. Those of skill in the art will appreciate that the invention is not limited in this respect.
Some aspects of this invention provide tagged MHC class II binding antigenic peptides and methods of using such peptides. In some embodiments, a tag conjugated to an antigenic peptide is useful for the isolation of the tagged peptide, either alone or when bound to an MHC class II molecule. Methods for isolating tagged peptides are well known to those of skill in the art and include, for example, affinity chromatography and ion exchange chromatography.
In some embodiments, an MHC class II binding peptide is provided or used that is conjugated to a tag. In some embodiments, the tag is an acidic tag. In some embodiments, the acidic tag is an acidic peptide tag, for example, a peptide tag comprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, or more contiguous acidic amino acid residues. In some embodiments, the acidic amino acid residues glutamic acid (Glu, E) or aspartic acid (Asp, D) residues. In some embodiments, the antigenic peptide is conjugated to an acidic tag that allows for the isolation of correctly loaded MHC class II molecules by anion exchange chromatography. In some embodiments, the tag is a tag comprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Asp residues, for example, a pY-D4 tag
(phosphor-tyrosine followed by four Asp residues), a pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag. In some embodiments, the acidic tag is a tag comprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Glu residues, for example, a pY-E6 (phosphor-tyrosine followed by six Glu), pY-E7, pY-E8, pY-E9, or a pY-E10 tag. In some embodiments, the acidic tag is an acidic detectable label, for example, an acidic fluorophore. In some embodiments, the acidic fluorophore is a cyanine dye tag, for example, a Cy5 tag, or a Cy5.5 tag. Other acidic tags suitable for peptide or protein isolation and/or purification are well known to those of skill in the art and the invention is not limited in this respect.
In some embodiments, tagged MHC class II binding peptides are provided that are reversibly tagged, e.g., that are tagged with a structure that can be cleaved, resulting in a release of the conjugated tag from the peptide. Methods of using such reversibly tagged peptides are also provided. In some embodiments, the tag is conjugated to the peptide via a cleavable linker. In some embodiments, the linker is a photocleavable linker. In some embodiments, the linker is a 2-nitro-phenyl-P-Ala (ΝΡβΑ) linker. In some embodiments, the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical. In some embodiments, the tagged peptide is a peptide conjugated to a part of a cleavable linker that remains after cleavage of the linker.
One advantage of a cleavable linker is that after isolation of the peptide-loaded MHC class II molecule with a method relying on the tag, the tag can be removed. Cleavage of a cleavable linker will, in some embodiments, leave part of the linker conjugated to the MHC class II binding peptide. However, some linkers can be designed in a manner that will result in complete removal of the linker from the MHC-class II binding peptide.
Tag removal by linker cleavage is particularly useful in embodiments, where the tag interferes or is suspected to interfere with the binding of the peptide-loaded MHC class II molecule to its target T-cell receptor. Exemplary cleavable linkers are described in more detail elsewhere herein. Additional cleavable linkers are known to those of skill in the art and the invention is not limited in this respect. In some embodiments, the cleavable linker is a photocleavable linker. Photocleavable linkers can be cleaved by irradiation with UV light. Photocleavable linkers are described herein and additional photocleavable linkers are well known in the art. See, for example, Pandori MW, Hobson DA, Olejnik J, Krzymanska- Olejnik E, Rothschild KJ, Palmer AA, Phillips TJ, Sano T., Photochemical control of the infectivity of adenoviral vectors using a novel photocleavable biotinylation reagent. Chem Biol. 2002 May;9(5):567-73.; Hahner S, Olejnik J, Ludemann HC, Krzymanska-Olejnik E, Hillenkamp F, Rothschild KJ. Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin. Biomol Eng. 1999 Dec 31 ; 16(1-4): 127-33; Olejnik J, Ludemann HC, Krzymanska-Olejnik E, Berkenkamp S, Hillenkamp F, Rothschild KJ.
Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS. Nucleic Acids Res. 1999 Dec 1;27(23):4626-31; Olejnik J, Krzymanska- Olejnik E, Rothschild KJ. Photocleavable aminotag phosphoramidites for 5 '-termini DNA/RNA labeling. Nucleic Acids Res. 1998 Aug l;26(15):3572-6. Olejnik J, Krzymanska- Olejnik E, Rothschild KJ. Photocleavable affinity tags for isolation and detection of biomolecules.Met ods, Enzymol. 1998;291: 135-54; Olejnik J, Sonar S, Krzymanska-Olejnik E, Rothschild KJ. Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules. Proc Natl Acad Sci U S A. 1995 Aug l;92(16):7590-4; all references incorporated herein by reference for disclosure of photocleavable linkers). In some embodiments, a peptide tagged via a cleavable linker is referred to as a reversibly tagged peptide.
In some embodiments, an MHC class II molecule loaded with a reversibly tagged peptide is isolated, for example, by affinity or ion exchange chromatography, and the tag is removed after isolation by cleavage of the linker. In some such embodiments, the MHC class II molecule loaded with a now untagged peptide is then isolated from the cleaved tag, for example, by size fractionation.
In some embodiments, an MHC molecule, for example, an MHC class II molecule conjugated to a chelant moiety is loaded with a tagged peptide. In some embodiments, the peptide tag is an acidic tag, as provided herein, and the chelant moiety is comprised in a peptide tag, for example, a His tag, as provided herein. For example, in some embodiments, an MHC class II molecule is provided that comprises a heavy chain conjugated to a His tag, for example, by C-terminal fusion, and that is loaded with a tagged antigenic peptide, for example, an antigenic peptide conjugated to an acidic tag, for example, a Cy5.5 tag) via a photocleavable linker. In some embodiments, a correctly peptide-loaded MHC class II molecule is isolated by anion exchange chromatography, the tag is cleaved from the antigenic peptide, for example, by UV irradiation, and the peptide-loaded MHC class II molecule thus generated is then assembled into an MHC multimer as described herein.
In some embodiments, an MHC molecule, for example, an MHC molecule loaded with an antigenic peptide is conjugated with a chelant moiety or a peptide tag after production of the MHC molecule or after loading the MHC molecule with the antigenic peptide, or after cleavage of a tag, if present, from a reversibly tagged antigenic peptide bound to the MHC molecule. In some embodiments, such post-production conjugation, for example, to a heavy chain of a peptide-loaded MHC class II molecule, is carried out by site- specific alkylation or a sortase-mediated transpeptidation reaction. In some embodiments, the antigenic peptide is tagged with a His tag, the tag is cleaved after peptide-loading, and a His tag is subsequently appended to a heavy chain of the MHC molecule. Using such "tag exchange" strategies, incompatible tags can be used subsequently, or the same tag can be employed at different positions. Methods for post-synthesis addition of tags to peptides and proteins are well known to those of skill in the art and include, but are not limited to, biotinylation and sortase-mediated protein labeling (for the latter see Popp et al., Site Specific Protein Labeling via Sortase -Mediated Transpeptidation Curr. Protoc. Protein Sci.
56: 15.3.1-15.3.9; 2009, incorporated herein by reference in its entirety for disclosure of sortase-mediated transpeptidation reactions).
To give a non-limiting example of an embodiment employing such a tag-exchange strategy: in some embodiments, an MHC class II molecule comprising a sortase target sequence is loaded with a His-tagged MHC class II -binding peptide and peptide-loaded MHC class II molecules are isolated form free peptide and empty MHC class II molecules by affinity chromatography and, optionally, size fractionation. After isolation, the His-tag is cleaved off the MHC class II binding peptide and, optionally, the tag-free MHC class II molecule loaded with the peptide is separated from the cleaved-off tag. In some such embodiments, the heavy chain of the MHC class II molecule is then tagged by performing a sortase-mediated transpeptidation reaction. In some such embodiments, the tag appended to the MHC heavy chain is a peptide tag. In some embodiments, the sortase- appended tag is a His-tag, thus effectively yielding MHC class II molecules in which the His tag was moved from the antigenic peptide to a heavy chain..
In some embodiments that include a charged tag, for example an acidic tag, that is conjugated to an MHC class II binding peptide, a peptide-loaded MHC class II molecule is isolated by ion exchange chromatography. In some embodiments including an acidic tag, anion exchange chromatography is used to isolate the tagged peptide or an MHC class II molecule loaded with the tagged peptide.
Other methods of producing peptide-loaded MHC multimers are known in the art (for example, see Altman et al., Science 274:94 96, 1996; Dunbar et al., Curr. Biol. 8:413 416, 1998; Crawford et al., Immunity 8:675 682, 1998). In all embodiments, non-denaturing conditions are preferred during isolation of empty and peptide-loaded MHC class II molecules.
Some aspects of this invention provide methods and reagents for the generation of "empty" MHC class II molecules. The term "empty" in the context of MHC class II molecules signifies that the MHC molecules are not loaded with an antigenic, MHC class II- binding peptide. Such empty MHC class II molecules are often instable and conventional methods of high-affinity antibody-mediated isolation are typically unsuitable for the preparation of such empty MHC class II molecules because of the denaturing conditions used in such methods.
Some aspects of this invention provide methods that allow gentle purification of fragile "empty" (without nominal peptide cargo) His tagged MHC II molecules by affinity chromatography on Ni2+ nitrilotriacetic acid (NTA) columns. In some embodiments of this invention, methods are provided that allow for the isolation of empty MHC class II molecules that retain the correct folding and dimerization properties. In some embodiments, such methods include a step of tagging a heavy chain comprised in an MHC class II molecule. In some embodiments, the tag is a peptide tag that can be used for isolation of the tagged protein by affinity chromatography or ion exchange chromatography. In some embodiments, the tag is a His tag, for example, a tag that comprises 3-12 Histidine residues. In some embodiments, MHC class II molecules comprising a His-tag labeled heavy chain are isolated by affinity chromatography. In some embodiments, the affinity chromatography uses an ΝΪ2+-ΝΤΑ resin. Methods for affinity chromatography for the isolation of tagged peptides and proteins are well known to those of skill in the art and it will be appreciated by those of skill that the invention is not limited in this respect.
Empty MHC multimers are useful in that they allow for standardized production of a core reagent that can then be customized for a specific application by loading a specific antigenic peptide of interest onto the multimer. In some embodiments, a chelant moiety is conjugated to an isolated protein after the protein has been synthesized, for example, post-translationally, or after in situ synthesis, for example, after Fmoc synthesis. In some such embodiments, the chelant moiety is conjugated to the protein, for example, the MHC molecule, for example, to a heavy chain of the MHC molecule by chemical or enzymatic modification. Methods for post-synthesis addition of tags to peptides and proteins are well known to those of skill in the art and include, but are not limited to, biotinylation and sortase-mediated protein labeling (for the latter see Popp et al., Site Specific Protein Labeling via Sortase -Mediated Transpeptidation Curr. Protoc. Protein Sci. 56: 15.3.1-15.3.9; 2009, incorporated herein by reference in its entirety for disclosure of sortase-mediated transpeptidation reactions).
Post-isolation addition of a chelant moiety is particularly useful in embodiments, where a chelant moiety would interfere with a synthesis or purification step, for example, in embodiments, where an MHC molecule is loaded with an antigenic peptide that comprises a chelant tag and the tag is used for isolation of peptide-loaded MHC molecules. In some such embodiments, the peptide tag can be cleaved from the antigenic peptide and a chelant tag can be added subsequently to the MHC molecule, for example, to an MHC heavy chain by methods well known to those of skill in the art.
Cell staining and detection methods
Some aspects of this invention provide methods for the staining, detection, and/or isolation of cells using a reversible protein multimer, for example, a reversible MHC multimer, as described herein. In some embodiments, the method comprises contacting a population of cells with a protein multimer, for example, an MHC multimer provided herein. In some embodiments, the multimer comprises a detectable label, for example, a fluorophore, either as the carrier molecule or conjugated to the multimer.
The term "detectable label," as used herein, refers to a molecule or moiety that can be detected, for example, by performing an assay known to those of skill in the art for its detection. A detectable label, accordingly, may be, for example, (i) an isotopic label (e.g., a radioactive or heavy isotope, including, but not limited to, 2H, 3H, 13C, 14C, 15N, 3 IP, 32P, 35S, 67Ga, 99mTc (Tc-99m), 11 lln, 1231, 1251, 169Yb, and 186Re), (ii) an affinity label (e.g., an antibody or antibody fragment, an epitope, a ligand or a ligand-binding agent) (iii) and enzymatic label that produce detectable agents when contacted with a substrate (e.g., a horseradish peroxidase or a luciferase); (iii) a dye, (e.g., a colored, luminescent,
phosphorescent, or fluorescent molecule, such as a chemical compound or protein).
Fluorophores, for example, fluorescent dyes and proteins, are of particular use for embodiments of this invention that involve detection or isolation of living cells. A fluorophore is a molecule or moiety that absorbs light of a specific wavelength and then re- emits light at a different specific wavelength, thus causing the molecule of moiety to be fluorescent. Other detectable labels are known to those of skill in the art and the invention is not limited in this respect. It will be appreciated that a detectable label may be incorporated into any part of the multimeric structure and in any manner that does not interfere with the stability or the function, for example, the binding activity of the multimer.
In some embodiments, the method includes a step of detecting the multimer bound to a cell, for example, to a surface receptor (e.g., a T-cell receptor) of a cell. The method performed to detect the multimer depends, of course, on the nature of the detectable label comprised in the multimer. For example, in some embodiments, where the detectable label is a fluorophore, suitable methods for detection are fluorescence microscopy, cytometry, or FACS.
In some embodiments, the method comprises a step of quantifying the number of detected cells, for example, quantifying the number of T cells expressing a specific TCR in a cell population, for example, in a cell population obtained from a subject. In some embodiments, the quantity of cells, for example, of T-cells expressing a specific TCR, is compared to a reference quantity. In some embodiments, the comparison of the quantity of T-cells expressing a specific TCR in a subject to a reference quantity is used to determine an immune reaction in the subject. In some embodiments, the reference quantity is a quantity measured or expected in a healthy subject or in healthy subjects, or a quantity measured in the subject prior to a clinical intervention, for example, prior to a vaccination, and a quantity in the subject that is higher than the reference is indicative of an immune response in the subject, whereas a quantity in the subject that is lower than the reference is indicative of depletion of a specific T-cell population.
Accordingly, MHC multimers as provided herein are useful, for example, for monitoring immune responses in subjects, either in response to a clinical intervention, for example, a vaccination, or as a result of a disease or condition, for example, a
hyperproliferative disease in the subject. In some embodiments, the clinical intervention is a vaccination against a tumor antigen. In some embodiments, the vaccination is a vaccination administered after surgical removal of a tumor expressing the tumor antigen. In some embodiments, the clinical intervention is an intervention aimed to suppress a function of the immune system, for example, by depleting a specific population of T-cells. In some embodiments, the subject is a subject having an autoimmune disease.
In some embodiments, the detection method further comprises a step of releasing the chelate complex bond comprised in the multimer employed, for example, by withdrawing the cation of the complex bond, or by contacting the multimers with an agent able to displace a chelant forming the chelate complex bond.
T cell staining methods
Interactions of T cell antigen receptors (TCRs) with MHC -peptide monomers are characterized by micromolar dissociation constants (KD) and half-lives in the range of seconds. The coordinate binding of CD8 to TCR-associated MHC class I-peptide complexes can considerably strengthen the binding interaction. According to some aspects of this invention, the use of MHC -peptide monomers that are conjugated to reversible multimers substantially increases the overall binding avidity and decreases the dissociation rate to half- lives in the order of hours. Accordingly, the use of MHC multimers as provided herein allows for the efficient staining, detection, and/or isolation of T cells bearing specific TCRs, for example, by fluorescent microscopy, flow cytometry, fluorescence activated cell sorting (FACS), or magnetic- activated cell sorting (MACS).
In some embodiments, MHC multimers and methods are provided that are useful for the staining of CD8+ T-cells. CD8 interacts preferentially with MHC class I molecules. Accordingly, MHC multimers useful for staining of CD8+ T-cells are preferentially MHC class I multimers. CD8 undergoes differentiation- and activation-dependent changes in the glycosylation and sialylation of its β chain, which can profoundly affect cognate and non- cognate MHC class I-peptide binding. Non-cognate CD8 binding to MHC class I-peptide multimers has been reported to increase non-specific multimer binding. Accordingly, in some embodiments, multimers are provided that contain the CD8 binding weakening mutation A245V in the MHC a3 domain. In some embodiments, an MHC class I multimer as provided herein (A245V mutated or not) is used in a staining procedure at a concentration of about 5-30nM (about 2.5 to 15μg/ml). At this concentration, non-specific staining is generally low.
Importantly, binding of MHC class I molecules to T-cell receptors can elicit T cell activation events, such as intracellular calcium mobilization, diverse tyrosine phosphorylation and endocytosis of MHC class I-peptide engaged TCR/CD8. For example, MHC class I- peptide complex driven cell activation can induce death of effector cytotoxic T-cells (CTLs) via FasL-dependent apoptosis or severe mitochondrial damage. This can lead to changes in T-cell populations that are contacted with MHC multimers, for example, for cell staining, detection, or isolation, for example, by selective depletion of stained T-cells. In some circumstances, TCR- activation-mediated cell depletion can render isolation of a non- activated T-cell population impossible. Some aspects of this invention provide reversible MHC multimers and methods for their use that avoid this problem by minimizing the time of high-avidity MHC/TCR interaction by re-monomerizing the multimers, thus minimizing undesired TCR activation-mediated effects on stained cells. However, it will be appreciated by those of skill in the art, that the reversible MHC multimers provided herein can also be used in methods that do not include a chelate complex bond release step, thus employing the reversible multimers in the manner conventional multimers would be employed. Accordingly, in some embodiments, methods are provided that exploit MHC multimer TCR activation to deplete specific T-cell populations by MHC-mediated TCR activation. In some such embodiments, reversible MHC multimers as provided herein are used to eradicate antigen- specific CD8+ CTLs.
MHC class II multimer binding to CD4+ T-cells can also lead to T-cell activation and death, for example, of CD4+ effector cells. Accordingly, reversible MHC class II multimers are useful in the staining of CD4+ T-cells and in the isolation of minimally manipulated or activated CD4+ T-cells.
Some aspects of this invention provide reversible MHC multimers and methods for the use of such multimers to analyze the state of activation or differentiation of T-cells, for example, CD8+ and/or CD4+ T-cells. As will be appreciated by those of skill in the art, homogenous populations of MHC multimers of defined structure are preferable over heterogeneous MHC multimer populations. In some embodiments, homogenous populations of MHC multimers, e.g. of dimers, trimers, tetramers, pentamers, hexamers, or decamers, are provided for use in such methods. In some embodiments, the MHC multimers comprise linkers of defined length and flexibility. In some embodiments, the MHC multimers comprise chelant groups in defined configurations, for example, in mono- di- or tetra-chelant configuration. In contrast to heterogeneous multimers, binding studies with defined, homogenous populations of multimers can reveal differentiation- and activation-dependent differences, for example, differentiation- and activation-dependent changes in glycosylation and sialylation of T cell surface molecules involved in antigen recognition of the cells under study which can affect, for example, CD8 participation in MHC class I molecule binding and/or aggregation of TCR and CD8.
