Pmhc multiplexers for detection of antigen-specific cells

A process for making a composition of pMHC Multiplexers, comprising the following steps:

• i) providing a collection of phage- or virus particles, each of which comprises a DNA or RNA molecule encoding a unique peptide (p);
• ii) transferring one of the phage- or virus particles into each of several containers, such as into each of several wells of a microtiter plate, where each of the containers comprise cells, growth medium and other conditions appropriate for the amplification of the phage- or virus particles and expression of the unique peptides in said cells;
• iii) adding a molecule comprising two reactive groups, A and X, where reactive group A is capable of forming a covalent bond to the surface of one of the phage- or virus particles, by reaction with a reactive group on the surface of one of the phage- or virus particles;
• iv) allowing the reaction of reactive group A with surface of a phage- or virus particle;
• v) optionally, partially lyse the cells;
• vi) releasing the unique peptides of the cells into the growth medium of the container;
• vii) adding beta2M and HC peptide, where one of the peptides carries a reactive group Y capable of reacting with reactive group X to form a covalent bond, or adding preformed MHC 1 complex carrying a reactive group Y capable of reacting with reactive group X to form a covalent bond;
• or adding alpha and beta subunits, where one of the subunits carries a reactive group Y capable of reacting with reactive group X to form a covalent bond, or adding preformed MHC 2 complex carrying a reactive group Y capable of reacting with reactive group X to form a covalent bond;
• viii) optionally, introducing denaturing conditions in the containers;
• ix) optionally, introducing renaturing conditions in the containers;
• x) allowing the reaction between reactive groups X and Y;
• xi) thereby leading to the formation of a pMHC complex and its chemical attachment to a phage particle by reaction of reactive group X with reactive group Y to form a covalent bond linking the pMHC complex to the surface of the phage- or virus particles, where the unique peptide (p) of the pMHC complex is encoded by the DNA or RNA comprised within the phage- or virus particle that the pMHC complex is attached to;
• where the above steps may be done in any sequence,
• thereby producing pMHC Multiplexers.

A process for making a composition of pMHC Multiplexers, comprising the following steps:

• i) providing a collection of phage- or virus particles, each of which comprises a DNA or RNA molecule encoding a unique peptide (p) and a protein fusion between a phage coat protein and a first dimerization domain peptide, such as the Acid Peptide;
• ii) transferring one of the phage- or virus particles into each of several containers, such as into each of several wells of a microtiter plate, where each of the containers comprise cells, growth medium and other conditions appropriate for the amplification of the phage- or virus particles and expression of the unique peptides in said cells, thereby leading to, in each container, the generation of multiple copies of the unique peptide (p) and multiple copies of the phage- or virus particle that carries within it the DNA or RNA that encodes the unique peptide, where the phage- or virus particle displays on its surface the first dimerization domain peptide;
• iii) optionally, partially lyse the cells;
• iv) releasing the unique peptides into the growth medium;
• v) adding the two proteins of a MHC complex-either separately or as a pre-formed complex-one of which is fused to a second dimerization domain peptide such as the Base peptide, capable of dimerizing with the first dimerization domain peptide;
• vi) optionally, introducing denaturing conditions in each of the containers;
• xii) optionally, introducing renaturing conditions in each of the containers;
• xiii) allowing the first and second dimerization domain peptides to bind to each other and leading to the formation of a pMHC complex and chemical attachment of the pMHC complex to the phage- or virus particle,
• where the above steps may be done in any sequence,
• thereby producing pMHC Multiplexers.

A process for making a composition of cell-based pMHC Multiplexers, comprising the following steps:

• i) Preparing a collection of encoding molecules, capable of being transcribed and/or translated into peptides or proteins, where each encoding molecule encodes the peptide of the pMHC complex of the a final MHC Multiplexer, or where each encoding molecule encodes the precursor peptide or precursor protein of the peptide of the pMHC complex being displayed in multiple copies in a final MHC Multiplexer;
• ii) introducing the collection of encoding molecules into dendritic cells or precursors of dendritic cells, thereby generating a collection of cells, each comprising one or more copies of an encoding molecule,
• iii) allowing the transcription and/or translation of said encoding molecules;
• iv) optionally, allowing the partial degradation and/or modification of peptide (p);
• v) allowing complexation of peptide (p) with MHC complex, to form pMHC complex, and allowing the transfer of the pMHC complex onto the surface of the dendritic cell;
• where steps ii), iii), iv), v), and vi) may be performed in any order, thereby producing a composition of pMHC Multiplexers.

