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WO2025109111A1 - Multimères d'activation de cellules immunitaires - Google Patents

Multimères d'activation de cellules immunitaires Download PDF

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Publication number
WO2025109111A1
WO2025109111A1 PCT/EP2024/083183 EP2024083183W WO2025109111A1 WO 2025109111 A1 WO2025109111 A1 WO 2025109111A1 EP 2024083183 W EP2024083183 W EP 2024083183W WO 2025109111 A1 WO2025109111 A1 WO 2025109111A1
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Prior art keywords
multimer
ligand
cells
antibody
mhc
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Lyn ZHU
Bjarke Endel HANSEN
Liselotte Brix
Dilek INEKCI
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Immudex ApS
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Immudex ApS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex

Definitions

  • the present disclosure relates to multimers capable of efficiently activating immune cells, such as T cells.
  • multimers comprising at least two different ligands capable of binding to and together activate said immune cell.
  • WO 2016/180721 discloses a method for polyclonal stimulation of T cells comprising contacting a population of T cells with said anti-CD3 antibody, optionally with an anti- CD28 antibody, wherein said anti-CD3 antibody and optionally said anti-CD28 antibody are linked to particles.
  • the present disclosure relates to multimers capable of efficiently activating immune cells, such as T cells.
  • the present invention combines ligands to two different immune cell receptors in the same multimer (e.g. a specific antigen recognized by the immune cell and a co-stimulatory molecule), thereby providing an efficient and robust activation of the immune cell.
  • each of the two ligands may be comprised separately in two different multimers that are provided together, such as provided simultaneously or subsequently in any order.
  • an immune cell activating multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell.
  • nucleic acid encoding the multimer as disclosed elsewhere herein.
  • a vector comprising the nucleic acid as disclosed elsewhere herein.
  • composition comprising the multimer as disclosed elsewhere herein, the nucleic acid as disclosed elsewhere herein and/or the vector as disclosed elsewhere herein.
  • composition comprising a) a first multimer comprising a first ligand of a first immune cell receptor; and b) a second multimer comprising a second ligand of a second immune cell receptor, wherein said first and second multimers are defined as disclosed elsewhere herein except that said first and second ligands are comprised in separate multimers.
  • a method for activating and optionally expanding one or more immune cells comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; and c) contacting said multimers or said composition, with said sample, thereby activating and optionally expanding said immune cells.
  • a method for isolating one or more immune cells of low quantity in a sample comprising the steps of: a) providing a sample comprising a population of immune cells in low quantity; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; c) contacting said multimers or said composition, with said sample, thereby activating and optionally expanding said immune cells; and d) isolating said activated, or said activated and expanded, immune cells, such as by FACS, thereby isolating said one or more immune cells of low quantity in said sample.
  • a method for assaying the ability of one or more immune cells in a sample to activate in response to a multimer as disclosed elsewhere herein comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more of said multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; c) contacting said multimers or said composition, with said sample; and d) measuring the activation and expansion of said immune cells, such as by FACS, thereby assaying the ability of said one or more immune cells to activate in response to said multimer.
  • compositions as disclosed elsewhere herein for use as a medicament.
  • compositions as disclosed elsewhere herein for use in a method of treatment of a disease associated with immune evasion, such as cancer.
  • a method of treatment of a disease associated with immune evasion, such as cancer, in a subject in need thereof comprising the step(s) of: a) administering the pharmaceutical composition as disclosed elsewhere herein to said subject in a therapeutically effective amount, or a) isolating immune cells, such as T cells, from said subject; b) contacting said immune cells with one or more multimers as disclosed elsewhere herein, or with a composition as disclosed elsewhere herein, thereby obtaining activated and expanded immune cells specific for said disease, such as specific for cells of said cancer; and c) transferring said activated and expanded immune cells back into said subject.
  • Figure 1 shows that HLA-A*0201/EBV specific CD8+ T cells upregulate cell surface activation markers, CD69 and CD137, upon stimulation with HLA- A*0201/EBV/aCD28/Dextramer.
  • MHC/aCD28/Dextramer stimulated cells were analyzed in flowcytometry and CD3+CD4- T cell population was analyzed for HLA- A*0201/EBV/Dextramer-PE labelling and CD8 expression. The results are further described in Example 1 .
  • FIG. 2 MHC/aCD28/Dextramer stimulated CD8+ T cells were divided into antigen specific HLA-A2*0201/EBV CD8+ T cells (Q2) and non-antigen specific CD8+ T cells (Q1 ) and analyzed for cell surface expression of CD69 and CD137 activation markers. The results are further described in Example 1 .
  • FIG. 3 The phenotype of TCR engineered T cells stimulated with Dextramer multimers comprising peptide-MHC/anti-CD28 Ab closely mimicked that of cells exposed to APCs. This similarity is demonstrated by comparative analysis of surface marker expression between cells stimulated with the peptide-MHC/anti-CD28 Ab Dextramer multimer and those stimulated with APCs.
  • MHC molecule includes a plurality of MHC molecules.
  • An antibody is herein to be understood as truncated or full-length antibodies (of isotype IgG, IgM, IgA, Ig E) , Fab, scFv or bi-Fab fragments or diabodies.
  • the antibody according to the present disclosure may be of any isotype, e.g., IgG, IgM, IgA, IgD, IgE or any subclass, e.g., lgG1 , lgG2, lgG3, lgG4.
  • antibodies produced recombinantly, or by other means, for use in the invention include any antibody fragment which can still bind antigen, e.g. an Fab, an F(ab)2, Fv, scFv.
  • the antibody, including an antibody fragment may be recombinantly engineered to include a hapten, e.g, a peptide.
  • the hapten may be a myc tag. Inclusion of a hapten in an antibody or antibody fragment facilitates subsequent binding of a binding agent, probe, or label.
  • linker derivatives comprising a primary antibody containing an antigen binding region. Some embodiments employ linker derivatives comprising a secondary antibody containing an antigen binding region which specifically binds to a primary antibody, e.g., the constant region of the primary antibody. In certain embodiments, the linker derivative is further conjugated to a polymer. Some embodiments employ linker derivatives comprising a tertiary antibody containing an antigen binding region which specifically binds to the secondary antibody, e.g., a constant region of the secondary antibody, or a hapten linked to the secondary antibody or a polymer conjugated to the secondary antibody. In certain embodiments, the tertiary antibody is further conjugated to a polymer.
  • antibody refers to an intact antibody, or a fragment of an antibody that competes with the intact antibody for antigen binding.
  • antibody fragments are produced by recombinant DNA techniques.
  • antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies.
  • Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and scFv.
  • Exemplary antibody fragments also include, but are not limited to, domain antibodies, nanobodies, minibodies ((scFv-CH 3 ) 2 ), maxibodies ((scFv-CH2-CH 3 ) 2 ), diabodies (noncovalent dimer of scFv).