It will be apparent to those of skill in the art that the MHC multimers provided herein can be employed alone or in combination with other binding and/or staining agents. For example, in some embodiments, MHC multimers provided herein are used to stain T-cells in combination with staining the cells for an additional antigen, for example, with a staining for intracellular cytokine staining.
In some embodiments, reversible MHC class I-peptide multimers, are provided that comprise a mutation in the a3 domain. In some embodiments, the mutation is a mutation that ablate CD8 binding, e.g. a D227K, T228A in human MHC and D227K, Q226A in mouse MHC molecules. In some embodiments, methods are provided that use such CD8 binding- deficient MHC multimers to stain, detect, and/or isolate CD 8 -independent T cells, which typically express high affinity TCRs.
For example, in some embodiments, CD8 binding-deficient MHC multimers are provided for the staining, detection, and/or isolation of CD8+ T cells expressing high-affinity TCRs specific for tumor antigens, for example, for MELAN-A/Mart-1, gplOO, or tyrosinase. It is known to those of skill in the art that such tumor-antigen specific T-cell tend to express low affinity TCRs and that infrequent CD8+ T cells specific for tumor antigens expressing high affinity TCRs efficiently kill tumor cells. In some embodiments, the use of a reversible MHC class I multimer as provided herein enables efficient identification and isolation of such rare cells with no or only minimal TCR activation, thus allowing for the isolation of native T- cell populations that cannot be isolated with conventional MHC class I multimers.
Further, in some embodiments, CD8 binding-deficient multimers are used to selectively induce FasL (CD95L) expression, resulting in apoptosis of antigen- specific CTLs.
Conditions and protocols for staining, detection, and isolation of cells using multimers are well known to those of skill in the art. In general, methods for the use of conventional MHC multimers are applicable to the reversible MHC multimers as provided herein, modified, where appropriate, to include a step of chelate complex bond release, as described in more detail elsewhere herein.
Staining with reversible MHC multimers can be performed through a wide range of temperatures. In some embodiments, staining is performed at a temperature between 0-37 °C.
In some embodiments, MHC multimer staining is performed at 37 °C. While staining at 37°C is rapid, and efficient staining of CD4+ T cells with reversible MHC class II multimers is often observed upon incubations at 37°C for extended periods of time, reversible MHC class I multimers efficiently effect TCR activation at this temperature.
In some embodiments, staining is performed at 0-4°C. It will be appreciated by those of skill, that MHC multimer binding at low temperatures (e.g., 0-4°C) tends to be slow, necessitating extended periods of time for staining as compared to staining at higher temperatures. In some embodiments, staining with MHC multimers is performed at ambient temperature, e.g. at 20-30°C, preferably at 22-25°C. In some embodiments, MHC staining is performed in the presence of EDTA (e.g., 5mM) and/or sodium azide (e.g., 0.02%) to inhibit cell activation. Under these conditions multimer binding is rapid. In some embodiments, staining is performed for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 20-45 minutes. In some embodiments, for example, in some embodiments using reversible MHC class II multimers, staining is performed for 30- 120min. Under these conditions, cognate MHC class II complexes binding to TCR (and CD4) are internalized and accumulate over time.
Multimer concentration is an important factor in achieving maximum staining efficiency, and, while exemplary MHC multimer concentrations are provided herein, it will be appreciated by those of skill that it is preferable to test a range of concentrations, for example, in the range of about 5-50nM (about 2.5-25 μg/ml), or, in the case of low affinity binding, in higher concentration ranges, for example, in the range of about 5-100nM (about 2.5-5(^g/ml).
In some embodiments in which a cell is contacted with an MHC multimer, for example, with an MHC class II multimer as provided herein, binding of the MHC multimer to the cell is facilitated by desialylation of the cell. Desialylation is a process by which sialyl groups on the cell surface are removed or modified. Methods and reagents for desialylating a cell are described in detail elsewhere herein and additional methods are well known to those of skill in the art. For example, in some embodiments, a cell is contacted with a desialylating agent in order to achieve desialylation. Desialylating agents are, in some embodiments, enzymes, while, in other embodiments, chemicals are used to effect desialylation. Enzymes known to desialylate cell surfaces are, for example, neuraminidases. Methods and conditions suitable for desialylation of cells by contacting them with a neuraminidase are well known to those of skill in the art.
In some embodiments, the cells are pre-treated with neuraminidase under conditions suitable to achieve desialylation of the cells (e.g., treatment with 0.03μ/ιη1 for 30 min at 37°C).
In some embodiments, staining is increased by inhibiting TCR down modulation with the protein kinase inhibitor dasatinhib. In some embodiments, scarce antigen- specific cells can be enriched by combination of fluorescence -based methods as described herein with a non-fluorescent-based isolation method, for example, with MACS using magnetic beads coated with an antibody against an epitope of the carrier molecule.
Isolation of cells with reversible mult inters
In some embodiments, methods for the use of multimers as described herein for isolating specific cells or cell populations are provided. In general, useful multimers for isolation methods comprise a detectable label and the methods include a step of staining the target cell population as described in more detail elsewhere herein. In some embodiments including the isolation of cells, a method of cell separation is employed that allows for the enrichment or the isolation of homogenous populations of cells based on the cells binding the employed multimer, for example, the employed MHC multimer. Such methods are well known in the art and include, for example, FACS and MACS.
In some embodiments, the method of isolating cells with a reversible multimer further includes a step of releasing the chelate bond comprised in the multimer. In some
embodiments, this step includes withdrawal of the central ion, for example, the central divalent cation, from the chelate complex. Method for ion withdrawal are well known in the art and include, in some embodiments, washing the cells with a solution that does not contain a significant amount of the divalent cation, or with a solution that comprises an agent that sequesters the divalent cation. In some embodiments, the step of chelate complex bond release includes contacting the chelate complex bond with an agent that displaces a chelant from the chelate complex bond. For example, if the chelate complex bond is formed by an NTA chelant and a histidine residue, the chelate complex bond can be released by contacting it with an imidazole residue, for example, with free imidazole. Imidazole is able to displace a chelant, in this case, the histidine chelant from the complex bond, thus releasing the chelate complex bond of the multimer. The result of this release is the re-monomerization of the proteins comprised in the multimer, for example, of MHC proteins in an MHC multimer.
In some embodiments, re-monomerization of the multimer after staining and/or isolation avoids detrimental effects on the cells and, in embodiments, where the cells are rare and/or sensitive to detrimental effects of staining, allows for efficient isolation of such cells that is cumbersome or impossible with conventional strategies.
Manipulation of T-cell populations
In some aspects, this invention provides methods for the manipulation of T-cells using reversible monomers. In some embodiments, the method includes a step of contacting a population of cells expressing a T-cell receptor with an MHC multimer as described herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC class I interaction to effect TCR-mediated T-cell activation. In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed ex vivo. In some embodiments, the contacting is performed in vivo. In some embodiments, the cells are contacted with an MHC multimer for a time long enough to activate high-affinity TCR expressing T-cells, but not to activate low affinity TCR expressing T-cells. In some embodiments, the cells are cells from a subject having an autoimmune disease. In some embodiments, the cells are contacted with an MHC multimer that is loaded with an antigenic peptide recognized by T-cells that mediate an autoimmune disease. In some embodiments, the method further comprises measuring the quantity of the T-cells targeted by the MHC multimer, for example, by methods for the identification or detection of T-cells provided herein or otherwise known in the art.
Isolated cell populations
Some embodiments of this invention provide isolated cells or cell populations, for example, isolated native, or non-activated T-cell populations obtained by using a reversible multimer or a method as provided herein. In some embodiments, an isolated cell is provided that has been contacted with a reversible multimer provided herein and isolated from a cell population based on the cell binding the multimer, for example, by a method for detection and/or isolation described in more detail elsewhere herein. In some embodiments, the cell is a T-cell. In some embodiments, the T-cell is a native T-cell, or a T-cell that has not undergone TCR-mediated cell activation. In some embodiments, the cell has been contacted with an agent releasing the chelate complex bond of the reversible multimer subsequent to its isolation. In some embodiments, the cell is a T-cell recognizing a tumor antigen. In some embodiments, the cell is a T-cell expressing a TCR that binds a tumor antigen with high affinity. In some embodiments, the cell is a therapeutically valuable cell. In some embodiments, the cell is expanded in vitro after isolation, and used in a therapeutic method. In some embodiments, the therapeutic method includes a step of administering the cell to a subject in need thereof, for example, a subject having a tumor or having an elevated risk of developing a tumor expressing a tumor antigen. In some embodiments, a subject at risk of developing a tumor expressing a tumor antigen is a subject which was diagnosed to have such a tumor and has undergone surgical removal of the tumor.
Further materials, methods, suitable conditions, and useful modifications for the use of reversible MHC multimers as described herein will be apparent to those of skill in the art. Methods for the use of conventional multimers can generally be applied to the use of the inventive multimers provided herein with modifications that do not amount to more than routine experimentation. Examples of such methods are described, for example, in Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer- Williams MG, Bell JI, McMichael AJ, Davis MM. Phenotypic analysis of antigen- specific T lymphocytes. Science 1996; 274: 94- 96.; Bakker AH, Schumacher TN. MHC multimer technology: current status and future prospects. Curr Opin Immunol 2005; 17: 428-433.; Xu XN, Screaton GR. MHC/peptide tetramer-based studies of T cell function. J Immunol Methods 2002; 268: 21-28.; Guillaume P, Baumgaertner P, Neff L, Rufer N, Speiser DE, Luescher IF. Novel soluble HLA- A2/Melan-A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in melanoma patients Jnt J Cancer 2009; in press.; Guillaume P, Legler DF, Boucheron N, Doucey MA, Cerottini JC, Luescher IF. Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis. J Biol Chem 2003; 278: 4500-4509.; Neveu B, Echasserieau K, Hill T, Kuus-Reichel K, Houssaint E, Bonneville M, Saulquin X. Impact of CD8-MHC class I interaction in detection and sorting efficiencies of antigen- specific T cells using MHC class I/peptide multimers:
contribution of pMHC valency. Int Immunol 2006; 18: 1139-1145.; Knabel M, Franz TJ, Schiemann M, Wulf A, Villmow B, Schmidt B, Bernhard H, Wagner H, Busch DH.
Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nat Med 2002; 8: 631-637.; Guillaume P, Baumgaertner P, Angelov GS, Speiser D, Luescher IF. Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC -peptide and antibody Fab' conjugates. J Immunol 2006; 177: 3903-3912.; Yao J, Bechter C, Wiesneth M, Harter G, Gotz M, Germeroth L, Guillaume P, Hasan F, von Harsdorf S, Mertens T, Michel D, Dohner H, Bunjes D, Schmitt M, Schmitt A. Multimer staining of cytomegalovirus phosphoprotein 65-specific T cells for diagnosis and therapeutic purposes: a comparative study. Clin Infect Dis 2008; 46: 96-105.; Chattopadhyay PK, Price DA, Harper TF, Betts MR, Yu J, Gostick E, Perfetto SP, Goepfert P, Koup RA, De Rosa SC, Bruchez MP, Roederer M. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 2006; 12: 972-977.; Cebecauer M, Guillaume P, Hozak P, Mark S, Everett H, Schneider P, Luescher, IF. Soluble MHC-peptide complexes induce rapid death of CD8+ CTL. J Immunol 2005; 174: 6809- 6819.; Cebecauer M, Guillaume P, Mark S, Michielin O, Boucheron N, Bezard M, Meyer, BH, Segura JM, Vogel H, Luescher IF.CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex -peptide dimers. J Biol Chem 2005; 280: 23820-23828.; Angelov GS, Guillaume P, Cebecauer M, Bosshard G, Dojcinovic D, Baumgaertner P, Luescher IF. Soluble MHC-peptide complexes containing long rigid linkers abolish CTL- mediated cytotoxicity. J Immunol 2006; 176: 3356-3365.; Batard P, Peterson DA, Devevre E, Guillaume P, Cerottini JC, Rimoldi D, Speiser DE, Winther L, Romero P.Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen- specific CD8+ T cells. J Immunol Methods 2006; 310: 136-148.; Fahmy TM, Bieler JG, Schneck JP. Probing T cell membrane organization using dimeric MHC-Ig complexes. J Immunol
Methods 2002; 268: 93-106.; van der Merwe PA, Davis SJ. Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol 2003; 21: 659-684.; Campanelli R, Palermo B, Garbelli S, Mantovani S, Lucchi P, Necker A, Lantelme E, Giachino C. Human CD8 co- receptor is strictly involved in MHC-peptide tetramer-TCR binding and T cell activation. Int Immunol 2002; 14: 39-44.; Luescher IF, Vivier E, Layer A, Mahiou J, Godeau F, Malissen B, Romero P.CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes. Nature 1995; 373: 353-356.; Kao C, Daniels MA, Jameson SC. Loss of CD8 and TCR binding to Class I MHC ligands following T cell activation. Int Immunol 2005; 17: 1607-1617.; Comelli EM, Sutton-Smith M, Yan Q, Amado M, Panico M, Gilmartin T, Whisenant T, Lanigan CM, Head SR, Goldberg D, Morris HR, Dell A, Paulson JC.Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans. J Immunol 2006; 177: 2431-2440.; Wooldridge L, Lissina A, Cole DK, van den Berg HA, Price DA, Sewell AK. Tricks with tetramers: how to get the most from multimeric peptide- MHC. Immunology 2009; 126: 147-164.; Xu XN, Purbhoo MA, Chen N, Mongkolsapaya J, Cox JH, Meier UC, Tafuro S, Dunbar PR, Sewell AK, Hourigan CS, Appay V, Cerundolo V, Burrows SR, McMichael AJ, Screaton GR.A novel approach to antigen- specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex. Immunity 2001 ; 14: 591-602.; Demotte N, Stroobant V, Courtoy PJ, Van Der Smissen P, Colau D, Luescher IF, Hivroz C, Nicaise J, Squifflet JL, Mourad M, Godelaine D, Boon T, van der Bruggen P. Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes. Immunity 2008; 28: 414-424.; Dimopoulos N, Jackson HM, Ebert L, Guillaume P, Luescher IF, Ritter G, Chen W.
Combining MHC tetramer and intracellular cytokine staining for CD8(+) T cells to reveal antigenic epitopes naturally presented on tumor cells. J Immunol Methods 2009; 340: 90-94.; Choi EM, Chen JL, Wooldridge L, Salio M, Lissina A, Lissin N, Hermans IF, Silk JD, Mirza F, Palmowski MJ, Dunbar PR, Jakobsen BK, Sewell AK, Cerundolo V.High avidity antigen- specific CTL identified by CD8-independent tetramer staining. J Immunol 2003; 171: 5116- 5123.; Pittet MJ, Rubio-Godoy V, Bioley G, Guillaume P, Batard P, Speiser D, Luescher I, Cerottini JC, Romero P, Zippelius A. Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells. J Immunol 2003; 171: 1844-1849.; Wooldridge L, Scriba TJ, Milicic A, Laugel B, Gostick E, Price DA, Phillips RE, Sewell AK. Anti-coreceptor antibodies profoundly affect staining with peptide- MHC class I and class II tetramers. Eur J Immunol 2006; 36: 1847-1855.; Scriba TJ, Purbhoo M, Day CL, Robinson N, Fidler S, Fox J, Weber JN, Klenerman P, Sewell AK, Phillips RE. Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining. J Immunol 2005; 175: 6334-6343.; Day CL, Seth NP, Lucas M, Appel H, Gauthier L, Lauer GM, Robbins GK, Szczepiorkowski ZM, Casson DR, Chung RT, Bell S, Harcourt G, Walker BD, Klenerman P, Wucherpfennig KW. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. J Clin Invest 2003; 112: 831-842.; Mallone R, Nepom GT.MHC Class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete. Clin Immunol 2004; 110: 232-242.; Reijonen H, Kwok WW.Use of HLA class II tetramers in tracking antigen- specific T cells and mapping T-cell epitopes. Methods 2003; 29: 282-288.; Vollers SS, Stern LJ. Class II major histocompatibility complex tetramer staining: progress, problems, and prospects.
Immunology 2008; 23: 305-313.; Arnold PY, La Gruta NL, Miller T, Vignali KM, Adams PS, Woodland DL, Vignali DA. The majority of immunogenic epitopes generate CD4+ T cells that are dependent on MHC class Il-bound peptide-flanking residues. J Immunol 2002; 169: 739-749.; Reche PA, Reinherz EL. Definition of MHC supertypes through clustering of MHC peptide-binding repertoires. Methods Mol Biol 2007; 409: 163-173.; Boniface JJ, Rabinowitz JD, Wulfing C, Hampl J, Reich Z, Altman JD, Kantor RM, Beeson C, McConnell HM, Davis MM. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands. Immunity 1998;9: 459-466.; Lovitch SB, Unanue ER. Conformational isomers of a peptide-class II major histocompatibility complex. Immunol Rev 2005; 207: 293-313.; Cameron TO, Cochran JR, Yassine-Diab B, Sekaly RP, Stern LJ. Cutting edge: detection of antigen- specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state. J Immunol 2001; 166: 741-745.; Yang J, James EA, Huston L, Danke NA, Liu AW, Kwok WW. Multiplex mapping of CD4 T cell epitopes using class II tetramers. Clin Immunol 2006; 120: 21-32.; Blanchet JS, Valmori D, Dufau I,
Ayyoub M, Nguyen C, Guillaume P, Monsarrat B, Cerottini JC, Romero P, Gairin JE.A new generation of Melan- A/MART- 1 peptides that fulfill both increased immunogenicity and high resistance to biodegradation: implication for molecular anti-melanoma immunotherapy. J Immunol 2001; 167: 5852-5861.; Schiavetti F, Thonnard J, Colau D, Boon T, Coulie PG.A human endogenous retroviral sequence encoding an antigen recognized on melanoma by cytolytic T lymphocytes. Cancer Res 2002; 62: 5510-5516.; Ali SA, Lynam J, McLean CS, Entwisle C, Loudon P, Rojas JM, McArdle SE, Li G, Mian S, Rees RC. Tumor regression induced by intratumor therapy with a disabled infectious single cycle (DISC) herpes simplex virus (HSV) vector, DISC/HSV/murine granulocyte-macrophage colony- stimulating factor, correlates with antigen- specific adaptive immunity. J Immunol 2002; 168: 3512-3519.;
Pardigon N, Darche S, Kelsall B, Bennink JR, Yewdell JW. The TL MHC class lb molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes. Int Immunol 2004; 16: 1305-1313.; Guilloux Y, Lucas S, Brichard VG, Van Pel A, Viret C, De Plaen E, Brasseur F, Lethe B, Jotereau F, Boon T.A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene. J Exp Med 1996; 183: 1173-1183.; Rakoff-Nahoum S, Kuebler PJ, Heymann JJ, E Sheehy M, M Ortiz G, S Ogg G, Barbour JD, Lenz J, Steinfeld AD, Nixon DF. Detection of T lymphocytes specific for human endogenous retrovirus K (HERV-K) in patients with seminoma. AIDS Res Hum Retroviruses 2006; 22: 52-56.; Liso A, Colau D, Benmaamar R, De Groot A, Martin W, Benedetti R, Specchia G, Martelli MP, Coulie P, Falini B. Nucleophosmin leukaemic mutants contain C- terminus peptides that bind HLA class I molecules. Leukemia 2008; 22: 424-426.; and Matsuki N, Ogasawara K, Takami K, Namba K, Takahashi A, Fukui Y, Sasazuki T, Iwabuchi K, Good RA, Onoe K.Prevention of infection of influenza virus in DQ6 mice, a human model, by a peptide vaccine prepared according to the cassette theory. Vaccine 1999; 17: 1161-1168. All references are incorporated herein in their entirety by reference for disclosure of methods and materials useful for the generation, isolation, and or purification of MHC multimers and for staining, detection, and/or isolation of cells using MHC multimers.