A process for making a composition of pMHC Multiplexers, comprising the following steps:

• i) preparing a collection of two or more phage genomes where each genome carries a DNA sequence encoding a promoter controlling the transcription of a DNA encoding a fusion-protein of a signal peptide and a unique peptide epitope, where said peptide epitope is capable of complexing with a MHC1 or MHC2 complex when it is not attached to the signal peptide;
• ii) introducing the collection of phage genomes of step (i) into the cells of a growing E. coli culture, by e.g. transformation, and growing the E. coli cells for several generations;
• iii) transferring aliquots of the supernatant, comprising on average less than 1 phage particle, to individual wells of a microtiter-plate comprising growing E. coli cultures;
• iv) growing the E. coli cultures, to produce phage particles;
• v) partially lysing the cells, to release peptides encoded by the phage genome and optionally releasing phage particles into the periplasm;
• vi) optionally, removing cells and cell debris by e.g. centrifugation and transfer of the supernatant to wells of another microtiter-plate;
• vii) adding a compound comprising at least two reactive groups (a) and (x), to the solution comprising the phage and peptide X, where (a) is capable of reacting with an amino acid residue of the phage coat protein, and where (x) is capable of reacting with a reactive group (y);
• viii) adding MHC1 complexes that have been modified on at least one surface-exposed amino acid residue with a moiety (y) capable of reacting with (x), and allowing reaction of (x) and (y), to covalently link the pMHC1 complexes to the phage coat;
• ix) denaturing the proteins of the MHC complexes attached to the phage coat;
• x) renaturing the proteins, thereby allowing the formation of pMHC1 complexes, where the peptide component is the peptide X present in the same well, thereby forming in each well a unique pMHC Multiplexer.

A process for making a collection of pMHC Multiplexers, comprising the following steps:

• i) preparing a collection of phagemids that all carry a DNA sequence encoding a peptide (p) in reading frame with and N-terminal to the pk VIII coat protein of phage M13, and where each of the DNA sequences encode a unique peptide (p) of between 7 and 25 amino acid residues;
• ii) introducing the collection of phagemids of step (i) into the cells of a growing E. coli culture, by e.g. transformation;
• iii) adding Helper Phage, to produce phage particles displaying the peptide (p) on pVIII coat protein;
• iv) adding a MHC complex, such as an peptide receptive MHC complex,
• thereby producing a collection of pMHC Multiplexers.

A process for making a collection of pMHC Multiplexers, comprising the following steps:

• i) preparing a collection of phagemids that all carry a DNA sequence encoding a peptide (p) in reading frame with a dimerization domain X, and where each of the DNA sequences encode a unique peptide (p) of between 7 and 25 amino acid residues, and carrying a DNA sequence encoding a dimerization domain Y fused to the pVIII coat protein, where the X and Y dimerization domains are capable of dimerizing to each other;
• ii) introducing the collection of phagemids of step (i) into the cells of a growing E. coli culture, by e.g. transformation;
• iii) adding Helper Phage, to produce phage particles displaying the peptide (p) on a phage coat protein;
• iv) adding a MHC complex, such as an empty MHC2 complex;
• thereby producing a collection of pMHC Multiplexers.

A process for making a collection of pMHC Multiplexers, comprising the following steps:

• i) preparing a collection of encoding molecules, capable of being transcribed and/or translated into peptides or proteins, where each encoding molecule encodes the peptide of the pMHC complex being displayed in multiple copies in the a final MHC Multiplexer, or where each encodes the precursor peptide or precursor protein of the peptide of the pMHC complex being displayed in multiple copies in a final MHC Multiplexer;
• and where the encoding molecules may be a collection of RNA molecules or a collection of DNA molecules, where the DNA molecules may be made by synthetic chemistry or may be made by enzymatic means such as by reverse transcription of an mRNA, followed by amplification e.g. by PCR, and where the DNA may be e.g. a wild type virus or a genetically modified recombinant virus e.g. belonging to the following group of viruses:
• adenovirus, retrovirus, herpes simplex virus, vaccinia virus, influenza virus, and alpha virus;
• and where the RNA may be a collection of mRNA molecules purified from a cell extract such as a cell extract from a cancer patient, e.g. a cell extract of the cells from a biopsy from the tumor of a cancer patient, or alternatively, cDNA may be prepared from a cell extract, amplified by e.g. PCR and then transcribed into mRNA, or mRNA may be made by transcription from (optionally recombinant) viral DNA or other vector DNA such as plasmids, or may be made from transcription of single-stranded or double-stranded oligonucleotides prepared synthetic chemistry;
• ii) introducing the collection of encoding molecules into a dendritic cell or a precursor of a dendritic cell, under conditions ensuring that one cell only receives one or more copies of the same encoding molecule, thereby generating a collection of cells, each comprising one or more copies of an encoding molecule, by e.g. electroporation, infection by e.g. virus, phagocytosis of another cell, e.g. a monocyte or bacteria, uptake of lipid nanoparticles or vesicles or other similar entities, or uptake of small lipid-comprising carriers, infection by e.g. bacterium, or transfection, e.g. using liposomes such as DOTAP;
• iii) optionally, adding activating or inhibiting molecule(s);
• iv) incubating;
• v) allowing the transcription and/or translation of said encoding molecules;
• vi) allowing the partial degradation and/or modification and complexation of peptide (p) with MHC complex, to form pMHC complex, and allowing the transfer of the pMHC complex onto the surface of the dendritic cell, thereby resulting in a display of the peptide in complex with MHC protein;
• where steps ii), iii), iv), v), and vi) may be performed in any order, thereby producing pMHC Multiplexers.