  • the antibody is PEGylated.
  • Monovalent antibodies The antibodies in the present invention can be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. A particular interesting example is an antibody binding the MHC complex distal to the interaction site with TCR, i.e.
  • An antibody in this example can be any truncated or full length antibody of any isotype (e.g. IgG, IgM, IgA or IgE), a bi-Fab fragment or a diabody.
  • the antibody could be bispecific with one arm binding to the a-chain and the other arm binding to the b-chain.
  • the antibody could be monospecific and directed to a sequence fused to the a-chain as well as to the b-chain.
  • the antibody is monospecific and binds to a surface of the complex that involves both the a- and b-chain, e.g. both the a2- and b2- domain or both the al and b1 - domain.
  • TCR T cell receptor
  • BCR B cell receptor
  • FACS Fluorescence-Activated Cell Sorting
  • the suffix “L“ added to CDX, means CDX ligand, and a ligand to a receptor may be named with the suffix L and the receptor name.
  • CD27L is the ligand for the immune receptor CD27, however CD27L may also be termed CD70.
  • the ligand to a receptor may also have a specific name and the suffix “L” is not used, e.g., CD80 is the ligand for the CD28 receptor.
  • None limiting and hereby included examples of how to activate an immune cell through a receptor includes receptor binding of ligand or binding of an antibody to the receptor (or surface ligands/antigens) thereby activating the “target” (the immune cell for activation) immune cell.
  • activation in the context of the present invention is used to describe changes in the target immune cells or in the target cell population upon encounter with a multimer of the invention, activation includes but is not limited to upregulating of activation markers such as surface molecules e.g., receptors on the surface of the immune cells to be activated (target cell), secretion of cytokines and/or expansion of the immune cells
  • activation markers such as surface molecules e.g., receptors on the surface of the immune cells to be activated (target cell), secretion of cytokines and/or expansion of the immune cells
  • expansion and the term “proliferation” may be used interchangeably, and in the context of the present invention expansion of the immune cell means generation of a cell population of e.g., T cells, which may be antigen specific depending on the activation means e.g., by ligand/antigen.
  • activating multimer means a multimer capable of activating e.g., immune cells.
  • regulation in the context of the present invention is used to describe changes in the target immune cells or in the target cell population upon encounter with a multimer of the invention, regulation includes but is not limited to upregulating or down regulation of target cell markers such as surface molecules e.g., receptors on the surface of the immune cells to be regulated (target cell), stimulation or inhibition of secretion of cytokines and/or stimulation or inhibition of expansion of the immune cells.
  • target cell markers such as surface molecules e.g., receptors on the surface of the immune cells to be regulated (target cell), stimulation or inhibition of secretion of cytokines and/or stimulation or inhibition of expansion of the immune cells.
  • inhibition in the context of the present invention is used to describe changes in the target immune cells or in the target cell population upon encounter with a multimer of the invention, inhibition includes but is not limited to down regulation of target cell markers such as surface molecules e.g., receptors on the surface of the immune cells to be regulated (target cell), inhibition of secretion of cytokines and/or inhibition of expansion of the immune cells.
  • target cell markers such as surface molecules e.g., receptors on the surface of the immune cells to be regulated (target cell)
  • inhibition of secretion of cytokines and/or inhibition of expansion of the immune cells.
  • CD1 refers to MHC Class I like molecules known as CD1 and includes CD1 a, CD1 b, CD1 c and CD1 d.
  • CD1 , “CD1 complexes”, “CD1 molecules”, “CD1 Monomers” and “CD1 constructs” are used interchangeably herein, and - if not further specified - comprises CD1 s loaded with or bound to CD1 specific ligands.
  • Cell population refers to one or more cells, such as two or more cells, such as multiple cells, such as a sample comprising cells. Said cells need not share any defining characteristics, such as cell cycle stage or tissue origin and can thus be any collection of multiple cells.
  • conjugated refers to two molecules that have been joined, such as through covalent or non-covalent interaction.
  • a marker such as an antibody
  • a marker may be conjugated to a fluorescent tag or a DNA oligonucleotide through a linker, such as a stretch of amino acids.
  • linker such as a stretch of amino acids.
  • DNA barcode oligonucleotide oligonucleotide barcode
  • nucleic acid barcode are used interchangeably and refer to unique oligo-nucleotide sequences typically ranging from 10 to more than 50 nucleotides.
  • the barcode may have shared amplification sequences in the 3’ and 5’ ends, and a unique sequence in the middle. This sequence can be revealed by sequencing and can serve as a specific barcode for a given molecule.
  • oligonucleotide label refers to a nucleic acid label that may comprise or consist of DNA, RNA, and/or artificial nucleotides such as PLA or LNA.
  • the oligonucleotide label may comprise one or more of the following components: a 5’ first primer region (forward), a barcode region (e.g. an oligonucleotide barcode), 3’ second primer region (reverse), random nucleotide region, connector molecule, stabilityincreasing components, short nucleotide linkers in between any of the above-mentioned components, adaptors for sequencing and annealing region.
  • the oligonucleotide label comprises at least a barcode region surrounded by primer regions, where the barcode region comprises a sequence of consecutive nucleic acids.
  • the oligonucleotide label comprises or consists of DNA, RNA, artificial nucleic acids and/or Xeno nucleic acid (XNA).
  • XNA Xeno nucleic acid
  • backbone molecule and “multimerization domain” are used interchangeably herein.
  • MHC molecule a MHC molecule as used everywhere herein is defined as any MHC class I molecule or MHC class II molecule as defined herein.
  • a “MHC Class I molecule” as used everywhere herein is used interchangeably with MHC I molecule and is defined as a molecule which comprises 1 -3 subunits, including a MHC I heavy chain, a MHC I heavy chain combined with a MHC I beta2microglobulin chain, a MHC I heavy chain combined with MHC I beta2microglobulin chain through a flexible linker, a MHC I heavy chain combined with an antigenic peptide, a MHC I heavy chain combined with an antigenic peptide through a linker, a MHC I heavy chain/ MHC I beta2microglobulin dimer combined with an antigenic peptide, and a MHC I heavy chain/ MHC I beta2microglobulin dimer combined with an antigenic peptide through a flexible linker to the heavy chain or beta2microglobulin.
  • the MHC I molecule chains can be changed by substitution of single or by cohorts of native amino acids, or by inserts, or deletions to enhance or impair the functions attributed to said
  • MHC Class I like molecules include CD1d, HLA E, HLA G, HLA F, HLA H, MIC A, MIC B, ULBP-1 , ULBP-2, and ULBP-3.
  • MHC Class II molecule as used everywhere herein is used interchangeably with MHC II molecule and is defined as a molecule which comprises 2-3 subunits including a MHC II alpha-chain and a MHC II beta-chain (i.e. a MHC II alpha/beta-dimer), an MHC II alpha/beta dimer with an antigenic peptide, and an MHC II alpha/beta dimer combined with an antigenic peptide through a flexible linker to the MHC II alpha or MHC II beta chain, a MHC II alpha/beta dimer combined through an interaction by affinity tags e.g.