The function and advantage of these and other embodiments of the present invention will be more fully understood from the examples below. The following examples are intended to illustrate the benefits of the present invention, but do not exemplify the full scope of the invention.
EXAMPLES
EXAMPLE 1
Some aspects of this invention provide a novel type of MHC multimers in which MHC class I-peptide monomers are conjugated to phycobilins (PE or APC) or quantum dots (Qdots) via chelate complexes of histidine (His) tags and Ni2+-nitrilotriacetic acid (NTA). Hexa (His6), dodeca (His12) or tandem hexa (2xHis6) histidine tags were fused to HLA- A*0201 (A2) heavy chain and A2-peptide monomers obtained in good yields by refolding. Mono, di and tetra NTA derivatives were synthesized and their interactions with His tagged monomers studied by surface plasmon resonance (SPR) and by CD8+ T cell staining experiments. The results described here indicate that the affinity (KD) increases in the order His6 > His12 >2xHis6 and mono > di > tetra NTA, respectively, spanning several orders of magnitudes. Staining experiments on influenza- specific CD8+ T cell clones and populations with NTA-His tag A2/Flus8_66 multimers indicated that: i) multimers containing 2xHis6 tagged complexes and short di-or tetra NTA moieties were equal or superior compared to conventional multimers; ii) di-NTA or tetra- NTA can be directly coupled to the phycobilin proteins or Qdots, which circumvents the use of biotin and streptavidin, and renders synthesis simpler and cheaper; iii) these reagents are molecularly better defined than conventional multimers and hence allow better analysis of binding data; iv) NTA-His tag built multimers dissociated rapidly in the presence of 100 mM imidazol (t1/2 < 1 min), which allows sorting of bona fide antigen-specific CD8+ T cells without inducing activation dependent cell death.
MATERIALS AND METHODS
Abbreviations used herein include: APC: allophycocyanine; β2ιη: beta-2- microglobulin; BSP: biotinylation sequence peptide; DIEA: di-isopropyl-ethyl-amine; EDIC: l-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GFC: gel filtration chromatography; Flu: influenza matrix; HOBt: 1-hydroxy-benzo-triazole; NHS: N-hydroxysuccinimide; NTA: nitrilotriacetic acid; PE: phycoerythrin; Qdot: quantum dot; SPR: surface plasmon resonance; TBTU: 0-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate.
Chemical synthesis
Protected amino acids and 2-chlorotrityl resin were obtained from Reactolab (Servion, Switzerland), TBTU and HOBt were from Multisynthec (Witten, Germany), maleimide-NTA from Dojindo Laboratories (Kumamoto, Japan). RP-HPLC analyses were performed on Waters HPLC station consisting of two 515 pumps and a Waters 996 photodiode array detector. The purity of all peptides was examined by analytical HPLC on a C18 reverse phase column (Uptisphere 5 μιη C18 particles, 250 x 4.6 mm) and which was eluted with a linear gradient rising from 100% of 0.1% TFA in H20 to 50% of 0.08% TFA in CH3CN in 30 min at a flow rate of 1 ml/min. peptides were purified on a semi preparative column
(Kromasil 15 μιη C18 particles, 250 x 20 mm) at a flow rate of 3 mL/min, with UV
monitoring at 214 nm. The purified peptides were characterized for correct Mr using matrix- assisted laser desorption ionization time-of-flight mass spectrometer (Micromass QTOF Ultima) (Waters Ltd, En Yvelines Cedex, France).
Synthesis of linear peptides - Synthesis of linear peptides was carried out manually in a syringe fitted with a sintered frit using Fmoc/tBu strategy. Coupling reactions were performed using 2 equiv of N-CC-Fmoc-protected amino acid relative to the resin loading, activated in situ with 2 equiv of TBTU, 2 equiv of HOBt and 4 equiv of DIEA in DMF (10 mL/g resin) for 1 h. Coupling completion was verified by Kaiser tests. N-a-Fmoc protecting groups were removed by treatment with a piperidine/DMF solution (1:4) (10 mL/g resin) for 5 min. The process was repeated twice, and the completion of deprotection was checked by the UV absorption of the piperidine washing at 299 nm. Peptides were obtained by cleavage of the resin with TFA/H20/TIPS (92.5/2.5/5) for 3 h and after filtering of the resin
precipitated by addition of ether, filtered off, dissolved in water, purified by semi-preparative HPLC and lyophilized.
Coupling of maleimide-NTA to linear peptides - Linear free SH containing peptides were dissolved in phosphate buffer (0.1M, pH 7.2) at a concentration of 0.1 M. Two equivalents of Mal-NTA (relative to SH) were added and the mixture stirred for 1 h under argon. The product was further purified by semi-preparative HPLC and analyzed by
Electrospray ionisation on a Micromass QTOF Ultima instrument. Alternatively, in the strategy shown in Fig. 13, the backbone peptide was reacted via in situ carboxyl activation with Na, Na-Bis[(tert-butyloxycarbonyl)methyl]-L-lysine tert-butyl ester (H2N-NTA(tBu)3), which was synthesized as follow: tert-butyl bromoacetate (1.59 ml, 10.8 mmol) and DIEA (2.30 ml, 13.5 mmol) were added sequentially to a solution of Νε-benzyloxycarbonyl-lysine tert-butyl ester (1.00 g, 2.7 mmol) in DMF (25 ml). The reaction vessel was purged with N2 and then continuously stirred overnight at 55°C. The volatiles were evaporated in vacuo at 60°C. Cyclohexane/ethylacetate (3: 1, 15 ml) solution was added to the partially solidified reaction mixture. The resulting slurry was filtered over sintered glass funnel and the precipitate washed three times with the same solvent (3x10 ml). The filtrate was
concentrated under reduced pressure and the resulting yellow powder dissolved in methanol (50 ml), the solution purged with N2 followed by addition of 10% Pd/C (20 mg). The reaction mixture was vigorously stirred for 6 hours under H2 atmosphere at room temperature. Pd/C was removed by filtration over celite and the volatiles removed under reduced pressure. The product was purified by silica chromatography with chloroform/methanol (3: 1) as the eluent. Yield: 1.03 g (2.4 mmol; 91%). The linear peptide H-K(aminocaproyl-biotin)-PEG- A-E*-A-E*-OH (*: Fmoc-E-OtBu) was synthesized on an ABI433 peptide synthesizer.
Double coupling of each Fmoc-protected amino acid were performed using DIPC and HOBt as coupling reagent. Fmoc was removed by 3x5 min treatment with piperidine 20% in DMF. Each cycle was followed by an acetylation (N-capping) to prevent the synthesis of truncated peptides. Final cleavage was performed in TFA/TIPS/H20 (92.5/5/2.5) for 2 h. The peptide was precipitated with cold ether, dissolved in water and purified by semi-preparative HPLC. Similarly, the synthesis of SH-NTA2 was performed assembling the linear C(Acm)-PEG- C(Trt)-G-C(Trt) on a chlorotrityl resin. After TFA treatment the linear peptide C(Acm)- PEG-C(SH)-G-C(SH) was coupled to maleimide-NTA as described previously. 1 μιηοΐ of lyophilized peptide was dissolved in 100 μΐ of AcOH 20%, the pH was adjusted to 4 with aqueous ammonia. 3 μιηοΐ of mercury(II) acetate were added and the mixture stirred for 1 h. 5 μιηοΐ of DTT were then added and the mixture stirred for 1 additional hour. The desired product was directly purified by semi-preparative HPLC and analyzed by mass spectrometry.
Surface plasmon resonance experiments - Affinity measurements were performed on a Biacore 3000 instrument equipped with SA coated chips. The eluent buffer (lOmM HEPES, 150mM NaCl, 50 mM EDTA at pH 7.4) and the dispensor buffer (lOmM HEPES, 150mM NaCl, 0.005% polysorbate 20, 3mM EDTA) were filtered and degassed prior to use. Two or five-fold dilutions of His-tagged A2/Fhi58-66 monomers were freshly prepared in eluent buffer before each experiment. Loading of biotin containing NTA peptides was performed in fresh solution of NTA-biotin peptides dissolved in eluent buffer. A RU (resonance unit) of 100 was used in all experiment. Loading of NTA with Ni2+ was performed by injecting NiC12 solution (500 mM in eluent buffer) and regeneration of the chip with imidazole (500mM in water) followed by a regeneration solution (lOmM HEPES, 150mM NaCl, 0.005%
polysorbate 20, 350mM EDTA at pH 7.4). All binding experiments were performed at a flow rate of 100 μΐ/min, starting with a 1 min injection of NiCl2 solution. Each sample was injected for 5 min followed by 5 min of undisturbed dissociation time. The regeneration procedure consisted of two subsequent 1-min injections of imidazole and regeneration solution. HLA-A2 Flu 58-66 multimers
HLA-A*0201-peptide complexes - HLA-A*0201 (A2) heavy chains containing the His tags shown in Fig. 2A expressed as inclusion bodies in E. Coli as described previously for the BSP containing heavy chain (3). The different heavy chains were refolded in the presence of 1ιβ2ηι and the influenza matrix peptide 5g_66 (GILGFVFTL, SEQ ID NO: 12) and purified on a Superdex S75 column as described (3).
Preparation of A2/Flu 58-66 multimers - Conventional streptavidin PE multimers were prepared as described (3). NTA- streptavidin PE multimers were prepared in two steps. First, streptavidin PE conjugate (Invitrogen) was incubated with NTA-biotin peptides (five- fold molar excess) at 4°C for lh followed by incubation for 30 min with NiS04 (10 mM). Excess of reagents were removed by means of min- spin columns (Zeba™ Spin Desalting Columns (Thermo scientific). His tagged monomeric A2/Flu complexes were mixed with Ni-NTA-biotin- streptavidin PE conjugate at a ten-fold molar excess and kept at 4°C until use. NTA2 -PE conjugates were prepared by first reacting PE (Sigma) (50 nM) in 0.1 M
phosphate buffer, pH 7.2 with 10 mM (or as indicated) SM(PEG)2 (Pierce) at room
temperature for 2h. Excess reagents were removed by centrifugation through spin columns (supplier). The resulting PE-maleimide conjugates were incubated under argon in 100 mM phosphate buffer, pH 7.0 with 50 mM cysteine-di-NTA at room temperature for l-2h (Fig. 3E). After incubation for 30 min with NiS04 (10 mM), excess reagents were removed by centrifugation through spin columns and the concentration of the resulting Ni 2+NTA2-PE was determined by Bradford.
Cells, staining procedures and flow cytometry
Cells under study - The HLA-A*0201 -restricted, influenza matrix peptides8_66- specific CD8+ T cell clones were obtained by limiting dilution cloning from bulk cultures. CD8+ PBMC from healthy donors were prepared by negative selection and were stimulated with Fhi58_66 peptides as described (4). The clones were re- stimulated in 24- well plates every 15 d in RPMI 1640 medium supplemented with 8 % human serum, rIL-2 (150 U/ml) (Hoffmann-la Roche Ltd, Basel, Switzerland) with PHA (1 μg/ml; Sodiag SA, Losone, Switzerland) and 1 x 106/ml irradiated alio geneic PBMC (3000 rad) as feeder cells. Bulk cultures were prepared by one or two peptide stimulations of CD8+ T cells obtained from PBMC from a
DR4+healthy donor (IFL). Multimer binding assays, flow cytometry and analysis. For binding studies CD8+ T cells (5 x 104) were incubated for 30-45 min at ambient temperature with graded
concentrations of the different A2/Flu58-66 complexes in 20 μΐ of FACS buffer (OptiMEM (Invitrogen AG, Basel, Switzerland) supplemented with 0.5% BSA (Sigma- Aldrich), 15 mM HEPES, 5 mM EDTA, and 5 mM NaN3). In some experiments cells were incubated an additional 20 min at 4°C with anti-CD8-FITC (Immuno Tools). After 30-fold dilution in FACS buffer, cell-associated fluorescence was measured on a LSRII flow cytometer (BD Biosciences). Background binding was determined on a A2/Mealn-A26-35 (ELAGIGILTV, SEQ ID NO: 13) clone (EM28- 9.24) and was subtracted from the cognate staining. Data were processed using the FlowJo software (Tree Star, Inc. Ashland, OR). For dissociation experiments CD8+T cells were incubated for 45 min at 4°C with 10 nM of multimers in FACS buffer, diluted 200 x fold in FACS buffer and after various periods of incubation at 4°C, cell-associated fluorescence was determined by flow cytometry (0 to 60 min). In some dissociation experiments with NTA multimers FACS buffer was supplemented with imidazol hydrochloride (50 or 100 mM).
Results and Discussion
To build sufficiently stable MHC-peptide multimers on Ni2+NTA - His tag chelate complexes, we examined the interaction of different His tags and NTA moieties. In the minimal subunit complex one NTA forms a coordinate complex with a Ni2+ ion, which in turn can bind two imidazoles, i.e. side chains of histidines (Fig. 1A). Since this subunit complex is not sufficiently stable, we prepared HLA-A*02010/Fhi58_66 monomers containing C-terminal a hexa-histidine (His6), a dodeca-histidine (His12) or double hexa-histidine tag (2xHis6) (Fig. 2A). On the other hand we synthesized mono, di- and tetra-NTA compounds, which contained biotin (Figs. 3A-C). Because the binding of biotin to streptavidin is exceedingly strong (KD ~10"15 M), we used streptavidin either conjugated to PE or immobilized on SPR sensor chips to stably bind the different biotinylated NTA derivatives. Addition of His tagged A2/Flu monomers to PE- streptavidin yielded multimers in which MHC-peptide complexes are conjugated via the NTA-His complexes (Fig. 1C). On the other hand this strategy allowed accurate SPR measurements of the different NTA-His tag interactions (Fig. 3A). Identification of suitable His tags and NT A linkers
To identify a suitable His tag we prepared A2 heavy chains containing C-terminally added His6, His12 and 2xHis6 (tandem) His tags (Fig. 2A). These heavy chains were refolded with P2m and Flu matrix58_66 peptide following established procedures (3). The refolding efficiency of the 2xHis6 tagged complex was nearly as high (98%) as the one of the BSP complex (Fig. 2B). For the His6 tagged complex the efficiency was approximately 85% and for the His12 tagged one only 60%. Similar results were obtained when using other peptides (e.g. Melan-A26_35 or NY-ESO-1157-105)·
We then synthesized the NTA linker shown in Figs. 3A-C and examined their binding of the His tagged A2/Flu5g_66 monomers by SPR. To this end streptavidin coated sensor chips were loaded with the biotinylated NTA compounds and the binding of the monomers measured by the changes in resonance units (RU). On mono-NTA (Fig. 3A) coated chips, the dissociation constant (KD) decreased dramatically from the His6, to the His12 and 2xHis6 tagged complexes (from 4100 to 34 nM) (Fig. 4B). On NTA2 (Fig. 3B) coated chips the KD values were lower still, reaching 12 nM for the 2xHis6 tagged A2/Flu complex. This increase in affinity was largely accounted for by decreased dissociation rates, i.e. the chelate complexes become increasingly more stable. These findings are consistent with reports showing that the affinity of His tags for Ni2+ NTA moieties dramatically increases with their valence and that NTA3 compounds bind His6 tags with sub-nanomolar KD (13-15).
We next examined staining of the Flu matrix- specific clone 81P1 by A2/Flu58-66 multimers containing the same His tags and streptavidin-PE saturated with biotin- Ni2+ NTA moieties. The 20°C binding isotherms of the multimers containing NTA2 were consistently higher than those obtained of mono NTA containing multimers (Fig. 5A). In both cases binding was strongest with multimers containing the 2xHis6 tag. Multimers containing this His tag and NTA2 containing exhibited higher binding than conventional multimers. While the binding values for NTA2 complexes with His12 containing complexes was only slightly lower, all other combinations exhibited substantially weaker binding and hence were not further investigated. Similar results were obtained on other Flu clones (data not shown).
To compare the binding of A2/Flu multimers containing NTA4 and NTA2, we performed similar binding experiments on cloned BCB 70 cells. The 20°C binding isotherms showed that NTA4 multimers bound more avidly than NTA2 or conventional multimers (Fig. 5B). While in the case of NTA4 the binding of multimers containing the His12 or 2xHis6 tag was essentially the same, for the NTA2 multimers those containing the 2xHis6 tag exhibited better binding compared to those containing the His12 tag.
It should be noted that staining results critically depend on the configuration of the NTA molecule. For example, we initially synthesized another biotin-NTA2 compound, in which lysine NTA was coupled via amide bonds to a linear peptide containing two orthogonal carboxyl side chains about 14 A apart (Fig. 13). Because this is longer (by about
4.4 A) compared to the other biotin-NTA2 (Fig. 3B, Fig. 12), we refer to this linker as short and the other as long. Binding isotherms on cloned 81P1 cells at 20°C cells indicated that A2/Flu multimers containing the short performed better than the multimers containing the long NTA2 moiety (Fig. 15). While multimers containing the short NTA2-biotin and 2xHis6 tagged A2/Flu complexes exhibited superior binding than conventional multimers (Fig. 15A), all multimers containing the long di-NTA showed inferior binding (Fig. 15B). Moreover, the binding hierarchy of multimers containing the differently tagged A2/Flu complexes and the short, respectively the long NTA2-biotin were remarkably disparate. We also tested multimers containing the commercial NTA-biotin (Fig. 3F) and observed substantially lower binding compared to multimers containing the long NTA-biotin (Fig 3 A) (data not shown).
Taken collectively, these results demonstrate that MHC class I-peptide multimers can be built on NTA-His tag chelate complexes that perform equal or better than conventional BSP multimers. The affinity and stability of Ni2+NTA-His tag complexes depends not only on the number of Ni2+NTA entities and histidines, but also on their configuration. This is primarily explained by the number of subunit Ni2+NTA-histidine chelate complexes that de facto can be formed (Fig. 1A). Our SPR binding studies indicated that complexes containing 2xHis6 tagged A2/Flu complexes have higher affinities (i.e. lower KD values) and slower dissociation kinetics (i.e. lower kQff values (Fig. 4B). This is consistent with previous reports and most likely explained by that two His6 tags joined by a flexible spacer can interact with more Ni2+NTA entities than the relative rigid His12 tag (). Our multimer binding studies on cells are consistent with this, although in the case of NTA4 containing multimer differences were marginal (Fig. 5B). On the other hand our staining results indicate that Ni2+NTA entities form more stable complexes with His tags when they have long flexible side chains, yet short intervening linkers (Figs. 12-15). Little is known on how binding parameters of
NTA-His tag interactions depend on the spatial configuration of the NTA moiety. One study showed that tri-NTA compounds most avidly bind to His6 tags when they contain minimal spacers (15). To better understand this relationship, we are currently testing additional di and tetra NTA molecules.