A process for making a collection of pMHC Multiplexer, comprising the following steps:

• i) preparing a collection of encoding molecules, capable of being transcribed and/or translated into peptides or proteins, where each encoding molecule encodes the peptide of the pMHC complex being displayed in multiple copies in the a final MHC Multiplexer, or where each encodes the precursor peptide or precursor protein of the peptide of the pMHC complex being displayed in multiple copies in a final MHC Multiplexer;
• and where the encoding molecules may be a collection of RNA molecules or a collection of DNA molecules, where the DNA molecules may be made by synthetic chemistry or may be made by enzymatic means such as by reverse transcription of an mRNA, followed by amplification e.g. by PCR, and where the DNA may be e.g. a wild type virus or a genetically modified recombinant virus e.g. belonging to the following group of viruses:
• adenovirus, retrovirus, herpes simplex virus, vaccinia virus, influenza virus, and alpha virus;
• and where the RNA may be a collection of mRNA molecules purified from a cell extract such as a cell extract from a cancer patient, e.g. a cell extract of the cells from a biopsy from the tumor of a cancer patient, or alternatively, cDNA may be prepared from a cell extract, amplified by e.g. PCR and then transcribed into mRNA, or mRNA may be made by transcription from (optionally recombinant) viral DNA or other vector DNA such as plasmids, or may be made from transcription of single-stranded or double-stranded oligonucleotides prepared synthetic chemistry;
• ii) introducing the collection of encoding molecules into a dendritic cell or a precursor of a dendritic cell, under conditions ensuring that one cell only receives one or more copies of the same encoding molecule, thereby generating a collection of cells, each comprising one or more copies of an encoding molecule, by e.g. electroporation, infection by e.g. virus, phagocytosis of another cell, e.g. a monocyte or bacteria, uptake of lipid nanoparticles or vesicles or other similar entities, or uptake of small lipid-comprising carriers, infection by e.g. bacterium, or transfection, e.g. using liposomes such as DOTAP;
• iii) adding activating or inhibiting molecule(s);
• iv) incubating;
• v) allowing the transcription and/or translation of said encoding molecules;
• vi) allowing the partial degradation and/or modification and complexation of peptide (p) with MHC complex, to form pMHC complex, and allowing the transfer of the pMHC complex onto the surface of the dendritic cell, thereby resulting in a display of the peptide in complex with MHC protein;
• where steps ii), iii), iv), v), and vi) may be performed in any order, thereby producing pMHC Multiplexers.

A process for making a collection of pMHC Multiplexers, comprising the following steps:

• i) preparing a collection of phagemids that all carry a DNA sequence encoding the Acid Peptide in reading frame with and N-terminal to the pIII coat protein of phage M13, and where each of the phagemids carry a unique DNA sequence leading to the expression of a unique peptide of between 7 and 25 amino acid residues;
• ii) preparing a DNA vector such as a plasmid that comprises a sequence encoding the beta2M protein and a fusion protein, Base Peptide-HC protein;
• iii) introducing the collection of phagemids and the vector of step (i) and (ii) into the cells of a growing E. coli culture, by e.g. transformation;
• iv) adding Helper Phage, to produce intracellular phage particles displaying the Acid Peptide on pIII coat protein;
• v) allowing the assembly of the Base Peptide-HC with the beta2M and the unique peptide, to form the pMHC complex thereof;
• vi) allowing the assembly of the Acid-Base dimer, thereby producing intracellular phage particles displaying a pMHC complex on the pIII coat protein;
• vii) adding redox buffer and then change to more oxidizing conditions, to form the Acid Peptide-Base Peptide dimer, thereby covalently attaching the pMHC complex to the phage coat protein,
• thereby producing pMHC Multiplexers.

A process for making a collection of pMHC Multiplexers, comprising the following steps:

• i) preparing a collection of encoding molecules, capable of being transcribed and/or translated into peptides or proteins;
• ii) introducing the collection of encoding molecules into a dendritic cell or a precursor of a dendritic cell by e.g. electroporation or infection;
• iii) adding activating or inhibiting molecule(s);
• iv) incubating;
• v) allowing the transcription and/or translation of said encoding molecules;
• vi) allowing the partial degradation and/or modification and complexation of peptide (p) with MHC complex, to form pMHC complex, displayed on a dendritic cell;
• where steps ii), iii), iv), and v) may be performed in any order, thereby producing pMHC Multiplexers.

A process for making a collection of pMHC Multiplexer, comprising the following steps:

• i) providing 1000 wells each comprising a dimerization domain Y, each domain Y being capable of binding to domain X, and being attached to a unique pMHC complex;
• ii) adding to each of the 1000 wells a unique DNA molecule, attached to a dimerization domain Y, capable of binding to domain X;
• iii) adding to each of the 1000 wells a SP1 protein, where each of the 12 subunits of the SP1 protein is attached to a dimerization domain X, said domain X being capable of binding to a dimerization domain Y;
• where steps (i) to (iii) can be performed in any order;
• iv) allowing the X and Y dimerization domain to form an XY dimer,
• thereby producing 1000 pMHC Multiplexers, each of which comprise a unique peptide (p) complexed to MHC protein, and comprising a unique DNA encoding said unique peptide (p).