  • MHC II alpha/beta dimer combined through an interaction by affinity tags e.g. jun-fos and further combined with an antigenic peptide through a flexible linker to the MHC II alpha or MHC II beta chain.
  • the MHC II molecule chains can be changed by substitution of single or by cohorts of native amino acids, or by inserts, or deletions to enhance or impair the functions attributed to said molecule.
  • the “MHC Class II molecule” can comprise only 1 subunit or 2 subunits if antigenic peptide is also included.
  • MHC Class II like molecules include HLA DM, HLA DO, l-A beta2, and l-E beta2.
  • MHC complexes MHC molecules
  • MHC Monomers MHC constructs
  • MHC-peptide complex defines any MHC I and/or MHC II molecule combined with antigenic peptide.
  • the MHC molecule may suitably be a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule.
  • a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule.
  • Such MHC complexes from different species have different names. E.g. in humans, MHC complexes are denoted HLA. The person skilled in the art will readily know the name of the MHC complexes from various species.
  • MHC molecule is intended to include all alleles.
  • HLA A, HLA B, HLA C, HLA D, HLA E, HLA F, HLA G, HLA H, HLA DR, HLA DQ and HLA DP alleles are of interest shall be included, and in the mouse system, H-2 alleles are of interest shall be included.
  • RT1 -alleles in the porcine system SLA-alleles, in the bovine system BoLA, in the avian system e.g. chicken-B alleles, are of interest shall be included.
  • MHC complexes such complexes and multimers thereof, which are capable of performing at least one of the functions attributed to said complex.
  • the terms include both classical and non-classical MHC complexes.
  • the meaning of “classical” and “non-classical” in connection with MHC complexes is well known to the person skilled in the art.
  • Non-classical MHC complexes are subgroups of MHC-like complexes.
  • MHC complex includes MHC Class I molecules, MHC Class II molecules, as well as MHC-like molecules (both Class I and Class II), including the subgroup non-classical MHC Class I and Class II molecules.
  • Non-covalent bond is a type of chemical bond that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions.
  • MR1 is used to describe the non-conventional MHC molecule known as MR1 binding the TCR of MAIT Cells.
  • MR1 , “MR1 complexes”, “MR1 molecules”, “MR1 Monomers” and “MR1 constructs” are used interchangeably herein, and - if not further specified - comprises MR1 s loaded with or bound to MR1 specific ligands, for example but not limited to the ligand 5-OP-RU.
  • one or more as used herein is intended to include one and a plurality i.e. more than one.
  • an immune cell activating multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell.
  • One embodiment of the invention relates to an immune cell activating multimer, said multimer comprising a plurality of a) a first ligand of a first immune cell receptor, specific for a T cell; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell.
  • the first ligand of a classical T cell may be an MHC peptide complex binding through the T cell receptor (TCR), and the first ligand may be any ligand for a non-classical T cell or an engineered T cell such as a TCR-T or CAR-T cell carrying an added TCR or a chimeric antigen receptor, respectively.
  • TCR T cell receptor
  • the ligand may be any molecule targeted by the T cells and such ligands may be cancer- associated, microbial or other disease-related antigens.
  • the first ligand of T cells and TCR-T cells may be any MHC molecule presenting a peptide of interest.
  • a first ligand of a non-classical T cell includes MR1 complexes (typically binding to MAIT cells) and CD1 molecules (typically binding to gamma-delta T cells or NKT cells).
  • the first ligand is selected from the group consisting of peptide-MHC class I, peptide-MHC class II, CD1 , CD1d, CD1c, CD1 a, CD1 b and MR1.
  • said multimer is capable of stimulating said immune cell. In some embodiments of the present disclosure, said multimer is capable of stimulating and activating said immune cell. In some embodiments of the present disclosure, said multimer is capable of stimulating, activating and causing cell expansion of said immune cell.
  • an immune cell regulating multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell.
  • an immune cell inhibiting multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell.
  • the multimer of the invention is capable of antigen specific activation of immune cells by using a multimer of the invention which is antigen specific.
  • a multimer of the invention which is antigen specific.
  • This may be obtained by including peptide MHC as first ligand, wherein the peptide complexed to the MHC molecule is specific for a certain antigen, only T cells specific for the MHC presented antigen will be activated.
  • Antigen specific activation may also be achieved for CAR T cells, the multimer of the invention then comprises a first ligand which is an antigen recognized by the CAR or the multimer comprises an antibody to the CAR on the T cells.
  • the first ligand may be an antigen recognized by a B-cell receptor or an antibody to the B cell receptor.
  • the present invention enables activation by specific antigen multimers resulting in activation of antigen specific immune cells.
  • Many interactions in the immune system are based on receptor-ligand interactions, securing a specific response by the immune cells comprising the receptor.
  • Preferably more than one first ligand e.g., MHC are used to activate the target immune cells.
  • first and second ligands are not limited to the order of addition of the ligands to the multimer. The terminology is used solely to distinguish between the two ligands. Accordingly, the first ligand can be attached to the backbone molecule before the second ligand, or the second ligand can be attached to the backbone molecule before the first ligand.
  • the first ligand and the second ligand are attached simultaneously to the backbone molecule in a one-pot fashion i.e. in a single reaction.
  • the invention relates to multimers comprising at least two ligands, wherein the ligands are not the same.
  • the multimer comprises a plurality of each of the two or more ligands.
  • the multimer of the present disclosure is capable of activating immune cells, such as T cells, to a significantly greater extent than using common reagents for activating immune cells, such as T cells, e.g. a commercially available colloidal polymeric nanomatrix conjugated to humanized recombinant CD3 and CD28 agonists, such as the product TransAct from the company Miltenyi Biotec.
  • the multimer as described herein is capable of activating immune cells, such as T cells to at least a 10%, such as at least a 20%, such as at least a 30%, such as at least a 40%, such as at least a 50%, such as at least a 60%, such as at least a 70%, such as at least a 80%, such as at least a 90%, such as at least a 100%, such as at least a 150%, such as at least a 200%, such as at least a 250%, such as at least a 300% greater extent than common immune cell activating reagents, such as TrAct.
  • immune cells such as T cells to at least a 10%, such as at least a 20%, such as at least a 30%, such as at least a 40%, such as at least a 50%, such as at least a 60%, such as at least a 70%, such as at least a 80%, such as at least a 90%, such as at least a 100%, such as at least a 150%, such as at least
  • the multimer of the present disclosure is capable of only activating specific antigen-specific immune cell specifically, i.e. it may be capable of efficiently and robustly stimulating, activating and causing cell expansion of immune cells in an antigen-specific manner. In some embodiments, the multimer of the present disclosure is capable of activating immune cells in an antigen-specific manner.