We performed staining experiments with multimers containing streptavidin Qdots6o5 loaded with biotin-NTA2 on 8 IP 1 cells. As shown in Fig 16 the staining of was similar for all monomers tested, i.e. the nature of the His tag had little effect on the staining. These
Qdots are larger than PE, contain more streptavidin on their surface and therefore more NTA2 groups; as discussed below, the density of NTA groups on a surface is another factor determining the stability of complexes with His tagged proteins.
NTA-His tag built multimers are reversible
We next assessed the dissociation kinetics of A2/Flu multimers built on NTA2-biotin- PE streptavidin in the presence of different concentrations of imidazol and/or EDTA. Cloned 81P1 cells were stained in the cold with multimers containing NTA2 and His12 or 2xHis6 tags, washed and incubated for different periods of time at 4°C in media containing imidazol. The staining of 2xHis6 tag containing multimers decreased more rapidly than the staining of His12 tag containing multimers (Figs. 6A, B). In the presence of 50 mM imidazole half maximal dissociation was observed after 8.1 and 54 min, respectively. In the presence of 50 mM imdidazol plus 20 mM EDTA the dissociation rate increased and half maximal dissociation was reached after 12.1 and 6.6 min, respectively. In the presence of EDTA alone
dissociations were very slow in both cases (data not shown). By contrast, rapid dissociations were observed in the presence of 100 mM imidazole, with half maximal dissociations after 1.3 and 0.7 min, respectively. At higher concentrations of imidazole the dissociation was further accelerated, but in some cases cell viability was affected.
Analogous dissociation experiments were performed on cloned BCB 70 cells with multimers containing NTA2-and NTA4 biotin- streptavidin A2/Flus8_66 multimers. For NTA2 multimers similar results were obtained as in the previous experiment (Figs. 6A and 7A). The dissociations for NTA4 multimers were slower. In the presence of 50 mM imidazol the half-life for the His12 containing multimer was in the range of hours and for the 2xHis6 containing one about 25 min (Fig. 7B). However, in the presence of 100 mM imidazol dissociations were much faster, with half-lives of 2 and 1.2 min for the His12 and 2xHis6 containing multimers, respectively. It is interesting to note that while the physical dissociation of 2xHis6 tagged molecules from Ni2+ NTA was slower compared to His12 tagged molecules (Fig. 4B), the inverse was true when the complexes were dissociated by addition of free imidazol (Figs. 6, 7).
Preparation, validation and application ofbiotinfree NTA2-PE MHC class I -peptide multimers.
Based on the finding that multimers containing 2xHis6 tagged MHC class I-peptide monomers and streptavidin-PE saturated with biotinylated short NTA2 were sufficiently stable to efficiently stain CD8+ CTL (Figs. 1C, 2-5), we coupled NTA2 directly to PE and produced multimers by loading these with 2xHis6 tagged MHC class I-peptide monomers. To this end PE, which has 24 surface exposed lysine residues, was reacted first with the water soluble maleimide-hydroxy-succinimide ester SM(PEG)2. The resulting maleimide conjugated PE was subsequently reacted with the NTA2-cysteine (see Fig. 3E), yielding stable PE-NTA2 conjugates by thioether formation. (Fig. 8).
To find out what degree of conjugation of PE with NTA2 was needed to obtain efficient NTA2-PE multimer staining, we reacted PE with different concentration of SM(PEG)2. The resulting PE maleimide derivatives were exhaustively alkylated with NTA2-cysteine (Fig. 3E) and the resulting NTA2-PE conjugates loaded with A2/Flu monomers carrying different His tags. The efficiency of all NTA2-PE multimers to stain cloned 81P1 CTL increased with the density of NTA2 groups on PE (Fig. 17). While at low degrees of conjugation the multimers containing the 2xHis6 tag exhibited superior binding, at high degrees the His12 tag containing multimer performed equally well and even the His6 containing multimer lagged only little behind. This is reminiscent to our experiments with Qdots (Fig. 16), but different from the multimer staining with NTA-biotin-streptavidin multimers in which those containing the 2xHis6 tag performed clearly better than those containing the His12 tag or a simple His6 tag (Fig. 5A). We argue that at high densities of NTA groups His tags can cooperatively interact with adjacent NTA moieties, whereas at low densities the binding strengths relies primarily on the interactions between individual His tags and NTA moieties.
We next performed binding isotherms on cloned 81P1 cells comparing conventional
A2/Flu BSP multimers with multimers containing NTA2-PE and His12 or 2xHis6 tagged monomers. At all temperatures tested, both NTA2 multimers exhibited a stable binding plateau above 4 nM multimer concentration. By contrast the binding of the BSP multimer increased over the whole concentration range tested (Fig. 9). This is explained by that NTA multimers are molecularly better defined than BSP multimers. While the NTA multimers consist of one PE conjugated with variable numbers of A2/Flu monomers, conventional multimers contain multiple complexes of different sizes and stoichiometries. This heterogeneity stems from the conjugation of PE with streptavidin (2, 3) and therefore the NTA-biotin- strep tavidin PE multimers are equally heterogeneous, as reflected by their binding isotherms (Fig. 5, Fig. 18). Importantly, because NTA multimers are better defined, they allow more conclusive binding analysis (e.g. Scatchard analysis) than conventional BSP multimers, i.e. can provide more information on given antigen- specific T cells.
So far all binding experiments were performed on Flu-specific CTL clones. Due to clonal variations such data may not be generally representative. We therefore repeated the binding studies on a population of Flu-specific CD8+ T cells derived from peptide stimulated PBMC of a DR4+ healthy donor. As shown in Fig. 18, the 20°C binding isotherms on this polyclonal population exhibited essentially the same picture as the one obtained on clones, namely: i) the binding of NTA4 multimer binding was higher than of NTA2 multimer; ii) multimers containing the 2xHis6 tag bound better than those containing the His12 tag in the case of NTA2, but not NTA4 reagents; iii) the NTA2-PE multimer exhibited clear saturation, whereas all streptavidin-PE containing ones did not; and iv) the NTA4 and NTA2-PE multimers exhibited the highest avidity, i.e. lowest concentrations for half maximal binding.
Finally we performed dissociation kinetics on cloned BCB 70 cells for multimers containing NTA2-PE and 2xHis6 tagged A2/Flu complexes. In the presence of 50 mM imidazol half maximal dissociation was reached at 4°C after about 12 min, at 20°C after 1.8 min and at 37°C after 1 min (Fig. 10). In the presence of 100 mM imidazol the dissociations were very rapid with half-lives below 1 min; especially at 20°C and 37°C the dissociations were too fast to be measured accurately by this flow cytometric analysis.
Reversible MHC class I-peptide complexes allow sorting of antigen-specific CD8+ T cells without inducing activation dependent cell death.
In previous studies we have shown that MHC-Tpeptide multimers induce extensive activation-induced death of CD8+ CTL and that this seriously compromises multimer sorting or cloning of antigen- specific CD8+ T cells (5, 6). To circumvent this, we previously made DTB (des-thio-biotin), a low affinity biotin variant, multimers which dissociate in the presence of free biotin (5). We previously demonstrated that antigen- specific CD8+ T cells cloned or sorted with DTB multimers are superior compared to cells sorted/cloned with conventional BSP multimers in terms of cell viability and functionality. To find out whether NTA built multimers would offer the same advantage, we first compared the dissociation kinetics of A2/Flu DTB multimers, with multimers containing NTA2-PE and 2xHis6 tagged A2/Flu complexes. As shown in Fig. 19 DTB- strep tavidin A2/Flu58-66 multimers in the presence of 2 mM biotin dissociated considerably slower than NTA2-PE-2xHis6 multimers in the presence of 100 mM imidazole. The differences were especially striking at 4°C and 20°C.
To directly examine the usefulness of A2/Flu NTA2-PE 2xHis6 multimers for FACS sorting, cloned BCB 70 cells were stained with this or conventional BSP multimers. FACS sorted cells were washed once with 100 mM imidazol and viable cells enumerated 1, 2 or 3 d afterwards. As shown in Fig. 11 A the percentage of viable cells of NTA2-PE multimer sorted cells was slightly lower compared to untreated cells or cells washed once with cold imidazol. By contrast cells sorted with conventional multimers exhibited only 42% viable cells after Id and merely 20% 3d after sorting. Although these experiments need to be extend by including sorting of polyclonal populations and by functional analysis, our results strongly argue that NTA2-PE 2xHis6 multimers are equivalent or superior to reversible DTB multimers; the beneficial effects of this we have described in detail in a previous study (5). The same conjugation strategy can be equally applied to prepare MHC class II multimers or antibody conjugates, i.e. is universally applicable for the preparation of reversible protein conjugates.
In sum, our results demonstrate that MHC class I-peptide multimers can be prepared based on NTA-His tag chelate complexes that perform equally well or better than
conventional BSP multimers. Importantly, because NTA complexes rapidly dissociate in the presence of imidazol, they allow isolation of bona fide antigen- specific CD8+ T cells by FACS or MACS, which is considerably advantage to BSP multimers. Because NTA built multimers contain neither biotin nor streptavidin, they are simpler and cheaper to prepare. REFERENCES
1. Altman JD, Davis MM. MHC-peptide tetramers to visualize antigen-specific T cells.
Curr Protoc Immunol. 2003;Chapter 17:Unit 17.3. 2. GuiUaume P, Dojcinovic D, Luescher IF. Soluble MHC-peptide complexes: tools for the monitoring of T cell responses in clinical trials and basic research. Cancer Immun. 2009;9:7.
3. GuiUaume P, Legler DF, Boucheron N, Doucey MA, Cerottini JC, Luescher IF. Soluble major histocompatibility complex -peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis. J Biol Chem. 2003 ;278:4500-9.
4. GuiUaume P, Baumgaertner P, Neff L, Rufer N, Wettstein P, Speiser DE, Luescher IF.
2010. Novel soluble HLA-A2/MELAN-A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in patients with melanoma. Int J Cancer 127:910-23.
5. GuiUaume P, Baumgaertner P, Angelov GS, Speiser D, Luescher IF. Fluorescence- activated cell sorting and cloning of bona fide CD 8+ CTL with reversible MHC-peptide and antibody Fab' conjugates. J Immunol. 2006;177:3903-3912.
6. Cebecauer M, GuiUaume P, Hozak P, Mark S, Everett H, Schneider P, Luescher IF.
Soluble MHC-peptide complexes induce rapid death of CD8+ CTL. J Immunol. 2005;
174:6809-19.
7. Knabel M, Franz TJ, Schiemann M, Wulf A, Villmow B, Schmidt B, Bernhard H, Wagner H, Busch DH. Reversible MHC multimer staining for functional isolation of T- cell populations and effective adoptive transfer. Nat Med. 2002; 8:631-7.
8. Neudorfer J, Schmidt B, Huster KM, Anderl F, Schiemann M, Holzapfel G, Schmidt T, Germeroth L, Wagner H, Peschel C, Busch DH, Bernhard H. Reversible HLA multimers (Streptamers) for the isolation of human cytotoxic T lymphocytes functionally active against tumor- and virus-derived antigens. / Immunol Methods. 2007; 320: 119-131.
9. Knecht S, Ricklin D, Eberle AN, Ernst B. Oligohis-tags: mechanisms of binding to ΝΪ2+-ΝΤΑ surfaces. J Mol Recognit. 2009;22:270-9.
10. Cao H, Lin R. Quantitative evaluation of His-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells. Biotechnol Prog. 2009;25:461-7.
11. Khan F, He M, Taussig MJ. Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on ni-nitrilotriacetic acid surfaces. Anal Chem. 2006; 78:3072-9. 12. Steinhauer C, Wingren C, Khan F, He M, Taussig MJ, Borrebaeck CA. Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments. Proteomics. 2006 6:4227-34.
13. Lata S, Reichel A, Brock R, Tampe R, Piehler J. High-affinity adaptors for switchable recognition of histidine-tagged proteins. JAm Chem Soc. 2005;127: 10205-10215.
14. Huang Z, Park JI, Watson DS, Hwang P, Szoka FC Jr. Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications. Bioconjug Chem. 2006;17: 1592-600.
15. Huang Z, Hwang P, Watson DS, Cao L, Szoka FC. Tris-Nitrilotriacetic Acids of Subnanomolar Affinity Toward Hexahistidine Tagged Molecules. Bioconjug Chem.
2009 Aug 3.
All references listed are incorporated herein in their entirety by reference.
EXAMPLE 2
The methods and materials described herein are universally applicable to generate reversible MHC multimers by conjugating a plurality of MHC molecules to a multivalent carrier molecule via a chelate complex bond. While the invention is not limited to specific MHC molecules, Tables 1 and 2 provide exemplary MHC molecules and exemplary antigenic peptides that can be used to produce empty or peptide-loaded reversible MHC molecules using the concepts, methods, and materials provided by aspects of this invention and described in more detail elsewhere herein. Further MHC molecules and antigenic peptides are known in the art and described, for example, in the Tetramer Collection of the Ludwig Institute for Cancer Research (see Guillaume P, Dojcinovic D, Luescher IF. LICR tetramer collection: Soluble tetrameric MHC/peptide complexes to identify and monitor tumor antigen-specific T cells. Cancer Immun 2009; URL:
http://www.cancerimmunity.org/tetramers/; both the publication and the online database are incorporated herein by reference in their entirety for disclosure of useful MHC molecules and antigenic peptides according to aspects of this invention).
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Tyrosinase 9-Jan MLLAVLYCL 132
369-377 YMDGTMSQV 133
Transaldolase 168-176 LLFSFAQAV 134 vaccinia virus
60-68 CLTEYILWV
C16/B22 135
WT1 126-134 RMFPNAPYL 136
235-243 CMTWNQMNL 137
HLA- influenza Ml
27-35 RT DVFAGK
A*0301 138 influenza NP 265-273 ILRGSVAHK 139
HLA- influenza Ml
58-66 GILGFVETL
A*2301 140
HLA- HER2 / neu
63-71 TYLPTNASL
A*2402 141
HIV nef 135-142 RYPLTFGW 142
MAGE-4 143-151 NYKRCFPVI 143
NY-ESO-1 158-166 LLMWITQCF 144
162-170 ITQCFLPVF 145
SAGE 715-723 LYATVIHDI 146
TERT 324-332 VYAETKHFL 147
Tyrosinase 206-214 AFLPWHRLF 148
WT1 235-243 CMTWNQMNL 149
417-425 RWPSCQKKF 150
HLA- influenza Ml
58-66 GILGFVETL
A*3101 151
HLA- gplOO / Pmel 17
182-191 HTMEVTVYHR
A*6801 152
MAGE- 3 115-123 ELVHFLLLK 153
HLA- hCMV pp65
417-426 TPRVTGGGAM
B*0702 154
HIV nef 129-138 TPGPGVRYPL 155
NY-ESO-1 60-72 APRGPHGGAASGL 156
98-109 TPMEAELARRSL 157
98-110 TPMEAELARRSLA 158
HLA- influenza Ml
58-66 GILGFVETL
B*1302 159
HLA- NY-ESO-1
88-96 LEFYLAMPF
B*1801 160
HLA- EBV NA-4
488-496 AVLLHEESM
B*3501 161 gplOO / Pmel 17 630-638 LPHSSSHWL 162 hCMV pp65 123-131 IPSINVHHY 163
HIV Gag-Pol 774-782 NPDIVIYQY 164
Figure imgf000077_0001
Figure imgf000078_0001
Protein Position Peptide :11Ι¾:
"H-2KT™
Figure imgf000078_0002
Figure imgf000079_0001
TABLE 1. Exemplary MHC class I molecules and antigenic peptides useful for the generation of reversible multimers.
Figure imgf000080_0001
TABLE 2. Exemplary MHC class II molecules and antigenic peptides useful for the generation of reversible multimers. Additional MHC molecules useful according to aspects of this invention include, but are not limited to MHC molecules comprising a mutant HLA-A*0201 chain, e.g. a chain comprising a D227K, a T228A, a D227K, a T228A, a T233A, and/or A245V mutation.
Additional MHC molecules useful according to aspects of this invention further include, but are not limited to HLA-A*1101, HLA-A*3001, HLA-A*3004, HLA-B*0801, HLA-B*2705, HLA-B*5101, HLA-Cw*0303, HLA-Cw*0401, HLA-Cw*0602, HLA-Cw*1402, H-2IAd, and H-2IEd molecules.
In some embodiments, chimeric MHC class I multimers are provided, for example, multimers in which the comprised heavy chains are in part human and in part murine.
Further, in some embodiments, peptides comprising modified amino acid residues are provided, for example, ABA, 4-azidobenzoic acid, or Dap, diamino-propionic acid. In some embodiments, peptides are provided that comprise or are conjugated to low molecular weight fluorescent dyes (see, e.g. www.attotech.com/), for example, for flow cytometry analysis.
EXAMPLE 3
Universally applicable methods for the preparation of immunopure MHC Il-peptide staining reagents are provided herein. A method for isolation of MHC II molecules that have stably bound a peptide of interest conjugated to a tag, which, in some embodiments, can subsequently be removed, for example, by cleavage of a linker connecting the tag to the peptide. Further, a method is provided that allows gentle purification of fragile "empty" (without nominal peptide cargo) His tagged MHC II molecules by affinity chromatography on Ni2+ nitrilotriacetic acid (NTA) columns. After isolation of correctly peptide loaded MHC Il-peptide complexes these can be directly converted to multimers by reaction with NTAn conjugated phycobilins (e.g. phycoerythrin) or quantum dots (Qdots).
Abbreviations used in this example include BSP (biotinylation sequence peptide), DR1 (DRB1*0101); DR4 (DRB1*0401); ESO (NY-ESO-1); GFC (gel filtration chromatography); HA (influenza hemagglutinin); LZ (leucine zipper); ΝΡβΑ (3-(2-nitrophenyl)-P-alanine); NTA (nitrilotriacetic acid); PE (phycoerythrin); and pY (phospho-tyrosine).
While MHC I-peptide complexes can be obtained by peptide driven refolding in good yields and high purity, soluble recombinant MHC class II proteins cannot and are typically produced by insect expression systems, e.g. Drosophila S2 cells or baculovirus and sf9 cells (1). Deletion of the transmembrane (TM) domains of the a and β chains results in their dissociation, which is re-established by addition of leucine zippers. In some embodiments, for multimer (e.g., "tetramer") formation, a biotinylation sequence peptide (BSP) sequence is added after the leucine zipper (e.g., after the acidic zipper) and enzymatic biotinylation and tetramerization is performed as for MHC I-peptide multimers (1-5). In some embodiments, "empty" (without nominal peptide cargo) MHC II molecules are isolated from culture supernatants by immunoaffinity chromatography and subsequently loaded with a peptide of interest. The efficiency of peptide loading depends on its binding strength to the MHC II molecule; if it is below a threshold, peptide loading is inefficient and the resulting complexes of limited stability. If this strategy is not feasible, peptides can be tethered to the N-terminus of the β chain via a flexible linker (6). This strategy works for some, but not all, complexes. Also, although the peptide is part of the molecule, in the case of weak binding peptides there is no knowing whether or not it is correctly bound in the peptide binding groove.