  • said immune cell is a CD3+ T cell. In some embodiments, said immune cell is a CD8+ T cell. In some embodiments, said immune cell is a CD4+ T cell. In some embodiments, said immune cell is a CD4+CD8+ (double positive) T cell. In some embodiments, said immune cell is a CD4-CD8- (double negative) T cell. In some embodiments, said immune cell is an MAIT cell (mucosal-associated invariant T cell). In some embodiments, said immune cell is an iNKT cell (invariant natural killer T cell). In some embodiments, said immune cell is a B cell. In some embodiments, said immune cell is an NK cell.
  • the immune cell is an engineered T cell, such as TCR-T. In some embodiment the immune cell is a CAR T cell. In a further embodiment, said immune cell is MART-1 specific T cells. In some embodiments, said immune cell is CMV specific T cells.
  • the first ligand binds to and stimulates a first immune cell receptor present on an immune cell.
  • Peptide-MHC multimers are described in detail in W002072631 , W020081 16468, W02009003492 and W02020127222, which are incorporated herein by reference in their entirety.
  • the MHC molecules according to the present disclosure are in one embodiment of human origin and/or animal origin.
  • the MHC molecules according to the present disclosure is of mammal origin (e.g., macaque origin, rodent origin, such as mouse or rat origin).
  • the MHC molecules according to the present disclosure is of non-mammalian vertebrate origin (such as from fish, bird, insect, amphibian, and/or reptile origin).
  • the MHC molecule according to the present disclosure is selected from the group consisting of human, non-human primates, Gorilla gorilla, Pan troglodytes, Macacca mulatta, Orangutang, Rodents, Mus musculus, Rattus norvegicus and Lagomorpha, such as rabbits and hares.
  • the MHC molecule is from a gorilla. In some embodiments of the present disclosure, the MHC molecule is from a chimpanzee. In some embodiments of the present disclosure, the MHC molecule is from a rhesus macaque. In some embodiments of the present disclosure, the MHC molecule is from a rodent. In some embodiments of the present disclosure, the MHC molecule is from a mouse. In some embodiments of the present disclosure, the MHC molecule is from a rat.
  • the MHC molecule is a human MHC molecule.
  • the MHC molecules of humans are designated human leukocyte antigens (HLA).
  • HLA human leukocyte antigens
  • HLA-A HLA-A
  • HLA-B HLA-C
  • HLA-A*01 HLA-A*02
  • HLA-A*11 are examples of MHC class I alleles that can be expressed from these loci.
  • Non-classical human MHC class I molecules, such as HLA-E (homolog of mouse Qa-1 b), CD1 and MR1 molecules are also encompassed by the present disclosure.
  • the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.
  • said peptide complexed to said MHC molecule exhibits nativelike properties in terms of recognition by TCRs and/or other MHC complex-engaging receptors. In some embodiments, said peptide-MHC complex thus exhibits native-like properties in terms of recognition by TCRs and/or other MHC complex-engaging receptors.
  • said peptide complexed to said MHC molecule exhibits nativelike properties in terms of T-cell recognition, NK cell recognition and/or recognition of other immune cells.
  • the peptide-MHC monomers in a multimer according to the present disclosure can be substantially identical peptide-MHC monomers, or the peptide-MHC monomers may be different.
  • each of the MHC monomers complexed to said peptides of said multimer are identical.
  • At least two, such as at least three, such as at least four, such as at least five, such as at least 10, such as at least 15, such as at least 20, such as at least 25, or such as each of said peptide-MHC monomers in a multimer are different.
  • the first ligand is an antibody.
  • the first ligand is an anti-CD3 antibody.
  • the anti-CD3 antibody may be an IgG antibody.
  • the anti-CD3 antibody is a mouse monoclonal lgG2a antibody.
  • Useful anti-CD3 antibodies are commonly known in the art and e.g. WO2016180721 , which is incorporated herein by reference in its entirety, discloses one such example: OKT3.
  • the first ligand is the anti-CD3 antibody OCT3.
  • the anti-CD3 antibody comprises a) a heavy chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 1 , a CDR2 region as set forth in SEQ ID NO: 2, and a CDR3 region as set forth in SEQ ID NO: 3; and a light chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 4, a CDR2 region as set forth in SEQ ID NO: 5, and a CDR3 region as set forth in SEQ ID NO: 6.
  • the immune cells to be regulated are B cells.
  • B cells recognize antigens via their membrane-bound immunoglobulin (mlg), which, in conjunction with two transmembrane proteins Igo and I gp (CD79A and CD79B, respectively), functions as a B cell receptor (BCR).
  • mlg membrane-bound immunoglobulin
  • Igo and I gp CD79A and CD79B, respectively
  • the first ligand may also be a BCR antigen or a B-cell receptor antibody.
  • the second ligand will in a preferred embodiment be the CD40L, an antibody to CD40, and/or IL-5.
  • the first ligand of BCR is a fragment antigen-binding region (FAB) or an F(ab’)2.
  • Some embodiment of the invention relates to a multimer comprising at least two different ligands attached to a backbone molecule, wherein the ligands each bind to its corresponding immune cell receptor on the immune cell to be activated and wherein at least one ligand is BCR antigen or BCR antibody.
  • the immune cells to be activated are CAR-T cells.
  • CAR-T cells comprise a CAR (Chimeric Antigen Receptor) on their surface, which recognise (target) specific antigens.
  • CAR-T cells are used in immunotherapy where they have shown to be effective in the treatment of certain cancers.
  • the first ligand may be a CAR-T cell antigen or a CAR antibody.
  • the first immune receptor is CAR on the T cells to be regulated, e.g., activated.
  • the CAR receptor is specific for an antigen selected from the group consisting of CD19, CD20, CD22, CD39, BCM (B-cell maturation antigen), PSMA (Prostate Specific Membrane Antigen), HER2, EGFR (epidermal growth factor receptor), ENTPD (ectonucleoside triphosphate diphosphohydrolase) and any antigen, such as tumor or other disease associated antigens targeted by CAR-T cells in CAR-T cell therapy.
  • the first ligand may also be an antibody to a CAR-T cell, specific to any of the above antigens.
  • Some embodiment of the invention relates to an immune cell activating multimer, said multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill.
  • said first and second immune cell receptors are present on the same immune cell, wherein the first immune cell receptor is a CAR on a T cell and wherein the first ligand is selected from the group consisting of CD19, CD20, CD22, CD39, BCM (B-cell maturation antigen), PSMA (Prostate Specific Membrane Antigen), HER2, EGFR (epidermal growth factor receptor), ENTPD (ectonucleoside triphosphate diphosphohydrolase) and any tumor or disease associated antigen.
  • the first immune cell receptor is a CAR on a T cell and wherein the first ligand is selected from the group consisting of CD19, CD20, CD22, CD39, BCM (B-cell maturation antigen), PSMA (Prostate Specific Membrane Antigen), HER2, EGFR (epidermal growth factor receptor), ENTPD (ectonucleoside triphosphate diphosphohydrolase) and any tumor or disease associated antigen.
  • ligand comprises antigens, and the terms may be used interchangeably throughout.
  • the second ligand binds to and stimulates a second immune cell receptor present on an immune cell, said immune cell also comprising the herein above mentioned first immune cell receptor, thereby providing a co-stimulatory signal to said immune cell.
  • the second immune cell receptor is thus a co-stimulatory immune cell receptor.
  • the second ligand may thus be any useful molecule able to bind and activate a co- stimulatory immune cell receptor.
  • the second ligand is an antibody. In some embodiments, the second ligand is an IgG antibody.
  • the second ligand is an antibody to an immune cell receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS-1 , CD2, CD28, CD40, CD134, CD152 (CTLA-4), CD40L, an ITAM, an ITIM, PD-1 , TIGIT, LAG-3, and TIM-4.
  • an immune cell receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS-1 , CD2, CD28, CD40, CD134, CD152 (CTLA-4), CD40L, an ITAM, an ITIM, PD-1 , TIGIT, LAG-3, and TIM-4.
  • the second ligand is an antibody to a second immune cell receptor wherein the antibody is specific to a second immune receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D.
  • a second immune receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D.
  • the second ligand is a fragment antigen-binding region (Fab region) to a second immune cell receptor wherein the Fab region is specific to a second immune receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1 ), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D
  • Fab region fragment antigen-binding region
  • ligands for stimulating co-stimulatory immune cell receptors are generally known in the art and may likewise be used as the second ligand. See e.g. Bryceson et al., 2006, which is incorporated herein for reference, for an overview of ligands for activation, co-activation, and co-stimulation of resting human NK cells. Antibodies to the targets of these ligands may likewise be used as the second ligand. In some embodiments, the second ligand is an anti-CD28 antibody.
  • Useful anti-CD28 antibodies are commonly known in the art and e.g. WO2016180721 , which is incorporated herein by reference in its entirety, discloses one such example: 15E8.
  • the second ligand is the anti-CD28 antibody 15E8.
  • the anti-CD28 antibody comprises a) a heavy chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 7, a CDR2 region as set forth in SEQ ID NO: 8, and a CDR3 region as set forth in SEQ ID NO: 9; and b) a light chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 10, a CDR2 region as set forth in SEQ ID NO: 11 , and a CDR3 region as set forth in SEQ ID NO: 12.
  • anti-CD28 antibodies include the “clone cd28.2” described in Pages et al., 1994, and Esensten et al., 2016, these references being incorporated herein by refence in their entirety.
  • the second ligand is the anti-CD28 antibody "clone cd28.2”.
  • Activation or regulation of an immune cell may be effectuated by binding of an antibody to the immune cell receptor or by binding a ligand to the immune cell receptor.
  • the second ligand to a second immune cell receptor is selected from the group consisting of ICAM 1-5, JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, QX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5.
  • the second ligand is a cytokine.
  • Said cytokine may be selected from any useful immune cell stimulating cytokines known to the skilled person.
  • the second ligand is selected from the group consisting of interferons, interleukins, and tumor necrosis factors.
  • said interferons are selected from the group consisting of IFN-alpha, IFN-beta, and IFN- gamma.
  • said interleukins are selected from the group consisting of IL-1 , IL-2, IL-3, IL-4, IL5, IL-6, IL-7, IL-8, IL-10, IL-11 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, -IL-29, IL-30, IL-31 , IL- 32, IL-33, IL-34 and IL-35.
  • said tumor necrosis factors are TNF- alpha or TNF-beta.
  • the second ligand is selected from the group consisting of a CSF, c-Kit, EPO, TGF- , CD40L, CD27L, CD30L, and MIF.
  • the second ligand is the cytokine IL-2 or IL-15.
  • the immune cells to be regulated e.g., activated, optionally expanded is a B cell and the first ligand of the multimer of the invention is an antigen or an antibody binding the BCR and the second ligand is the cytokine IL-5.
  • each of said first ligands such as each of said peptide-MHC monomers
  • is associated with one or more multimerization domains such as a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable of binding said peptide-MHC monomers.
  • a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable
  • each of said second ligands such as each of said anti-CD28 antibodies, is associated with one or more multimerization domains, such as a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable of binding said peptide-MHC monomers.
  • a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable of binding said
  • each of said first and second ligands such as each of said peptide-MHC monomers and said anti-CD28 antibodies, is associated with one or more multimerization domains, such as a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable of binding said peptide- MHC monomers.
  • a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules
  • multimerization domain can be any type of molecule that is directly or indirectly associated with one or more MHC molecules and/or peptide-MHC monomers.
  • a multimerization domain is a molecule, a complex of molecules, or a solid support, to which one or more MHC molecules and/or peptide-MHC monomers can be attached.
  • a multimerization domain can consist of one or more carriers and/or one or more scaffolds and may also contain one or more linkers connecting carrier to scaffold, carrier to carrier, and/or scaffold to scaffold.
  • the multimerization domain may also contain one or more linkers that can be used for attachment of MHC molecules and/or peptide-MHC monomers and/or other molecules to the multimerization domain.
  • a multimerization domain will in one embodiment refer to a functionalized polymer (e.g., dextran) that is capable of reacting with MHC molecules and/or peptide- MHC monomers, thus covalently attaching the MHC molecules and/or peptide-MHC monomers to the multimerization domain, or that is capable of reacting with scaffold molecules (e.g., streptavidin), thus covalently attaching streptavidin to the multimerization domain; the streptavidin then may bind the MHC molecules and/or peptide-MHC monomers.
  • a functionalized polymer e.g., dextran
  • scaffold molecules e.g., streptavidin
  • Multimerization domains include IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, dextran, modified dextran, streptavidin dextran, avidin dextran, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs that can bind MHC molecules and/or peptide-MHC monomers and other molecules, such as identified in detail herein elsewhere.
  • Non-limiting examples of suitable multimerization domain(s) are polysaccharides including dextran molecules, carboxy methyl dextran, dextran polyaldehyde, carboxymethyl dextran lactone, modified dextran, streptavidin dextran, avidin dextran and cyclodextrins, pullulans, schizophyllan, scleroglucan, xanthan, gellan, O-ethylamino guaran, chitins and chitosans indlucing 6-0- carboxymethyl chitin and N-carboxymethyl chitosan, derivatised cellolosics including carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose, hydroxy- ethyl cellulose, 6-amino-6-deoxy cellulose and O-ethyl- amine cellulose, hydroxylated starch, hydroxypropyl starch, hydroxyethyl starch, carrageenans, alginates, and
  • Extract is a complex, branched polysaccharide made of glucose molecules joined into chains of varying lengths.