The staining of antigen- specific CD4+ T cells often is weak and the frequency of stained cells ex vivo very low, usually necessitating prior in vitro peptide stimulation to permit conclusive detection. There are significant differences in multimer staining of CD8+ and CD4+ T cells, such as 1) the staining with MHC II multimers is usually higher at 37°C than at lower temperatures, which is not the case for MHC I multimers (2, 3, 7); 2) Efficient CD4+ T cell staining requires longer incubation periods, which is explained, at least in part, by accumulation of MHC II multimers by endocytosis; therefore agents that affect cell vitality and cyto skeleton function inhibit CD4+ T cell staining (7, 8). 3) The avidity of MHC II multimer binding is usually lower than the binding of MHC I multimers, mainly because CD8 greatly strengthens MHC-peptide binding to CD8+ T cells, whereas CD4 does not (9, 10). MHC II multimer staining therefore typically requires higher concentrations (up to 100 nM, i.e. about 50 μ^ηύ) (2-5). While MHC I-peptide complexes obtained by refolding are highly pure and conformationally uniform, MHC II-peptide complexes obtained by peptide loading of "empty" MHC II proteins or containing tethered-on peptides often are not, which can seriously impair MHC II multimer staining. The commonly used purification of MHC II molecules by immuno-affinity chromatography is not only tedious and expensive, but prone to yield protein denaturation. This decreases the fraction of MHC II molecules that can be loaded with a given peptide and thus the active reagent fraction of multimers prepared with these monomers (Fig.26). We have previously demonstrated that the quality of MHC II multimers can be dramatically increased when these are produced with molecularly defined monomers, i.e. monomers that contain only the peptide of interest (2, 3). In these studies added a histidine tag (His6) N-terminally of the peptide of interest and after peptide loading isolated correctly loaded complexes by affinity chromatography on Ni2+ NTA (nitrilotriacetic acid) columns. While this method is efficient, it has the disadvantage that the peptides are modified with a His tag, which in some cases may affect their binding to MHC molecules and/or their T cell recognition.
Here we report that molecularly defined MHC II-peptide complexes can be isolated by anion exchange chromatography when adding an acidic tag on the peptide of interest.
After purification this tag can be removed by means of a photo-cleavable linker. This allowed the use of His tagged MHC II proteins which offered two important advantages: 1) the protein can be purified by gentle affinity purification on Ni2+-NTA columns, thereby significantly reducing denaturation of fragile "empty" MHC II molecules. 2) After peptide loading and isolation of immunopure, bona fide (i.e. without peptide tag) MHC II-peptide monomers can be directly converted in multimers. In addition we report that enzymatic de- sialylation of cells increases their MHC II multimer staining by several-fold, which is a simple means to obtain better staining results. Peptide synthesis
Protected amino acids and tentagel resin for solid phase peptide synthesis were obtained from Rapp-polymere (Tubingen, Germany and Bachem AG, Bubendorf,
Switzerland). The coupling reagents TBTU and HOBt were purchased from Multisynthec (Witten, Germany), Cy5.5-maleimide from GE Healthcare and Fmoc-3-amino-(2- nitrophenyl) -propionic acid from Peptech Corporation (USA). Reverse phase HPLC analyses were performed on a Waters system consisting of two Waters 515 pumps and a Waters 996 photodiode array detector. The purity of all peptides was assessed by analytical HPLC.
Analytical HPLC columns (Uptisphere 5 μιη C18 particles, 250 x 4.6 mm) was eluted at flow of 1 ml/min, and semi preparative HPLC columns (Kromasil 15 μιη C18 particles, 250 x 20 mm) at 3 ml/min, with a linear gradient of acetonitril rising in lh from 0 to 75% on 0.1% TFA in H20. The mass of purified peptides was measured by mass spectrometry (MS) on a MALDI-TOF mass spectrometer. Peptide syntheses were performed using 2 equiv of N-CC-Fmoc-protected amino acid relative to the resin loading, activated in situ with 2 equiv of TBTU, 2 equiv of HOBt and 4 equiv of DIEA in DMF (10 ml/g resin) for 1 h. Coupling completion was verified by the Kaiser test. N-CC-Fmoc protecting groups were removed by treatment with a piperidine/DMF solution (1:4) (10 ml/g resin) for 5 min. Crude peptides were obtained by treating the resin with a solution of TFA/H20/TIPS (92.5/2.5/5) for 3 h at ambient temperature. Peptides were precipitated with addition of anhydrous ether, filtered off, dissolved in water, lyophilized and purified by reverse phase HPLC. For the synthesis of Cy5.5 labelled peptides, precursors peptide containing a free thiol were dissolved in DMSO at 10 mM and reacted with one equivalent of Cy5.5-maleimide. After 2h of stirring the mixture was directly subjected to by semi-preparative HPLC.
Photocleavage under UV irradiation
Peptides containing the photocleavable amino-acid ΝΡβΑ in water (50 μΜ) or MHC II-peptide complexes in PBS (2-20 μΜ) and irradiated in open 96 well plates with a UV lamp (Vilber Luormat) containing two 15 W mercury fluorescent tubes emitting at 365 +/- 40 nm at a distance of 10 cm. Samples irradiated for different periods of time were directly analyzed by HPLC and mass spectroscopy.
MHC II-peptide complexes
Extracellular coding parts of DR alpha and beta chains were PCR amplified using ctttagatctcgaccacgtttcttggagc (SEQ ID NO: 297) as the 5' primer and
ctttgaattccttgctctgtgcagattcag (SEQ ID NO: 298) as the 3' primer from cDNA preparations (Qiagen) of total RNA extracted from human PBMCs, digested with appropriate restriction enzymes and cloned in pMT A BiP/V5/His vector-derived cassette (Invitrogen) containing sequences for appropriate leucine zippers and the AviTag (Avidity). The AviTag
(GLNDIFEAQKIEWHE, SEQ ID NO: 299) was encoded in an oligo (CTTT CTG GAT ATC TCA TTC GTG CCA TTC GAT TTT CTG AGC CTC GAA GAT GTC GTT CAG ACC GCC ACC, , SEQ ID NO: 300) used to extend the basic leucine zipper sequence
(TTAPSAQLKKKLQALKKKNAQLKWKLQALKKKLAQ, SEQ ID NO: 301) separated by a flexible linker (GGGSGGS, SEQ ID NO: 302). Oligos for introduction of a single His6 (CTTTGATATCTCAATGATGGTGATGATGGTGGCCGGTGCGCTGAGCCAGTTCCTT TTCC, SEQ ID NO: 303) or a double His6
(CTTTGATATCTCAGTGGTGGTGGTGGTGGTGGCTGCCGCTGCCGCCGCCGCTGCC GCCGCCATGATGGTGATGATGGTGGCCGGTGCG, SEQ ID NO: 304) were designed to follow the acidic leucine zipper (TTAPSAQLEKELQALEKENAQLEWELQALEKELAQ, SEQ ID NO: 305) on the DRa chain separated by a flexible linker mentioned above. All constructs were verified by sequencing.
Soluble "empty" DR1 (DRA, DRB1*0101), DR4 (DRA, DRB1*0401) and DR52b
(DRA, DRB3*0202) molecules were produced in Drosophila melanogaster D. Mel-2 cells (a serum-free medium adapted variant of S2 cells) grown in Sf900 II serum-free medium (Invitrogen) at room temperature (22-26 °C). Cells were simultaneously transfected with three plasmids (the plasmid for DRA, DRB and pBS-PURO, a plasmid conferring puromycin resistance (a gift from K. Karjalainen, Nanyang Technological University, School of
Biological Sciences) using Cellfectin (Invitrogen), Singapore). For DR1 and DR4, a population of transfected cells was used, whereas for DR52b high-yielding clones were obtained by limiting dilution. The cells were grown in roller bottles (BD Falcon) rotating at 6 rev/min at room temperature to 5-10*106/ml and protein production was induced by addition of 1 mM CuS04 for 3-5 days. The yields of purified protein were 2-5 mg per liter of medium.
For immuno-affinity purification a column was used containing Sepharose 4B coupled with L243 (anti-DRalpha) antibody. Soluble "empty" DRs were eluted with 50 mM glycine/HCl buffer pH 11.5 and the eluate was immediately brought to pH 8.0 by addition of 2 M Tris HCl pH 6.8. For peptide loading soluble "empty" molecules were brought to pH 5.5 (DR1 and DR4) or pH 6.0 (DR52b) by addition of 100 mM citric acid and incubated with a peptide of interest (1-5 μΜ final concentration) for 24 h at 28 °C (DR52b) or 37 °C (DR1, DR4) in the presence of 0.2% octyl β-D-glucopyranoside (Sigma), protease and phosphatase inhibitor cocktails (Roche). In some cases peptide loaded DR molecules were biotinylated with recombinant BirA biotin transferase (Avidity) according to the supplier' s
recommendations. Complexes were purified by GFC on a Superdex S200 column. (GE
Healthcare Life Sciences). In the case of MHC II-peptide complexes containing His tagged peptides (e.g. DR4 peptide-NPpA-SGSGHHHHHH, SEQ ID NO: 306), samples were passes through a HisTrap HP column (GE Healthcare Life Sciences), which after washing was eluted with 200 mM imidazole, which subsequently was removed by GFC in PBS on a Superdex S200 column (GE Healthcare Life Sciences) or by ultrafiltration. In the case of complexes containing Cy5.5 or pY-D4 tagged peptides samples were dialyzed in 5 mM Tris- HC1 pH 9.0, 50 mM NaCl and loaded on a Mono Q 5/50 GL column (GE Healthcare Life Sciences), which was eluted with a NaCl gradient rising win 30 min from 0 to 0.5 M NaCl on 20 mM Tris, pH 9.0 at a flow rate of 1 ml and collecting Purified MHC II-peptide complexes were concentrated on an Amicon Ultra-4 filter concentrator, 10,000 MWCO (Millipore) to 1- 2 mg/ml as assessed by the Bradford protein assay (Bio-Rad). Degree of biotinylation and purity were assessed by the avidin shift assay and was routinely >90%. Briefly, two different amounts of biotinylated concentrated complexes (typically 2 and 5 μg) are mixed or not with 10 μg of avidin (Pierce) and run on 12% SDS-PAGE (non-boiling, non-reducing). After staining in Gelcode blue (Pierce), gels are scanned and quantified using the ImageQuant TL software (GE Healthcare Life Sciences).
ELISA assays
For monitoring of Cy5.5 tagged peptides 10 μΐ aliquots of fractions were
supplemented (10 x) with blocking buffer (PBS, 1% BSA, 0.5% Tween 20) and loaded in 1:2 serial dilutions (100 μΐ/well) in blocking buffer into two wells of 96 well plates (MaxiSorp, Nunc), one of which was coated with anti-DR antibody L243, and the other with anti-Cy5.5 antibody (Sigma). After washing bound complexes were revealed with a biotinylated anti-
BSP antibody (a gift from Dr. Gennaro DiLibero, Basel University Hospital) and a secondary antibody conjugated with streptavidin-alkaline phosphatase (Sigma). The plates were developed with p-nitrophenly-phosphate (pNPP) SigmaFAST substrate (Sigma) and absorbance read at 405 nm on an ELISA plate reader. The concentration of His6 tag was assessed likewise, i.e. biotinylated MHC II-peptide complexes were trapped on streptavidin- coated plates (Maxisorp, Nunc) and His tag detected by means of Ni-NTA alkaline phosphatase (His-Detector, KPL).
Peptide competition assay
For each test peptide eight wells of a 96-well plate (Maxisorp, Nunc) were filled with 50 ul a citrate saline buffer (50 mM citrate, 200 mM NaCl, pH 5.5) containing 1 μg recombinant empty DR1 protein (1 μg), 0.2% octyl-glucoside, complete protease inhibitors (Roche) and 0.2 μΜ biotin-HA306-3is peptide. Competitor peptides were added to final concentrations of 1000, 300, 100, 10, 3, 1, 0.3 and 0.1 μΜ. After overnight incubation at 37°C the samples were diluted 4-fold with PBS supplemented with 0.1% BSA and 0.05% Tween 20 and 100 μΐ applied into 96 well plates previously coated with L243 antibody (2 μg/ml); after 1 h of incubation at room temperature the plates were washed, incubated with streptavidin-alkaline phosphatase (Sigma) (1: 10,000); after 1 h the plates were washed and developed with pNPP SigmaFAST substrate (Sigma) and absorbance read at 405 nm.
Cells under study
DR4-restricted, HA306-3 is- specific CD4+ T cell clones were obtained by limiting dilution cloning of a DR4 HA306-318 multimer-sorted population of a CD4+ T cell line generated from PBMC of a healthy donor (HD137) that was propagated by stimulation with 2 μΜ HA306-3i8 peptide and γ-irradiated CD4" PBMC in RPMI-1640, 10% human serum AB, supplemented with 100 U/ml of hIL-2. The clones were propagated by phyto-hemagglutinin (PHA) (Oxoid) stimulations every 2-3 weeks. DR1 -restricted, ESO-specific CD4+ T cell lines were derived and maintained as described previously (3). ESO-specific CD4+ cells from A2/DR1 mice were obtained as follows: groups of mice (n= 4-5) were immunized by injections (s.c. at the base of the tail) of 50 μΐ of emulsion containing the indicated peptides (50 μg of peptide emulsified in 50 μΐ PBS and 50 μΐ complete Freund's adjuvant (DIFCO); 7- 8 days later the draining lymph nodes were removed and homogenized by passing through a cell strainer (BD Falcon) to obtain single cell suspensions for further analysis.
Tetramers and staining
Biotinylated DR-peptide complexes were multimerized by mixing with small aliquots of streptavidin-PE (Invitrogen) up to the calculated 4: 1 stoichiometric equivalent. Cells were stained in 50 μΐ of FACS buffer (PBS, 0.5% BSA, 2 mM EDTA, 0.05% sodium azide) for 1 hour at 37°C. In the case of neuraminidase treatment, cells were incubated with 0.03 U/ml of neuraminidase from V. cholerae (Roche) in complete medium and washed twice prior to tetramer staining. In some experiments fluorescent antibodies (e.g. anti-CD4) were added after multimer staining and incubated at 0-4°C for 15 min. After 2 washes the cells were suspended in 300 μΐ buffer and analyzed by flow cytometry on a FACSCalibur (BD).
Propidium iodide (Invitrogen) was added just before acquisition for exclusion of dead cells. Human cells were fixed in 2% formaldehyde (Polysciences) in FACS buffer analysis. Data analysis was performed with FlowJo 7.6 software (TreeStar).
Preparation and evaluation of immunopure biotin-streptaxidin multimer s
In order to produce immunopure MHC II-peptide complexes, we used a His6 tag linked via a short flexible GSG linker (H6-GSG-) to the N-terminus of the peptide of interest, which allowed isolation of pure complexes by affinity chromatography on Ni2+ NTA columns (2, 3). Although in these studies the added His6 tag had only modest effects on T cell recognition and/or MHC binding, this may not be the case in other applications. In order to avoid this, we introduced 3-(2-nitrophenyl)-P-alanine (ΝΡβΑ) between the H6-GSG moiety and the peptide of interest (Fig. 20). This tag (H6-GSG-NPpA) can be added N- or C- terminally to the peptide of interest (Fig. 21). Upon UV irradiation at 365 nm the ΝΡβΑ residue is cleft, resulting in the removal of the His tag after its use for affinity purification of MHC II-peptide complexes (Fig. 21 A). ΝΡβΑ has been used for the preparation of conditional MHC I-peptide ligands and its usefulness is well documented (11, 12). As shown for immunopure DR4/HA 06- i8 complexes carrying the His tag either N-terminally (His6- GSG- ΝΡβΑ-ΗΑ306-3ΐ8 or C-terminally (HA306-3i8 NPpA-GSG-H6) the tag is cleft rapidly upon UV irradiation at 365 +/-40 nm, with 50% cleavage achieved after 0.51 and 0.57 min, respectively (Figs. 21B, C). It should be noted that this cleavage is only about 90% complete; higher cleavage yields can be achieved by using two ΝΡβΑ groups (12).
When the His tag is added C-terminally at the peptide, it emerges after photo-cleavage as an amide, i.e. its C-terminal carboxyl group is an amide (Fig. 21A). Conversely when the tag is added N-terminally the peptide carries after photolysis an N-terminal 2-nitroso- phenacetyl-P-acetoyl moiety (Fig. 21A). To compare the staining of immunopure DR4-HA tetramer prepared by using either the N- or C-terminal His tag with and without photo- cleavage, we performed 37°C binding isotherms on four DR4-restricted, HA-specific Thl clones. On all four clones the binding of immunopure multimers was considerably more avid as compared to the conventional DR4/HA multimer (Figs. 22A-D). The differences were particularly striking on the low affinity clones 15 and 8. The multimer containing the N- terminally tagged HA peptide (His6-GSG-NPpA-HA3o6-3is) exhibited the most efficient binding on all clones tested. After photolytic removal of the His tag the staining efficiency decreased on clone 2, less on clones 9 and 15, but remained unchanged on clone 8.
Conversely, multimers containing the C-terminally tagged HA peptide (ΗΑ3ο6-3ΐ8-ΝΡβΑ- GSG-¾) exhibited modestly increased (clone 2) or the same binding (clones 8, 9, 15) compared to the multimers in which the tag was removed by photolysis. Importantly, after UV irradiation mediated removal of the tag both multimers (i.e. those carrying His6-GSG- ΝΡβΑ-ΗΑ3ο6-3ΐ8 and HA3o6-3is NPpA-GSG-¾) stained all four clones very similar, arguing that the small modifications of the peptide by the photo-cleavage little affect multimer staining (Figs. 21 A, 22A-D). Taken collectively these results demonstrate that purification of correctly loaded MHC Il-peptide monomers very significantly increases multimer binding. As we have shown previously this is critical for the detection of antigen- specific CD4+ T cells directly ex vivo (2, 3). Moreover, our results indicate that the presence of an N-terminal His6 tag can artificially increase multimer staining (Fig. 22). This is explained, at least in part, by increased peptide binding to the restricting MHC II molecule (Fig. 27 and refs. 2, 3). This we observed for the N-terminally, but not (or barely) for the C-terminally His tagged peptide (Fig. 22). This observation is consistent with reports showing that adding tags N-terminal to MJC II binding peptides such as invariant chain derived KEY tags (13, 14) or photoreactive groups (15) can considerably increase their binding. In newly synthesized MHC II molecules the peptide binding site is occupied by the CLIP sequence of the invariant chain (Ii), which on its way to the plasma membrane "clings" to invariant regions, mainly of the β2 domain, situated in front of the N-terminal portion of the peptide binding groove (16). One might argue that thus increased peptide binding could be a means to increase multimer staining. This, however, is risky as this engagement of this invariant region on MHC II proteins may provoke extended conformational changes, which may alter the fidelity of the multimer staining (17). We therefore strongly advocate the use of photo-cleavable tags, which can be removed after purification of correctly loaded MHC Il-peptide monomers.