  • the straight chain consists of a1->6 glycosidic linkages between glucose molecules, while branches begin from a1->3 linkages (and in some cases, a1->2 and a1->4 linkages as well).
  • said first ligands such as said peptide-MHC monomers
  • said second ligands such as said anti-CD28 antibodies
  • said first and second ligands are linked to a backbone molecule.
  • the backbone molecule comprises polysaccharides, including glucans such as dextran. In some embodiments, said backbone molecule comprises dextran such as modified dextran, streptavidin dextran and avidin dextran.
  • the multimerization domain is a streptavidin-dextran conjugate, wherein the streptavidin is covalently linked to the dextran, and wherein the dextran backbone size is selected from the group consisting of 70 kDa, 250 kDa, 500 kDa and 2000 kDa.
  • the multimerization domain is a streptavidin-dextran conjugate, wherein the streptavidin is covalently linked to the dextran, and wherein the number of streptavidins per dextran is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 and 40.
  • the multimerization domain is a streptavidin-dextran conjugate, wherein the streptavidin is covalently linked to the dextran, and wherein the number of streptavidins is selected from the group consisting of from 2 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35 and 35 to 40 steptavidins per dextran or any combination of these intervals.
  • the multimerization domain is a Streptavidin-dextran conjugate, wherein the streptavidin is covalently linked to the dextran, and wherein the number of streptavidins is at least 2, such as at least 5, such as at least 10, such as at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40 streptavidins per dextran.
  • the multimerization domain is a streptavidin-dextran or avidindextran conjugate that has been created by first activating dextran with divinyl sulfone, and subsequently coupling streptavidin and/or avidin to the activated dextran.
  • the multimerization domain consists of or comprises carboxy dextran.
  • the multimerization domain consists of or comprises aminodextran, functionalized with primary amine.
  • the multimerization domain consists of or comprises an antibody- dextran conjugate.
  • the multimerization domain consists of or comprises a soluble dextran backbone.
  • the dextran is a linear and/or linear branched dextran. In another embodiment the dextran is a bead or beadlike structure In one embodiment the first ligand and the second ligands are covalently linked to dextran. In another embodiment the first ligand and the second ligands are non- covalently linked to dextran. In another embodiment the first ligand is non-covalently linked to dextran and the second ligand is covalently linked to dextran. In a further embodiment the first ligand is covalently linked to dextran and the second ligand is non- covalently linked to dextran.
  • the backbone molecule is a protein. In some embodiments, the backbone molecule is a streptavidin. In some embodiments, the backbone molecule is a coiled-coil polypeptide. In some embodiments, the backbone molecule is a streptactin.
  • two or more of said ligands are coupled to the backbone covalently or non-covalently, such as through one or more connector molecules, including a streptavidin-biotin binding and/or avidin-biotin; or and/or via the MHC heavy chain and/or via light chain (B2M).
  • said first ligand is said peptide-MHC monomer and said second ligand is said anti-CD28 antibody.
  • the multimer may comprise more ligands than said first and said second ligand.
  • the multimer comprises one or more additional ligands each capable of binding additional immune cell receptors different to the first and second immune cell receptors.
  • Said additional immune cell receptors are preferably present on the same immune cell as the first and second immune cell receptors.
  • the multimer comprises the peptide-MHC monomer as disclosed elsewhere herein, the anti-CD3 antibody as disclosed elsewhere herein and the anti-CD28 antibody as disclosed elsewhere herein.
  • the multimer comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 1 1 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least 17, such as at least 18, such as at least 19, such as at least 20, such as at least 21 , such as at least 22, such as at least 23, such as at least 24, such as at least 25, such as at least 25, such as at least 26, such as at least 27, such as at least 28, such as at least 29, such as at least 30 molecules of said first ligand.
  • the multimer comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 1 1 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least
  • the multimer comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 1 1 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least
  • the multimer comprises no more than 30, such as no more than 29, such as no more than 28, such as no more than 27, such as no more than 26, such as no more than 25, such as no more than 24, such as no more than 23, such as no more than 22, such as no more than 21 , such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11 , such as no more than 10, such as no more than 9, such as no more than 8, such as no more than 7, such as no more than 6, such as no more than 5, such as no more than 4, or such as no more than 5 molecules of said first ligand, such as of said peptide-MHC monomer and/or said anti-CD3 antibody.
  • the multimer comprises 2 to 30, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, or such as 10 to 20 molecules of said first ligand, such as of said peptide-MHC monomer and/or said anti-CD3 antibody, or any combination of these intervals.
  • the multimer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 molecules of said first ligand, such as of said peptide-MHC monomer and/or said anti-CD3 antibody.
  • the multimer comprises at least 1 , at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least 17, such as at least 18, such as at least 19, such as at least 20, such as at least 21 , such as at least 22, such as at least 23, such as at least 24, such as at least 25, such as at least 25, such as at least 26, such as at least 27, such as at least 28, such as at least 29, such as at least 30 molecules of said second ligand.
  • at least 1 at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least 17,
  • the multimer comprises at least 1 , at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least 17, such as at least 18, such as at least 19, such as at least 20, such as at least 21 , such as at least 22, such as at least 23, such as at least 24, such as at least 25, such as at least 25, such as at least 26, such as at least 27, such as at least 28, such as at least 29, such as at least 30 molecules of said anti-CD28 antibody.
  • at least 1 at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least
  • the multimer comprises no more than 30, such as no more than 29, such as no more than 28, such as no more than 27, such as no more than 26, such as no more than 25, such as no more than 24, such as no more than 23, such as no more than 22, such as no more than 21 , such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11 , such as no more than 10, such as no more than 9, such as no more than 8, such as no more than 7, such as no more than 6, or such as no more than 5 molecules of said second ligand, such as of said anti-CD28 antibody.
  • the multimer comprises 1 to 30, such as 1 to 2, such as 2 to 3, such as 3 to 4, such as 4 to 5, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, or such as 10 to 20 molecules of said second ligand, such as of said anti- CD28 antibody, or any combination of these intervals.
  • the multimer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 molecules of said second ligand, such as of said anti-CD28 antibody, or any combination of these intervals.
  • the multimer comprises a total number of molecules of said first and second ligands of at the most 50, such as at the most 49, such as at the most 48, such as at the most 47, such as at the most 46, such as at the most 45, such as at the most 44, such as at the most 43, such as at the most 42, such as at the most 41 , such as at the most 40, such as at the most 39, such as at the most 38, such as at the most 37, such as at the most 36, such as at the most 35, such as at the most 34, such as at the most 33, such as at the most 32, such as at the most 31 , such as at the most 30, such as at the most 29, such as at the most 28, such as at the most 27, such as at the most 26, such as at the most 25, such as at the most 24, such as at the most 23, such as at the most 22, such as at the most 21 , such as at the most 20, such as at the most 19, such as at the most 18, such as at the most 17, such as at the
  • the multimer comprises a total number of molecules of said first and second ligands of at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 1 1 , such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least 17, such as at least 18, such as at least 19, such as at least 20, such as at least
  • At least 21 such as at least 22, such as at least 23, such as at least 24, such as at least 25, such as at least 25, such as at least 26, such as at least 27, such as at least 28, such as at least 29, such as at least 30, such as at least 31 , such as at least 32, such as at least 33, such as at least 34, such as at least 35, such as at least 36, such as at least 37, such as at least 38, such as at least 39, such as at least 40, such as at least 41 , such as at least 42, such as at least 43, such as at least 44, such as at least 45, such as at least 46, such as at least 47, such as at least 48, such as at least 49, or such as at least 50.
  • at least 41 such as at least 42, such as at least 43, such as at least 44, such as at least 45, such as at least 46, such as at least 47, such as at least 48, such as at least 49, or such as at least 50.
  • the multimer comprises a total number of molecules of said first and second ligands of from 5 to 50, such as from such 8 to 45, as from 10 to 40, such as from 10 to 35, such as from 12 to 30, such as from 15 to 30, such as from 15 to 25 or such as from 15 to 20.
  • total number of molecules of said first and second ligands denotes the total number of the first ligand in a given multimer added together with the total number of the second ligand in said same multimer.
  • the multimer according to the invention comprises a first ligand, a second ligand and at least one additional different ligand, such additional ligand may be selected from the group of first ligands and/or the group of second ligands.
  • the multimer of the invention may comprise two different ligands selected from the group of first ligands and one ligand selected from the group of second ligands and vice versa.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising a plurality of; a) a first ligand to a T cell, such as the T cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising a plurality of; a) a first ligand to a T cell, such as the T cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell and wherein and the second ligand is selected from the group consisting of;
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • a ligand selected from the group consisting of ICAM 1 -5, JAM-A, NKG2DL, ICOS- L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1 A, GITRL, IgA, TRAF2 and TRAF5; and
  • a cytokine such as IFN-alpha, IFN-beta, IFN-gamma, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, -IL-29, IL-30, IL-31 , IL-32, IL-33, IL- 34, IL-35, TGF-p, TNF-alpha and TNF-beta.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising a plurality of; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell, wherein the first ligand is a MHC-peptide and the second ligand is selected from the group consisting of;
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1 ), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • a ligand selected from the group consisting of ICAM 1 -5, JAM-A, NKG2DL, ICOS- L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1 A, GITRL, IgA, TRAF2 and TRAF5; and III.
  • a cytokine such as IFN-alpha, IFN-beta, IFN-gamma, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, -IL-29, IL-30, IL-31 , IL-32, IL-33, IL- 34, IL-35, TGF-p, TNF-alpha and TNF-beta.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising a plurality of; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell, wherein the first ligand is an anti-CD3 antibody, and the second ligand is selected from the group consisting of;
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2, TRAF5; and
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising a plurality of; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell, wherein the first ligand is a CAR-T cell ligand, preferably CD19 or BCM, and the second ligand selected from the group consisting of:
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising plurality of; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell, wherein the first ligand is a B cell receptor antigen or an antibody to the B cell receptor and the second ligand selected from the group consisting of an antibody to CD40, CD40L and IL-5.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising plurality of; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell, wherein the multimer comprises a plurality of MHC-peptide and optionally a plurality of an anti-CD3 antibody and wherein the second ligand selected from the group consisting of:
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising a plurality of; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell, wherein the multimer comprises a plurality of a MHC-peptide comprising a peptide complexed to an MHC molecule and optionally a plurality of an anti-CD3 antibody and an anti-CD28 antibody.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor; and c) a third ligand of a third immune cell receptor, wherein i. said first, second and third ligands are different; ii. said first, second and third immune cell receptors are different; and ill. said first, second and third immune cell receptors are present on the same immune cell.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor; and c) a third ligand of a third immune cell receptor, wherein i. said first, second and third ligands are different; ii. said first, second and third immune cell receptors are different; and ill. said first, second and third immune cell receptors are present on the same immune cell, wherein the activating multimer comprises a plurality of a T cell receptor ligands and wherein the second and third ligand is selected from the group consisting of:
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor; and c) a third ligand of a third immune cell receptor, wherein i. said first, second and third ligands are different; ii. said first, second and third immune cell receptors are different; and iii. said first, second and third immune cell receptors are present on the same immune cell, wherein the activating multimer comprises a plurality of MHC-peptide and wherein the second and third ligand is selected from the group consisting of:
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor; and c) a third ligand of a third immune cell receptor, wherein i. said first, second and third ligands are different; ii. said first, second and third immune cell receptors are different; and iii. said first, second and third immune cell receptors are present on the same immune cell, wherein the activating multimer comprises a plurality of anti-CD3 antibody and wherein the second and third ligand is selected from the group consisting of:
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1 ), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and III.
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor; and c) a third ligand of a third immune cell receptor, wherein i. said first, second and third ligands are different; ii. said first, second and third immune cell receptors are different; and ill. said first, second and third immune cell receptors are present on the same immune cell, wherein the activating multimer comprises a plurality of CAR-T cell receptor ligands, preferably CD19 or BCM, and wherein the second and third ligand is selected from the group consisting of:
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and
  • One preferred embodiment relates to an immune cell activating multimer, said multimer comprising; a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor; and c) a third ligand of a third immune cell receptor, wherein i. said first, second and third ligands are different; ii. said first, second and third immune cell receptors are different; and iii. said first, second and third immune cell receptors are present on the same immune cell, wherein the first ligand is a B cell receptor antigen or an antibody to the B cell receptor and the second ligand and/or third ligand is selected from the group consisting of; an antibody to CD40, CD40L and IL-5.
  • an immune cell regulating multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and iii. said first and second immune cell receptors are present on the same immune cell, wherein the first ligand is a plurality of a MHC-peptide monomer or a plurality of an anti-CD3 antibody and wherein the second ligand is selected from the group consisting of
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, CD80, CD86, 0X40 (CD134), CD152 (CTLA-4), CD160, CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, BTLA, SLAM family, CD40L, an ITAM, an ITIM, PD-1 , PD-L1 , TIM-1 , TIM-3, TIM-4, TIGIT, LAG-3, LAIR1 and NKG2D; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2, TRAF5, PD-L1 , HVEM, CECAM-1 , PTPN6, CD28, PD1 , EPO, SCF and EPO; and
  • an immune cell inhibiting multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell, wherein the first ligand is a plurality of a MHC-peptide monomer or a plurality of an anti-CD3 antibody and wherein the second ligand is selected from the group consisting of
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of CTLA4 (CD152), PD1 , BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1 (CD305), CD80, CD86, PD-L1 and TIGIT; and
  • the immune cell activating multimer is an artificial antigen presenting scaffold, that is decorated with specific MHC-peptide complexes and CD28 engaging antibodies or ligands.