Preparation of immunopure DR4-HA306-3i8 monomers using Cy5.5 tagged peptides. A major shortcoming of conventional MHC II multimer synthesis is that one does not know what fraction of the MHC II molecules have bound the peptide of interest. This fraction can be highly variable, depending on the MHC II molecule, its purification and on the peptide and loading procedures. Because the effective multimer fraction decreases with the fraction of correctly peptide loaded monomers (Fig. 26) a frequent, probably the most frequent failure of poor or undetectable MHC II multimer staining is inadequate peptide loading. In order to able to directly monitor MHC II peptide loading, we used Cy5.5 as tag for labelling antigenic peptides. Cy5.5 (and Cy5) is a blue fluorescent low molecular dye that can be coupled to peptides via amides or thioethers (Fig. 23A, www.gelifesciences.com). To establish a proof a principle, we loaded DR4 with Cy5.5 labelled HA306-318 peptide (HA306-3i8-GSGC-Cy5.5, SEQ ID NO: 307) and analyzed the reaction mixture by GCF on Superdex S200 column recording the OD of the eluent at 675 nm (Cy5.5) and 280 nm
(protein) (Fig. 23B). From the integrated peak surfaces and the molar extinction coefficients, the ratio of Cy5.5 (i.e. peptide) and protein (i.e. DR4) was calculated to be 0.42, i.e. 42% of the input DR4 molecules had bound the blue peptide. Analogous experiments were performed with DR52b and Cy5.5 labelled NY-ESO-1 peptide 123-137, in which case peptide loading was remarkably poor (< 10%) and no significant staining was observed with the corresponding multimers (data not shown and ref. 2 ).
As for His6 tagged peptides, the content of Cy5.5 (or Cy5) labelled peptide in MC II- peptide complexes can also be detected by ELISA, using Cy dye specific antibodies (Fig. 23C). Moreover and importantly Cy5.5 contains four negatively charged sulfonyl groups (S03 ), i.e. is strongly acidic. This allows quantitative separation of the Cy5.5-peptide loaded DR4 molecules from other DR4 molecules by anion exchange chromatography (Fig. 23D). Although this method allows direct assessment of peptide loading efficiency and isolation of immunopure MHC II-peptide complexes, it has disadvantages, such as i) the synthesis of Cy5.5 labelled peptides is expensive and tedious and ii) the removal of Cy5.5 by photolysis by means of a ΝΡβΑ linker is not feasible due to quenching.
Preparation of immunopure MHC II-peptide NTA multimers using the pY-D4-tag
Isolation of "empty" MHC II molecules from culture supernatants by immuno- affinity chromatography is not only costly and often inefficient, but is a major cause for extensive protein denaturation. In order to obtain efficient MHC protein recovery, high affinity antibodies are used, which for elution require the use of extreme pH (e.g. 11.5) at which empty MHC II molecules start to denature. To circumvent this we added at the DRA chain C-terminal of the leucine zipper a His tag, which allows gently and universal purification of empty MHC II molecules by affinity chromatography on commercially available Ni2+ NTA columns (Fig. 24). This necessitated the use of another tag to be used for the purification of correctly peptide loaded MHC IT peptide complexes. Encouraged by the finding that a negatively charged tag allows purification of correctly loaded MHC complexes, we searched for a related strategy, namely for a negatively charged tag that can be readily synthesized and then be removed by photolysis. As new tag we used phospho-tyrosine-Asp4 (pY-D4), which can be detected by anti-pY antibodies in ELISA and having six negative charges is expected to allow separation of loaded MHC II molecules by anion exchange chromatography (Fig. 24). Moreover and importantly, after peptide loading, purification of correctly peptide loaded complexes and removal of the pY-D4 tag by photolysis, the His tag can be used again to directly form fluorescent staining reagents by reaction with NTAn-PE or NTAn-Qdots (as described for MHC I-peptide complexes in the first technical report).
To validate the feasibility of this strategy, we passed a supernatant from S2 cells secreting soluble His6 tagged DR4 over a Ni2+-NTA column. After washing the column, it was eluted wit 200 mM imidazole, whereby DR4 eluted in a sharp peak, which according to GFC on a Superdex S200 column and SDS-PAGE was pure (Figs. 25A-C). This material was then loaded with pY-D4-GSG-NPpA-HA 06-3i8 peptide and subjected to anion exchange chromatography on Mono-Q column, which was eluted with the indicated NaCl gradient and on the OD of the eluate was monitored at 280 nm and the content of DR4 and pY, respectively, of the fractions determined by ELISA (Fig. 25D). This analysis showed that
DR4 containing the acidic pY-D4-GSG-NPpA-HA306-3i8 peptide eluted after DR4 molecules carrying no or different peptides, similarly as observed for the Cy5.5 tagged HA peptide (Fig. 23D). Because phospho-tyrosine a priori is susceptible to enzymatic de-phosphorylation (although with complete phosphates inhibitors, e.g. from Roche, this can be prevented) we also tested para-sulfate-tyrosine (e.g. Y(S04)-D4) as tag, but observed lesser shifts of the acidic peptide containing DR4 complexes (data not shown). In order to further to extend and optimize the separation of correctly loaded MHC II-peptide complexes we have evaluated variations in the conditions of the anion exchange chromatography (e.g. different buffers and NaCl gradients) as well other acidic tags (pY-E4_8) as well as other MHC II molecules (e.g. DR1 and DR52b) and other peptides (e.g. NY-ESO-l123- 137) . From these experiments it is apparent that correctly loaded MHC II peptide complexes can be separated from other MHC II molecules by anion exchange chromatography; however for general usage the best suitable acidic tags and chromatography conditions may differ somewhat from those described here.
Over a dozen of different reagents were investigated for their ability to increase MHC II multimer staining. It was determined that brief pre-treatment of the cells with
neuraminidase increases multimer binding by 2-5-fold. This was observed on a range of DR4-restricted, HA-specific CD4+ T cell clones (Fig. 28) and on populations of HA peptide stimulated PBMC from healthy donors. Because MHC II multimer staining tends to be weak, this observation suggests pre-treatment of the cells with neuraminidase, an enzyme that removes sialic acid residues on cellular surface proteins (18) increases MHC class II multimer staining efficiency. REFERENCES
1. Guillaume P, Dojcinovic D, Luescher IF. Soluble MHC-peptide complexes: tools for the monitoring of T cell responses in clinical trials and basic research. Cancer Immun.
2009;9:7.
2. Ayyoub M, Dojcinovic D, Pignon P, Raimbaud I, Schmidt J, Luescher I, Valmori D.
Monitoring of NY-ESO-1 specific CD4+ T cells using molecularly defined MHC class II/His-tag-peptide tetramers. Proc Natl Acad Sci U S A. 2010;107:7437-42.
3. Maha Ayyoub, Pascale Pignon, Danijel Dojcinovic, Isabelle Raimbaud, Lloyd J. Old, Immanuel Luescher and Danila Valmori 2010. Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor- specific antigen NY-ESO-1 using DRB1*0101 tetramers. Clin. Cancer Res. accepted.
4. Danke NA, Koelle DM, Yee C, Beheray S, Kwok WW. Autoreactive T cells in healthy individuals. J Immunol. 2004;172:5967-72.
5. Cecconi V, Moro M, Del Mare S, Dellabona P, Casorati G. Use of MHC class II tetramers to investigate CD4+ T cell responses: problems and solutions. Cytometry A.
2008;73: 1010-8.
6. Cunliffe SL, Wyer JR, Sutton JK, Lucas M, Harcourt G, Klenerman P, McMichael AJ, Kelleher AD. Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability, and allows identification of antigen- specific CD4+T cells in peripheral blood mononuclear cells. Eur J Immunol. 2002;32:3366- 75.
7. Cameron TO, Cochran JR, Yassine-Diab B, Sekaly RP, Stern LJ. Cutting edge:
detection of antigen-specific CD4+ T cells by HLA-DRl oligomers is dependent on the T cell activation state. J Immunol. 2001: 166:741-5.
8. Wooldridge L, Lissina A, Cole DK, van den Berg HA, Price DA, Sewell AK. Tricks with tetramers: how to get the most from multimeric peptide-MHC. Immunology
2009;126: 147-64.
9. Luescher IF, Vivier E, Layer A, Mahiou J, Godeau F, Malissen B, Romero P. CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes. Nature 1995;373:353-6.
10. Hampl J, Chien YH, Davis MM. CD4 augments the response of a T cell to agonist but not to antagonist ligands. Immunity 1997;7:379-85. 11. Toebes M, Coccoris M, Bins A, Rodenko B, Gomez R, Nieuwkoop NJ, van de Kasteele W, Rimmelzwaan GF, Haanen JB, Ovaa H, Schumacher TN. Design and use of conditional MHC class I ligands. Nat Med. 2006;12:246-51.
12. Celie PH, Toebes M, Rodenko B, Ovaa H, Perrakis A, Schumacher TN. UV-induced ligand exchange in MHC class I protein crystals. J Am Chem Soc. 2009 ; 131: 12298-304.
13. Kallinteris NL, Lu X, Blackwell CE, von Hofe E, Humphreys RE, Xu M. Ii- Key/MHC class II epitope hybrids: a strategy that enhances MHC class II epitope loading to create more potent peptide vaccines. Expert Opin Biol Ther. 2006 6: 1311-21.
14. Kallinteris NL, Lu X, Wu S, Hu H, Li Y, Gulfo JV, Humphreys RE, Xu M. Ii- Key/MHC class II epitope hybrid peptide vaccines for HIV. Vaccine. 2003 21 :4128-32.
15. Luescher IF, Allen PM, Unanue ER. Binding of photoreactive lysozyme peptides to murine histocompatibility class II molecules. Proc Natl Acad Sci U S A. 1988 85:871-4.
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17. Rotzschke O, Lau JM, Hofstatter M, Falk K, Strominger JL. A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules. Proc Natl Acad Sci U SA. 2002;99: 16946-50.
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The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference for the purposes or subject matter referenced herein. In case of a conflict between a reference incorporated herein and the instant disclosure, the teaching of the instant disclosure shall control.
EXAMPLE 4
PE-NTA-A2/peptide multimers - a new type of staining reagent based on oxime ligation.
To generate PE-NTA multimers, 1 nmol Phycoerythrine (PE) was first reacted in phosphate buffer, pH 7.4, with 20 mM of sulfo-SFB (sulfo-succinimidyl-formylbenzoate) for 4 h at room temperature. The resulting activated-PE was subsequently dialyzed in 2 L PBS over 2 days and then coupled in phosphate buffer (pH 7.2) with a large excess of H2N-0- NTA2 or H2N-0-NTA4 aminooxy-containing peptide. After overnight reaction at 4 °C, bioconjugates were loaded with Ni and dialyzed in 2 L PBS over 2 days. PE-NO-NTA2 or PE-NO-NTA4 obtained were conjugated with A2/peptide-2xHis6 complexes to generate biotin SA free PE-NTA2 or PE-NTA4 multimers. Staining is a function ofPE substitution degree.
The staining characteristics of the multimers generated via oxime chemistry were investigated. Figure 29A illustrates a scheme of PE activation with sulfo-SFB and conjugation with H2N-0-NTA2 via oxime ligation. Figure 29B: PE (1 nM) was activated with the indicated concentrations of sulfo-SFB and conjugated with an excess of NTA peptide. 0.5 μg of PE-NO-NTA2 were mixed with 5 μg of A2/Flu58-66-2xHis6 monomers. Flu matrix 58-66-specific BC74cells were stained with 8 nM of the different conjugates at room temperature for 30 min and analyzed by flow cytometry. SFB: succinimidyl-p- formylbenzoate.
Figure 30 shows staining isotherms on Flu stimulated PBMC. Figure 30A: PE activated with 1, 10 or 20 mM sulfo-SFB was coupled to NTA2 peptide via oxime ligation and subsequently mixed with A2/Flu-2xHis6 A2/Flu58-66 monomers. Flu matrix peptide stimulated PBMC were stained with different concentrations of the conjugates and analyzed by flow cytometry. The numbers indicate MFI of specific (above the bar in each graph) and non-specific (below the bar) staining. The right hand numbers indicate the input amounts of monomers (upper number) and PE-NTA2 (lower number). Figure 30B: Data plotted using GraphPad Prism software.
Figure 31 shows exemplary NTA linkers that were synthesized and tested. The bioNTA4 and oxNTA2 linkers were determined to be of particular interest for the generation of MHC I and MHC II multimers and were used for further testing as described below. The bioNTA2 and bioNTA4 compounds contain biotin- NT A2 or4, all others contain N terminal Cys(SH)-PEG2 on NTA2 or NTA4 to be used for conjugation with maleimido-PE. The non- biotin compounds contain N-terminal imines and NTA2 linked via a PEG2 Spacer, a PEG4 spacer, or a dipeptide linker (bottom).
PE conjugates with these linkers were obtained and the performance of these conjugates was tested. The conjugates comprised either A2/Flu58-66 monomers, which were tested in staining of Flu peptide stimulated PBMC, or comprised DR4/Flu HA306-318 monomers, which were tested in staining of Flu-Specific CD4+ T cells (Figure 32). From these initial tests, the biotin-NTA4 and SA-PE multimers appear to perform similar to conventional BSP multimers, but result in better stainings of both CD8+ and CD4+ T cells. In addition, the multimers described herein are fully reversible, as explained in more detail elsewhere herein, which allows sorting of antigen-specific T cells without causing activation dependent T cell death. The data presented here indicates that the PEG2-NTA2-comprising PE-NTAmers coupled to PE via oxime bond formation exhibit an improved staining performance as compared to multimers obtained by conjugation via maleimides. This may in par be due to an increased degree of conjugation associated with the use of oxime-chemistry. Comparison of conventional, PE-Cys-PEG2-NTA2 and PE-HNO-NTA2 multimers staining.
Figure 33A shows the results of an experiment in which cloned, Flu matrix 58-66- specific CD8+ 81P1 cells were incubated at 20°C for 30 min with graded concentration of conventional multimers (circles), PE-Cys-PEG2-NTA2 NTAmers (squares ), or PE-HNO- NTA2 NTAmers (triangles). Cell-associated PE fluorescence was then assessed by flow cytometry. The binding data of Figure 33A were subjected to Scatchard analysis (Figure 33 B), and KD and Bmax were calculated from the results of the Scatchard Analysis (Figure 33C).
Reducing of background staining by milk supplements.
It was found that the background staining observed when using multimers generated via oxime chemistry can be reduced by using a protein supplement, for example, milk protein, during the staining procedure (Figure 34). Cloned, A2/Flu matrix58-66 specific 81P1 cells were incubated with a A2/Melan-A -specific clone at 20°C for 30 min with the indicated concentrations of conventional (column 1) or PE-HNO-NTA2 containing multimers (columns 2, 3, 4, 5 and 6) in the absence (row 1) or presence of the indicated concentrations of dried milk powder (rows 2-4). Milk powder efficiently decreased the background staining of oxime-chemistry multimers.
Binding titration of NTAmers to determine best dilution for ex vivo staining.
Cloned, Melan A LAU 959 46 (Figure 35 A) or EBV LAU 1013 specific cloned cells
(Figure 35B) were mixed with fresh PBMC and incubated at 20°C for 30 min with graded dilution of PE-NTA2 NTAmer (row 2), SA-PE multimers containing SA-PE biotin-NTA2 (row 3) or SA-PE biotin-NTA4 (row 4).
Ex vivo staining with BSP and NTA multimers on fresh PBMC was also compared. Figure 36 shows ex vivo flow cytometric analysis of PBMC stained with A2/peptide multimers containing BSP (conventional), PE-oxime NTA2, or SA-PE biotin-NTA4. PBMCs were obtained from EBV+ (BCL 7) individuals (Figure 36A), CMV+ (BCL 8) individuals (Figure 36B), and from two melanoma patients: LAU 616 (Figure 36C) and LAU 1164 (Figure 36D).
EXAMPLE 5
Use of desthiobiotin for purification of correctly peptide loaded MHC Il-peptide complexes
In some embodiments, multimeric MHC II staining reagents described herein are generated by loading monomeric MHC molecules with a peptide of interest and purifying correctly peptide-loaded monomers for further processing and assembly to multimers. For example, in some embodiments, a hexahistidine (His6) tag is added N-terminally at the peptide and the complexes are purified on Ni2+ NTA columns. In some embodiments, an N- terminal polyacidic tag is used, which allows purification of complexes by anion exchange chromatography. In some embodiments, a desthiobiotin (DTB) tag is N-terminally added to the peptide using conventional solid phase peptide synthesis (SPPS) and the target complexes are purified on streptactin columns using elution with free DTB. The column can be completely regenerated by washing with 2-(4'-hydroxyazobenzene) benzoic acid (HABA) and the Tris, pH 9.0. The recovery yields are close to 100%, and pMHC II complexes fully active.
The use of DTB is less complex than using a poly-acidic tag and anion exchange chromatography, and has the advantage that a His tag can be used on the MHC II protein. In some embodiments, this allows i) gentle and universal purification of MHC II proteins from supernatants and ii) diverse conjugate formation based on the His tag- NTA chelate complex conjugation strategies described herein. An overview of an exemplary strategy for NTA-His tag multimer preparation using DTB is outlined in Figure 37. Protein expression in Drosophila cells
Serum-free adapted Drosophila melanogaster cell line (D.mel-2) were transfected concomitantly with two pMT-derived plasmids containing cloned extracellular parts of the a and beta chain of the MHC class II allele, followed by leucine zipper sequences and terminating with a Avi-Tag (beta chain) or a tandem His-tag (a chain). A pBS-derived plasmid containing the puromycin resistance gene is co-transfected to allow for antibiotic selection. After selection in 10 μg/ml puromycin (Sigma), cells were cloned by limiting dilution and clones screened for high expression. Expression levels in supernatants of DR1, DR4 and DP4 proteins with C-terminal tandem His-tags range from 1 to 5 mg/ml as determined by ELISA.
Recombinant MHC class II protein purification on IDA columns
Clarified and 0.22 μιη filtered supernatants from 3-5 day conditioned Sf900 II SFM media (Invitrogen) from insect cell culture were flowed through a column of 25-50 ml Chelating Sepharose FF (GE Healthcare Life Sciences) at 1.5-2 ml/min (previously equilibrated in PBS) and washed with 10 mM imidazole in PBS. Tandem His-tagged recombinant MHC class II proteins were eluted with 200 mM imidazole in PBS and the column regenerated with 20 mM EDTA in 50 mM Tris pH 8.0. The eluate was further concentrated in an Amicon cell concentrator (Millipore) and buffer exchanged with a HiLoad 26/10 gel filtration column (GE Healthcare Life Sciences) against the loading buffer (100 mM sodium citrate pH 6.0).
Peptide loading and purification of "immunopure" complexes
Loading of purified recombinant MHC class II protein with 100 μΜ desthiobiotin (DTB) or polyglutamate-tagged (pY-E8) HA308-318 or NY-ESO-1119-143 peptide at 100 μΜ was performed at 37 °C for 24 h. To remove the excess peptide, the sample was passed through the HiLoad 26/10 column twice in PBS (for DTB-tagged complexes) or 50 mM Tris pH 9.0 (for pY-E8-tagged complexes) and then applied to a StrepTrap HP 1 ml (for DTB- tagged complexes) or a MonoQ 4.6/100 (for pY-E8-tagged complexes). After a 5 CV wash, DTB-containing complexes were eluted with 50 mM desthiobiotin in PBS (pH corrected to 7.4) while the pY-E8 complexes were eluted with a NaCl gradient (0-1 M NaCl in 20 CV) at 400 mM NaCl. Flow rates were 1 ml/min for the StrepTrap column and 2 ml/min for the MonoQ column (both columns from GE Healthcare Life Sciences). The StrepTrap column is regenerated with 1 mM HABA (Sigma) in PBS and equilibrated in 30 CV of PBS. The eluted proteins were diluted with an equal volume of PBS (DTB-tagged peptide) or 50 mM Tris pH 9.0 (pY-E8-tagged peptide) before being concentrated to 0.5-1 mg/ml in an Amicon- 5 filter concentrator (Millipore). Aliquots of 10-25 μg were snap frozen in liquid nitrogen and stored at -80 C. Optional tag removal by UV. In case the tag is linked to the peptide with a photocleavable amino-acid derivative (βΝΡΑ), it can be removed by irradiation with a 30 W UV lamp (365 nm) by irradiating for 10 min in V-bottom 96-well plates (1 μg MHC -peptide complex in 100 ul PBS).
Multimerization
Purified MHC class II-peptide complexes with C-terminal tandem His-tags are added to NTA-derivatized PE and SA-PE conjugates in a well of a V-bottom 96-well plate, vigorously mixed and incubated at least 2 hours at 4°C before use in staining a cell sample.
Use ofDTB to isolate MHC-II peptide complexes
The structure of biotin and desthiobiotin are provided in Figure 38. We found out that DTB binds to StreptActin with sufficient affinity to permit efficient retention and can be readily displaced by free DTB. Importantly, StreptActin columns could be completely regenerated by washing with HABA, which displaces DTB, and subsequently with Tris, pH 9.0, which removes HABA. Contrary to previous reports, the regeneration of streptavidin columns after elution with free DTB was incomplete (with HABA and/ high or low pH). While streptactin columns are used for purifying Strep tag-containing molecules and monomer streptamer columns for purifying biotinylated molecules, we report here that StreptActin columns allow efficient purification of DTB tagged molecules and complete regeneration of the columns.
Evaluation of DTB tag peptide purification efficiency
A DTB tagged model peptide was generated and purification performance was evaluated (Figure 39). The structure of a low molecular weight colored peptide used for quantification of the purification efficiency is described in Figure 39A. Figure 39B shows a compilations of the efficiencies of retention, elution and regeneration for 3 different columns. Figure 39C shows purification yields on StreptActin superflow high capacity column for three consecutive rounds of purification/regeneration. Regeneration buffer: HABA 0.1 M followed by Tris 0.2 M, pH 9.
DTB tag MHC I monomer purification efficiency was evaluated next (Figure 40). BindingAVashing buffer: Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8. Elution Buffer: Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8, 50 mM DTB. Regeneration Buffer: Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8, 1 mM 2-(4'-hydroxyazobenzene) benzoic acid (HABA). For purification evaluation, 499 μg of pMHC I monomers carrying a DTB C terminal on the heavy chain were applied onto 2 ml of StreptActin High Capacity sepharose (IBA).
Flowthrough, washing and elution were quantified by at OD 350 nm measurements (Figure 41). Elution was with 50 mM DTB with a recovery of 307 μg (85 % yield).
Purification of pMHC I DTB complexes on StreptActin sepharose was also evaluated . Figure 42A shows a chromatogram of a DTB-tagged protein purified on a StreptActin sepharose column. Figure 42B shows the purification parameters for DTB tagged MHC class I-peptide complexes.
An exemplary scheme of DTB-HA peptide loading and purification is described in Figure 43. An exemplary structure of DTB-HA and the corresponding DR42xHis DTB-HA complexes are depicted in Figure 43A. A schematic of the purification and regeneration cycle of DR42xHis DTB-HA on a StreptActin sepharose column is illustrated in Figure 43B.
DR4-DTB-HA peptide complexes were generated and purified on a streptactin column. After peptide-loading, DR4-2xHis DTB-HA306-318 complexes were passed over a StreptActin column, washed and eluted with 50 nM DTB as indicated by arrows (Figure 44A), and the OD of the effluent at 280 nm was monitored. Pure DR4-HA complexes eluted in a sharp peak (indicated by a box in Figure 44 A). DR4 2xHis DTB-HA306-318 complexes were analyzed by SDS-PAGE (12%) before (lane 1) and after (lane 2) purification on
StreptActin sepharose (Figure 44B). SDS-PAGE was performed under non-reducing, non- boiling (NR, NB, left gel image) or reducing and boiling conditions (R, B, right gel image). Each lane was loaded with 10 μg protein and the gel was stained with Coomassie blue. DTB- tag based purification resulted in highly pure peptide-loaded MHC preparations. Surprisingly, the purity of DTB-tagged peptide-loaded MHC molecules obtained by StreptActin column purification was observed to be superior to that of purification of polyanionic peptide tagged MHC molecules, which yield highly pure peptide-loaded MHC molecules. Staining of CD4+ DR4-restricted Flu-specific T cells with DTB immunopure mult inters.
Staining performance of PE-NTA2, biotin-NTA4-SA-PE, and conventional BSP multimers was compared. As described in more detail elsewhere herein, PE-NTA2 and biotin-NTA4-SA-PE multimers contain the NTA moieties shown in Figure 45.
MHC-peptide NTAmer preparation
5 μg DR4 2xHis DTB-HA complexes were incubated at 4 °C for 2 -16 h with 1.4 μg PE NTA2, or 2.8 μg SA-PE NTA4 in wells of a V-bottom 96 well plate; then each incubation was diluted with EDTA-free FACS buffer (0.5% BSA in PBS with 0.05% sodium azide). Staining of cells was performed in 50 μΐ volume at room temperature (RT) or 37°C for l-2h or as indicated. The PE-NTA2 generated contained an estimated 7-10 monomers per conjugate. The BSP and biotin-NTA4 multimers were prepared with the same SA-PE (from Caltag).
Cloned Flu HA3o6-3i8-specific CD4+ T cells (clone 23-1) were incubated at room temperature (RT) or 37°C. DR4/HA306-3i8 BSP, biotin-NTA4-SA-PA, or PE-NTA2 multimers made with DTB- strep tactin purified monomers and after washing were analyzed by flow cytometry (Figure 46A, "multimer" refers to conventional BSP multimers). The background staining (black histograms) was assessed by parallel staining on a DR4-restricted /Flu matrix 61-72- specific clone. Five percent of cloned 23-1 clone cells were added to fresh PBMC and analyzed in the same manner (Figure 46B).
HA-specific CD4+ T cell clones 9(A) or 8(B) were incubated for 1 hour at 37°C with graded concentrations of DR4/HA3o6-3is multimer (as in previous experiments), washed and analyzed by flow cytometry (Figure 47). A DR4-restricted Flu matrix specific clone was used to evaluate nonspecific binding, which was subtracted. Scatchard analysis was performed and the KD (dissociation constant) and Bmax (maximal binding) values are described in the tables on the right.
The PE-NTA2 multimers exhibited efficient binding/staining, particularly on clones that exhibited poor BSP multimer staining. Similarly, the biotin-NTA4-SA-PE multimers stained with higher efficiency than BSP multimers. It is important to note, however, that significant clonal variations were observed.
DR4/HA 06-3i8 SA-PE NTA4 multimers exhibited less background staining than PE NTA2 multimers (Figure 48). HA peptide- stimulated PBMC were incubated at room Temperature (A, B) or 4°C, (C). Optimal multimer concentrations (14 μg/ml for PE NTA2, 28 μg/ml for SA-PE NTA4) were used in (A) and (B). In (C), three different concentrations of the PE NTA2 reagent were compared. Non- stimulated PBMCs were used as a control.
The PE-NTA2 multimers tended to exhibit increased background staining on control PBMCs. Background staining was efficiently suppressed by addition of 0.5% milk powder (as used, e.g., in Western blotting).
SA-PE NTA4 DR4/HA3o6-3 i8 multimers were able to detect more antigen-specific cells than BSP multimers over a wide range of concentrations (Figure 49). Stimulated PBMCs were incubated for 1 h at 37°C (A) or room temperature (C) with 10 μg/ml of the indicated HA peptide-loaded multimers and analyzed after washing by flow cytometry.
Nonspecific background (control) was determined on TT634-653 peptide stimulated PBMCs. Specific multimer staining was assessed in cells prepared and stained as in (A) over at different concentrations of multimers (B). For comparison the frequencies of IFNy+ T cells was assessed by ICS and flow cytometry with (+) or without (-) peptide stimulation.
Reversibility of multimer staining
Biotin-NTA4, but not BSP multimers, can be rapidly removed from stained, antigen- specific cells (Figure 50). Peptide- stimulated PBMCs were stained with optimal
concentrations (28 μ^πύ for biotin-NTA4-SA-PE and 16 agl \ for BSP multimer) DR4-HA multimers at 4°C for 1 h and washed with cold EDTA-free FACS buffer. After the initial acquisition (0 min), imidazole (100 mM final concentration) was added and data acquisitions (10,000 CD3+ CD4+events) was performed at 1, 3, 5 and 10 min by flow cytometry. Control cells stained with biotin-NTA4-SA-PE multimer were not treated with imidazole (A).
Specific binding observed in (A) was plotted versus the time elapsed (B). Biotin-NTA4-SA- PE multimers were observed to be stable in the absence and rapidly reversible in the presence of imidazole. This prevents multimer staining-induced death of antigen- specific T-cells, for example, CD4+ T cells or CD8+ T cells. As described elsewhere herein.
Comparative staining of a DRl/ESOug-m cell line by DRl/NY-ESOU9-i43 BPS, PE-NTA2 and biotin-NTA4-SA-PE multimers.
The staining reagents were prepared as described above from DTB/StreptActin purified DRl/NY-ESOn9-143 monomers. CD4+ cells were incubated with three different multimer dilutions (1:2, 1:4 and 1:8, corresponding to 14 μg/ml, 7 μg/ml, and 3.5 μg/ml for the SA-PE NTA4 and the BSP multimers, and to 7 μg/ml, 3.5 μg/ml and 1.75 μg/ml for the PE NTA2 multimers (Figure 51 white histograms). Mouse splenocytes (C57BL/6J) were used as a negative control (black histograms). The DRl/ESOu9-143 cell line was derived from an HLA-DR1 transgenic mouse immunized with NY-ESOu9-i43 peptide, and was maintained in IMDM-10, 50 μΜ beta-mercaptoethanol, pen/strep, with 50 U/ml of rmIL-2.
EXAMPLE 6
Additional Tags
Additional tags for purification of pep tide-loaded MHC monomers were evaluated
(Figure 52). Earlier elution on anion-exchange chromatography of pY-E8-HA peptide loaded than DR4-x (x= no/ any peptide) from anion exchange chromatography column (mono-Q) upon elution with a linear gradient of NaCl is shown in Figure 52. The gradient shown as a light line rises from 0 to 1 M NaCl. Figure 52 B shows an ELISA of DR4 (light line) and pY (phospho-tyrosine)(dark line).
Additional acidic tags that were successfully employed for the isolation of correctly peptide loaded MHC II monomers includes YP-D4-SGSG-*-HA, YS-D6-SGSG-*-HA, YP- E8-GSG-HA, YP-E8-GSG-HA, YP-DDGGDDGGDD-SGS-HA, P - P04 2",-S - SO3".
Of the tags tested, the pY-E8 tag provided the best results. While anionic tags are suitable for the isolation of peptide-loaded MHC monomers, the disadvantages of using such tags include: i) difficulties in peptide synthesis, ii) the high NaCl concentration needed to elute the complexes may partially denature them; iii) an additional desalting step is need to remove the high NaCl concentrations. While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles "a" and "an", as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."
The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean " either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as " only one of or
"exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of, when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited.

Claims

1. A protein multimer, comprising
(a) a multivalent carrier molecule, and
(b) a plurality of proteins bound to the carrier molecule,
wherein at least one of the proteins is conjugated to the carrier molecule via a non- covalent bond with a dissociation constant ΙμΜ > KD > lOfM.
2. The protein multimer of claim 1, wherein the dissociation constant is KD > O. lpM.
3. The protein multimer of claim 1, wherein the dissociation constant is KD > lpM.
4. The protein multimer of claim 1, wherein the dissociation constant is KD > ΙΟρΜ.
5. The protein multimer of claim 1, wherein the dissociation constant is KD≥ ΙΟΟρΜ.
6. The protein multimer of claim 1, wherein the dissociation constant is KD≥ InM.
7. The protein multimer of claim 1, wherein the dissociation constant is KD≥ ΙΟηΜ.
8. The protein multimer of any one of claims 1-7, wherein the dissociation constant is KD < ΙΟΟηΜ.
9. The protein multimer of any one of claims 1-7, wherein the dissociation constant is KD < ΙΟηΜ.
10. The protein multimer of any one of claims 1-6, wherein the dissociation constant is KD < InM.
11. The protein multimer of any one of claims 1-5, wherein the dissociation constant is KD < ΙΟΟρΜ.
12. The protein multimer of any one of claims 1-4, wherein the dissociation constant is KD < ΙΟρΜ.
13. A protein multimer, comprising
(a) a multivalent carrier molecule, and
(b) a plurality of proteins bound to the carrier molecule, wherein at least one of the peptides or proteins is conjugated to the carrier molecule via a chelate complex bond.
14. The protein multimer of claim 13, wherein the chelate complex bond is a bond with a dissociation constant 5μΜ > KD≥ lfM.
15. The protein multimer of any one of claims 1-14, wherein the protein is an MHC molecule and the protein multimer is an MHC multimer.
16. The protein multimer of claim 15, wherein the chelate complex bond comprises a chelant conjugated to the MHC molecule, and a chelant conjugated to the carrier molecule.
17. The protein multimer of claim 16, wherein the chelant conjugated to the carrier molecule is of a different structure than the chelant conjugated to the MHC molecule.
18. The protein multimer of any one of claims 15-17, wherein the MHC molecule comprises an MHC a chain.
19. The protein multimer of any one of claims 15-18, wherein the MHC molecule further comprises an MHC β chain or a β2 microglobulin chain.
20. The protein multimer of claim 18, wherein the MHC molecule is an MHC class I molecule.
21. The protein multimer of claim 19, wherein the MHC molecule is an MHC class II molecule.
22. The protein multimer of any one of claims 1-21, wherein the chelant conjugated to the MHC molecule is C-terminally conjugated to the MHC a chain.
23. The protein multimer of claim 21, wherein the chelant conjugated to the MHC molecule is C-terminally conjugated to the MHC β chain or the β2 microglobulin chain.
24. The protein multimer of any one of claims 15-21, wherein the chelant conjugated to the MHC molecule is a peptide comprising a chelant moiety.
25. The protein multimer of claim 24, wherein the peptide comprising a chelant moiety is fused to a polypeptide chain comprised by the MHC molecule.
26. The protein multimer of claim 24 or 25, wherein the peptide comprising a chelant moiety comprises a poly-Histidine sequence.
27. The protein multimer of claim 26, wherein the poly-Histidine sequence comprises 3-24 His residues.
28. The protein multimer of any one of claims 15-27, wherein the chelant conjugated to the MHC molecule comprises a His6 tag, a His12 tag, or a 2x His6 tag.
29. The protein multimer of any one of claims 15-28, wherein the chelant conjugated to the carrier molecule comprises an NTA moiety.
30. The protein multimer of claim 29, wherein the NTA moiety is bound to the carrier molecule in mono-NTA configuration.
31. The protein multimer of claim 29, wherein the NTA moiety is bound to the carrier molecule in poly-NTA configuration.
32. The protein multimer of claim 29, wherein the NTA moiety is bound to the carrier molecule in di-NTA, or tetra-NTA configuration.
33. The protein multimer of any one of claims 29-32, wherein the NTA moiety is bound to a linker.
34. The protein multimer of claim 33, wherein the linker comprises a maleimide moiety or derivative.
35. The protein multimer of claim 33, wherein the linker comprises an oxime moiety or derivative.
36. The protein multimer of claim 33, wherein the linker is between about 9A and about 23A long.
37. The protein multimer of claim 33 or 36, wherein the linker is covalently bound to the carrier molecule.
38. The protein multimer of claim 33 or 36, wherein the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule.
39. The protein multimer of claim 38, wherein the ligand is biotin and the binding molecule is streptavidin.
40. The protein multimer of any one of claims 15-39, wherein the chelate complex bond further comprises a divalent cation.
41. The protein multimer of claim 40, wherein the divalent cation is an Ni2+, Cu2+, Zn2+, Co2+, Cd2+, Sr2+ , Mn2+, Fe2+, Mg2+, Ca2+, or Ba2+ ion.
42. The protein multimer of any one of claims 1-41, wherein the carrier molecule is a fluorophore,, a phycobilin, phycoerythrin or allophycocyanine, a quantum dot (Qdot), a microsphere (e.g., a fluorescent microsphere (e.g. fluorospheres® type), a magnetic particle, or a nanoparticle.
43. The protein multimer of any one of claims 22-42, wherein the MHC molecule is an empty MHC molecule or a peptide-loaded MHC molecule.
44. The protein multimer of claim 43, wherein the peptide-loaded MHC molecule is chosen from the MHC molecules disclosed in Table 2.
45. The protein multimer of claim 43, wherein the MHC molecule comprises an HLA- A*0201 heavy chain.
46. The protein multimer of any one of claims 1-45, wherein the MHC molecule is loaded with an antigenic peptide.
47. The protein multimer of claim 46, wherein the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL (SEQ ID NO: 308).
48. The protein multimer of any one of claims 1-47, wherein the multimer is a tetramer.
49. The protein multimer of claim 46, wherein the antigenic peptide is conjugated to a tag.
50. The protein multimer of claim 48,wherein the tag is conjugated to the peptide via a
cleavable linker.
51. The protein multimer of claim 50, wherein the linker is a photocleavable linker.
52. The protein multimer of claim 51, wherein the linker is an ΝΡβΑ linker.
53. The protein multimer of claim 50, wherein the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
54. The protein multimer of claim 50, wherein the tag is an acidic peptide tag.
55. The protein multimer of claim 54, wherein the acidic peptide tag comprises a plurality of acidic amino acid sequences.
56. The protein multimer of claim 54, wherein the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag.
57. The protein multimer of claim 54, wherein the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
58. The protein multimer of claim 50, wherein the tag is a desthiobiotin (DTB) tag.
59. A multivalent chelant, comprising
a water-soluble carrier molecule, and
a plurality of chelant moieties conjugated to the carrier molecule.
60. A multivalent chelant, comprising
a carrier molecule or structure, and
a plurality of chelant moieties conjugated to the carrier molecule or structure, wherein the carrier molecule or structure has a diameter of less than 0. Inm, less than 0.2nm, less than 0.25nm, less than 0.3nm, less than 0.4nm, less than 0.5nm, less than 0.6nm, less than 0.7nm, less than 0.75nm, less than 0.8nm, less than 0.9nm, less than Inm, less than l. lnm, less than 1.2nm, less than 1.3nm, less than 1.4nm, less than 1.5nm, less than 1.6nm, less than 1.7nm, less than 1.8nm, less than 1.9nm, less than 2nm, less than 2.5nm, less than 3nm, less than 4nm, less than 5nm, less than 6nm, less than 7nm, less than 8nm, less than 9nm, or less than lOnm. In some embodiments, the diameter of the carrier molecule is less than 20nm, less than 30nm, less than 40nm, less than 50nm, less than 60nm, less than 70nm, less than 80nm, less than 90nm, less than lOOnm, less than 200nm, less than 300nm, less than 400nm, less than 500nm, less than 600nm, less than 700nm, less than 800nm, less than 900nm, or less than Ιμιη.
61. The multivalent chelant of claim 60, wherein the chelant moieties are nitrilotriacetic acid (NTA) moieties.
62. The multivalent chelant of claim 61, wherein the NTA moieties are in mono-NTA configuration.
63. The multivalent chelant of claim 61, wherein the NTA moieties are in poly- NTA configuration.
64. The multivalent chelant of claim 62, wherein the NTA moieties are in di-NTA, or tetra- NTA configuration.
65. The multivalent chelant of any one of claims 60-63, wherein the NTA moieties are bound to a linker.
66. The multivalent chelant of claim 65, wherein the linker comprises a maleimide moiety.
67. The multivalent chelant of claim 65, wherein the linker comprises an oxime moiety.
68. The multivalent chelant of claim 65, wherein the linker is between about 9A and about 23 A long.
69. The multivalent chelant of claim 65 or 68, wherein the linker is covalently bound to the carrier molecule.
70. The multivalent chelant of claim 65 or 68, wherein the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule.
71. The multivalent chelant of claim 70, the ligand is biotin and the binding molecule is strep tavidin.
72. The multivalent chelant of any one of claims 60-71, wherein the carrier molecule is a fluorophore, a phycobilin, phycoerythrin or allophycocyanine, or a quantum dot (Qdot).
73. The multivalent chelant of any one of claims 60-72, wherein a plurality of the chelant moieties conjugated to the carrier molecule form chelate complex bonds to a plurality of chelant moiety-conjugated monomeric molecules, thus forming a multimer of the monomeric molecule.
74. The multivalent chelant of claim 73, wherein the monomeric molecule is a polyprotein.
75. The multivalent chelant of claim 73, wherein the monomeric molecule is a small molecule compound.
76. The multivalent chelant of claim 73, wherein the monomeric molecule is a
polynucleotide.
77. The multivalent chelant of any one of claims 73-76, wherein the monomeric molecule is a ligand of a receptor.
78. The multivalent chelant of any one of claims 77, wherein the receptor is a cell-surface receptor.
79. The multivalent chelant of any one of claims 77, wherein the receptor is a T-cell receptor.
80. The multivalent chelant of any one of claims 73-78, wherein the monomeric molecule is an MHC molecule.
81. The multivalent chelant of any one of claims 73-80, wherein the monomeric molecule is an MHC class I molecule.
82. The multivalent chelant of any one of claims 80 or 81, wherein the MHC molecule comprises an HLA-A*0201 heavy chain.
83. The multivalent chelant of any one of claims 80, wherein the MHC molecule is an MHC class II molecule.
84. The multivalent chelant of any one of claims 80-83, wherein the MHC molecule is loaded with an antigenic peptide.
85. The multivalent chelant of any one of claims 84, wherein the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL (SEQ ID NO: 309).
86. The multivalent chelant of any one of claims 59-85, wherein the multivalent chelant is a tetravalent chelant.
87. The multivalent chelant of claim 84 or 85, wherein the tag is an acidic peptide tag.
88. The multivalent chelant of claim 87, wherein the acidic peptide tag comprises a plurality of acidic amino acid sequences.
89. The multivalent chelant of claim 88, wherein the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, pY-D10, pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
90. The multivalent chelant of claim 84 or 85, wherein the tag is a desthiobiotin (DTB) tag.
91. A method, comprising
contacting a monomeric chelant moiety-conjugated MHC molecule with a water-soluble carrier molecule conjugated to a plurality of chelant moieties under conditions suitable for formation of a chelate complex between the chelant moieties conjugated to the MHC molecule and the chelant moieties conjugated to the carrier molecule.
92. The method of claim 91, wherein the chelant moieties conjugated to the carrier molecule are NTA moieties.
93. The method of claim 92, wherein the NTA moieties are in mono-NTA configuration.
94. The method of claim 92, wherein the NTA moieties are in poly-NTA configuration.
95. The method of claim 94, wherein the NTA moieties are in di-NTA, or tetra-NTA configuration.
96. The method of any one of claims 93-95, wherein the NTA moieties are bound to a linker.
97. The method of claim 96, wherein the linker is between about 9A and about 23 A long.
98. The method of claim 96 or 97, wherein the linker is covalently bound to the carrier molecule.
99. The method of any one of claims 96-97, wherein the linker is covalently bound to a ligand of a binding molecule, and wherein the binding molecule is covalently bound to the carrier molecule.
100. The method of claim 99, wherein the ligand is biotin and the binding molecule is strep tavidin.
101. The method of any one of claims 91-100, wherein the carrier molecule is a fluorophore.
102. The method of claim 101, wherein the carrier molecule is a phycobilin.
103. The method of claim 102, wherein the carrier molecule is phycoerythrin or
allophycocyanine.
104. The method of any one of claims 91-100, wherein the carrier molecule is a quantum dot (Qdot).
105. The method of any one of claims 91-100, wherein the carrier molecule is a magnetic particle.
106. The method of any one of claims 91-100, wherein the carrier molecule is a nanoparticle.
107. The method of any one of claims 91-103, wherein the carrier molecule is PE conjugated to strep tavidin.
108. The method of any one of claims 91-107, wherein the carrier molecule conjugated to a plurality of chelant moieties is generated by incubating a carrier molecule conjugated to a plurality of binding molecules with an excess of chelant-conjugated ligand under conditions suitable for the ligand to bind the binding molecule.
109. The method of claim 108, wherein the molar ratio of carrienligand is between 1:2 and 1: 10.
110. The method of claim 109, wherein the molar ratio of carrienligand is 1:5.
111. The method of any one of claims 108-110, wherein the incubating is performed at a temperature between 2-16 °C.
112. The method of claim 111, wherein the incubating is performed at about 4 °C.
113. The method of any one of claims 91-112, wherein the method comprises a step of incubating the carrier molecules contacted with the ligand with N1SO4.
114. The method of any one of claims 91-113, wherein the method comprises a step of contacting the carrier molecule conjugated to a plurality of chelant moieties with a molar excess of the MHC molecule conjugated to a chelant.
115. The method of claim 114, wherein the excess is 2-20 fold.
116. The method of claim 114, wherein the excess is 10-fold.
117. The method of any one of claims 91-116, wherein the MHC molecule is an MHC class I molecule.
118. The method of claim 117, wherein the MHC molecule comprises an HLA-A*0201 heavy chain.
119. The method of any one of claims 91-116, wherein the MHC molecule is an MHC class II molecule.
120. The method of any one of claims 91-119, wherein the MHC molecule is loaded with an antigenic peptide.
121. A method, comprising
contacting an MHC molecule conjugated to a first chelant with a ligand molecule conjugated to a second chelant under conditions suitable for formation of a chelate complex between the first and the second chelant.
122. The method of claim 121, wherein the first chelant comprises a peptide.
123. The method of claim 122, wherein the peptide is C-terminally fused to a polypeptide chain comprised by the MHC molecule.
124. The method of claim 122 or 123, wherein the peptide comprises a poly-Histidine sequence.
125. The method of any one of claims 122-124, wherein the poly-Histidine sequence comprises between 3 and 24 His residues.
126. The method of claim 125, wherein the chelant conjugated to the MHC molecule comprises a His6 tag, a His12 tag, or a 2x His6 tag.
127. The method of any one of claims 121-126, wherein the second chelant is NTA.
128. The method of claim 127, wherein the NTA is in mono-NTA configuration.
129. The method of claim 127, wherein the NTA is in poly-NTA configuration.
130. The method of claim 129, wherein the NTA is in di-NTA, or tetra-NTA configuration.
131. The method of any on of claims 127-129, wherein the NTA is bound to a linker.
132. The method of claim 131, wherein the linker is covalently bound to the ligand molecule.
133. The method of claim 131 or 132, wherein the linker is between about 9A and about 23 A long.
134. The method of any one of claims 121-133, further comprising
contacting the ligand molecule with a multivalent binding molecule under conditions suitable for the ligand to bind the multivalent binding molecule.
135. The method of claim 134, wherein the ligand is biotin and the binding molecule is strep tavidin.
136. The method of any one of claims 134 or 135, wherein the binding molecule is conjugated to a carrier molecule.
137. The method of any one of claims 121-136, wherein the carrier molecule is a fluorophore.
138. The method of claim 137, wherein the carrier molecule is a phycobilin.
139. The method of claim 138, wherein the carrier molecule is phycoerythrin or
allophycocyanine.
140. The method of any one of claims 121-136, wherein the carrier molecule is a quantum dot (Qdot).
141. The method of any one of claims 121-136, wherein the carrier molecule is a magnetic particle.
142. The method of any one of claims 121-136, wherein the carrier molecule is a
nanoparticle.
143. The method of any one of claims 121-142, wherein the MHC molecule is an MHC class I molecule.
144. The method of claim 143, wherein the MHC molecule comprises an HLA-A*0201 heavy chain.
145. The method of any one of claims 121-142, wherein the MHC molecule is an MHC class II molecule.
146. The method of any one of claims 121-145, wherein the MHC molecule is loaded with an antigenic peptide.
147. The method of any one of claims 121-146, wherein the MHC molecule is loaded with a peptide comprising the sequence GILGFVFTL or a peptide comprising a sequence provided in Table 2.
148. A method comprising,
providing an MHC molecule bound to an antigenic MHC molecule-binding peptide that is conjugated to a tag via a cleavable linker,
removing the tag from the antigenic peptide, and
conjugating a chelant moiety to a heavy chain of the MHC molecule.
149. The method of claim 148, further comprising, contacting a multivalent chelant molecule with the MHC molecule under conditions suitable for the chelant moiety conjugated to the MHC molecule to form a chelate complex bind with a chelant moiety of the multivalent chelant molecule.
150. A method, comprising
contacting a population of cells with a protein multimer according to any preceding claim, wherein the multimer comprises a chelate bond and a detectable label, and
detecting a cell binding the multimer.
151. The method of claim 150, wherein the protein multimer is an MHC multimer.
152. The method of claim 150 or 151, wherein the detectable label is a fluorophore.
153. The method of any one of claims 150-152, wherein detecting is by fluorescent microscopy or cell sorting.
154. The method of claim 150 or 151, wherein the detectable label is a magnetic particle.
155. The method of any one of claims 150-154, wherein detecting is by isolating the cell binding the multimer.
156. The method of any one of claims 150-155, wherein detecting comprises quantifying a number of cells binding the multimer.
157. The method of any one of claims 150-156, wherein detecting comprises quantifying a number of cells binding the multimer as a ratio to a number of cells of the population of cells that do not bind the multimer.
158. The method of any one of claims 150-157, further comprising reversing the binding of the multimer to the cell binding the multimer by contacting the cells binding the multimer with a monomeric chelant moiety competing for the chelate complex bond comprised by the multimer.
159. The method of claim 158, wherein the monomeric chelant moiety is an imidazole molecule.
160. A method, comprising
(a) contacting a population of cells with a protein multimer according to any preceding claim, wherein the multimer comprises a chelate bond and a detectable label,
(b) optionally, detecting a cell binding the multimer, and
(c) isolating the cell binding the multimer.
161. The method of claim 160, wherein the multimer is an MHC multimer.
162. The method of claim 160 or 161, wherein the method further comprises reversing the binding of the multimer to the cell binding the multimer by contacting the isolated cell binding the multimer with a monomeric chelant moiety competing for the chelate complex bond comprised by the multimer.
163. The method of claim 162, wherein the monomeric chelant moiety is an imidazole molecule.
164. The method of any one of claims 160-163, wherein the isolated cell is a T-cell.
165. The method of claim 164, wherein the T-cell does not undergo CD8/TCR- mediated activation during the contacting, optionally, during the detection, and during the isolation step.
166. The method of claim 164 or 165, wherein the isolated T-cell is part of an isolated, native T-cell population.
167. A method, comprising, contacting a population of cells expressing a T-cell receptor with an MHC multimer of any one of claims 13-51 under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC class I molecule interaction to activate a T-cell expressing the T-cell receptor and binding the MHC multimer.
168. A method, comprising, contacting a population of cells expressing a T-cell receptor with an MHC multimer of any one of claims 13-51 under conditions suitable for the multimer to bind to the T-cell receptor and to render the T-cell non-responsive to a naturally occurring antigen.
169. The method of claim 167 or 168, further comprising contacting the population of T- cells with an agent able to release the chelate complex bond of the MHC multimer after the cells were contacted with the multimer.
170. A cell or cell population, comprising
a cell contacted with the protein multimer of any preceding claim, wherein the multimer comprises a chelate complex bond.
171. The cell of claim 170, wherein the multimer is an MHC multimer.
172. The cell of claim 170, wherein the cell is or has been contacted with an excess of monomeric chelant moieties competing for the chelate complex bond comprised by the multimer.
173. A kit, comprising
an MHC multimer as provided in any preceding claim.
174. The kit of claim 173, wherein the MHC multimer is loaded with an antigenic peptide.
175. The kit of claim 173, wherein the MHC multimer is an empty MHC multimer.
176. The kit of claim 175, wherein the kit further comprises at least one antigenic peptide that can be loaded onto the empty MHC multimer.
177. The kit of claim 175, wherein the kit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different antigenic peptides.
178. The kit of claim 176, wherein the antigenic peptides are antigenic peptides of tumor antigens.
179. An isolated peptide-loaded MHC molecule, comprising
an MHC heavy chain, and
an antigenic peptide, wherein the peptide is conjugated to a tag for ion exchange chromatography.
180. The isolated peptide-loaded MHC molecule of claim 179, wherein the MHC molecule is an MHC class II molecule.
181. The isolated peptide-loaded MHC molecule of claim 179 or 180, wherein the tag is an acidic tag.
182. The isolated peptide-loaded MHC molecule of claim 181, wherein the acidic tag is an acidic cyanine dye.
183. The isolated peptide-loaded MHC molecule of claim 181, wherein the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences.
184. The isolated peptide-loaded MHC molecule of claim 183, wherein the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag.
185. The isolated peptide-loaded MHC molecule of claim 183, wherein the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
186. The isolated peptide-loaded MHC molecule of any one of claims 179-185, wherein the MHC molecule further comprises a heavy chain that is conjugated to a chelant moiety.
187. The isolated peptide-loaded MHC molecule of any one of claims 179-186, wherein the molecule comprises a combination of a heavy chain and an antigenic peptide discloses in
Table 2.
188. The isolated peptide-loaded MHC molecule of any one of claims 179-187, wherein the tag is conjugated to the peptide via a cleavable linker.
189. The isolated peptide-loaded MHC molecule of claim 188, wherein the linker is a photocleavable linker.
190. The isolated peptide-loaded MHC molecule of claim 189, wherein the linker is an ΝΡβΑ linker.
191. The isolated peptide-loaded MHC molecule of claim 188, wherein the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
192. The isolated peptide-loaded MHC molecule of any one of claims 179-191, wherein the MHC molecule is comprised in an MHC multimer.
193. A method, comprising contacting an empty MHC molecule with an antigenic peptide conjugated to a tag under conditions suitable for the antigenic peptide to bind the MHC molecule.
194. The method of claim 193, wherein the MHC molecule is an MHC class II molecule.
195. The method of claim 194, wherein the tag conjugated to the MHC class II binding antigenic peptide is an affinity tag that is not a polyhistidine tag.
196. The method any one of claims 193-195, wherein the tag is an acidic tag.
197. The method of claim 196, wherein the acidic tag is an acidic cyanine dye.
198. The method of claim 196, wherein the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences.
199. The method of claim 198, wherein the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag.
200. The method of claim 198, wherein the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.
201. The method of any one of claims 193-200, wherein the tag is conjugated to the peptide via a cleavable linker.
202. The method of claim 201 wherein the linker is a photocleavable linker.
203. The method of claim 202, wherein the linker is an ΝΡβ A linker.
204. The method of claim 201, wherein the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
205. The method of claim 193 , wherein the tag is a part of a cleavable linker that remains after cleavage of the linker.
206. A method, comprising
contacting an MHC class II type alpha heavy chain with an MHC class II type beta heavy chain under conditions suitable for the alpha and the beta chain to form a heterodimeric MHC class II molecule, wherein at least one of the MHC class II heavy chains is conjugated to a tag, and
isolating the MHC class II molecule, wherein the isolating comprises a step of affinity chromatography.
207. The method of claim 206, wherein the tag is a protein or peptide tag.
208. The method of claim 207, wherein the tag is a poly-His tag.
209. The method of claim 208, wherein the His tag comprises 3-12 His residues.
210. The method of claim 208, wherein the affinity chromatography is ΝΪ2+-ΝΤΑ chromatography.
211. The method of any one of claims 206-210, wherein the contacting is performed by expressing both heavy chains in a cell.
212. The method of any one of claims 206-211, wherein the cell is an insect cell.
213. The method of any one of claims 206-212, wherein the MHC class II molecule is "empty" (not loaded with an antigenic, MHC class II binding peptide).
214. The method of any one of claims 206-213, wherein the method further comprises contacting the MHC class II molecule with an MHC class II binding antigenic peptide.
215. The method of claim 214, wherein the MHC class II binding antigenic peptide is conjugated to a tag.
216. The method of claim 215, wherein the tag conjugated to the MHC class II binding antigenic peptide is an affinity tag that is not a polyhistidine tag.
217. The method of claim 215, wherein the tag is an acidic tag.
218. The method of claim 217, wherein the acidic tag is an acidic cyanine dye.
219. The method of claim 217, wherein the acidic tag is a peptide tag comprising a plurality of acidic amino acid sequences.
220. The method of claim 219, wherein the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag.
221. The method of claim 219, wherein the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-ElO tag.
222. The method of any one of claims 215-221, wherein the tag is conjugated to the peptide via a cleavable linker.
223. The method of claim 222 wherein the linker is a photocleavable linker.
224. The method of claim 223, wherein the linker is an ΝΡβΑ linker.
225. The method of claim 222, wherein the linker is a peptide linker that comprises an amino acid sequence that can be cleaved by a protease or by a chemical.
226. The method of claim 215, wherein the tag is a part of a cleavable linker that remains after cleavage of the linker.
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