  • MHC-peptide binds the specific T Cell Receptor (TCR) and delivers a TCR specific signal (signal 1 ) and the CD28 engaging antibodies/ligands bind CD28 providing a co-stimulatory signal through CD28 (Signal 2).
  • TCR T Cell Receptor
  • Signal 1 the CD28 engaging antibodies/ligands
  • CD28 providing a co-stimulatory signal through CD28
  • One embodiment relates to an immune cell activating multimer, said multimer comprising a plurality of a) a first ligand of a first immune cell receptor, wherein the first ligand is MHCp (MHC- peptide); and b) a second ligand of a second immune cell receptor, wherein the second ligand is an antibody or a ligand to CD28, and wherein said first and second immune cell receptors are present on the same immune cell.
  • MHCp MHC- peptide
  • the immune cell activating multimer is an artificial antigen presenting scaffold, that is decorated with specific ligands (ligand 1 ) able to bind TCR and one or more second ligands (ligand 2) able to bind other immune receptors on the surface of a T cell important for T cell activation.
  • ligand 1 binds the specific T Cell Receptor (TCR) and deliver a TCR specific signal (signal 1 ) and the second ligand bind another immune receptor on same cell providing a costimulatory signal (signal 2).
  • the multimer may comprise one or more additional useful modifications.
  • the first and/or the second ligands may comprise one or more additional useful modifications.
  • the multimer, the first and/or the second ligand comprises at least one DNA barcode oligonucleotide.
  • said at least one DNA barcode oligonucleotide barcode comprises a primer region, a barcode sequence that specifies the MHC-peptide specificity, and a capture region.
  • said at least one oligonucleotide barcode comprises two primer regions, a barcode sequence that specifies the MHC-peptide specificity, and optionally a capture region.
  • the at least one oligonucleotide barcode is coupled to the backbone, covalently or non-covalently, such as through one or more connector molecules, including a streptavidin-biotin binding and/or a avidin-biotin binding.
  • the multimer, the first and/or the second ligand of the present disclosure comprises one or more labels. In one embodiment the multimer, the first and/or the second ligand of the present disclosure comprises at least two labels. These labels can all be different or identical, or some the labels can be identical and some different. In some embodiments of the present disclosure, all labels are different or at least two of the labels are different. In some embodiments of the present disclosure, all labels are identical or at least two of the labels are identical.
  • the one or more labels comprise at least one fluorescent label and/or at least one oligonucleotide label.
  • the one or more labels are directly attached to the multimer, the first and/or the second ligand. In one embodiment the one or more labels are indirectly attached to the multimer, the first and/or the second ligands, such as via one or more marker molecules carrying one or more labels.
  • the one or more labels may be used for combinatorial use of labelling.
  • the one or more labels may result in positive selection of said multimer or alternatively in negative selection of said multimer.
  • the one or more labels may comprise one or more covalently attached labels and/or one or more non-covalently attached labels.
  • the one or more labels may be covalently attached to “polypeptide a” of the MHC molecule and/or peptide-MHC monomer, covalently attached to “polypeptide b” of the MHC molecule and/or peptide-MHC monomer, covalently attached to the peptide and/or covalently attached to the one or more multimerization domains.
  • the one or more labels may be non-covalently attached to “polypeptide a” of MHC molecule and/or peptide-MHC monomer, non-covalently attached to “polypeptide b” of the MHC molecule and/or peptide-MHC monomer, non-covalently attached to the peptide and/or non- covalently attached to the one or more multimerization domains.
  • the one or more labels may be covalently and/or non-covalently attached to the multimerization domain via a molecule, wherein the molecule e.g., may be selected from the group consisting of an antibody, an aptamer, a protein, a sugar residue, and a nucleotide such as DNA.
  • the one or more labels are attached to the multimer, the first and/or the second ligand via a streptavidin-biotin linkage.
  • Label is used interchangeable with labeling molecule.
  • Label as described herein is an identifiable substance that is detectable in an assay and that can be attached to a molecule creating a labeled molecule. The behavior of the labeled molecule can then be studied. Labels may be organic or inorganic molecules or particles.
  • labels include, but are not limited to, polymers, nucleic acids, DNA, RNA, oligonucleotides, peptides, fluorescent labels, phosphorescent labels, enzyme labels, chemiluminescent labels, bioluminescent labels, haptens, antibodies, dyes, nanoparticle labels, elements, metal particles, heavy metal labels, isotope labels, radioisotopes, stable isotopes, chains of isotopes and single atoms, or combination thereof.
  • the labelling compound may suitably be selected from fluorescent labels such as 5-(and 6)- carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeston Red, Green fluorescent protein (GFP) and analogues thereof, and conjugates of R-phycoerythrin or allophycoerythrin and e.g.
  • fluorescent labels such as 5-(and 6)- carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido
  • Cy5 or Texas Red and inorganic fluorescent labels based on semiconductor nanocrystals (like quantum dot and QdotTM nanocrystals), and time-resolved fluorescent labels based on lanthanides like Eu 3+ and Sm 3+ .
  • the peptide-MHC monomer or the multimer according to the present disclosure comprises at least one oligonucleotide label.
  • nucleic acids labels are disclosed in WO 2015/188839 and WO 2015/185067, which are hereby incorporated by reference in their entirety.
  • the label is an oligonucleotide, such as a nucleic acid molecule comprising or consisting of DNA, RNA, and/or artificial nucleotides such as PLA or LNA.
  • the oligonucleotide label comprises one or more of the following components: a 5’ first primer region (forward), a barcode region, 3’ second primer region (reverse), random nucleotide region, connector molecule, stabilityincreasing components, short nucleotide linkers in between any of the above-mentioned components, adaptors for sequencing and annealing region.
  • the oligonucleotide label comprises at least a barcode region surrounded by primer regions, where the barcode region comprises a sequence of consecutive nucleic acids.
  • the oligonucleotide label comprises or consists of DNA, RNA, artificial nucleic acids and/or Xeno nucleic acid (XNA).
  • XNA Xeno nucleic acid
  • at least two different labels are attached to the peptide-MHC monomer or the multimer according to the present disclosure, such as one or more fluorescent labels and an oligonucleotide label.
  • each fluorescent label may be identical. In embodiments of the present disclosure, wherein multimers comprising at least two peptide-MHC monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label may be different. In embodiments of the present disclosure, wherein multimers comprising at least two peptide-MHC monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each oligonucleotide label may be identical.
  • each oligonucleotide label may be different. In embodiments of the present disclosure, wherein multimers comprising at least two peptide-MHC monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label and each oligonucleotide label may be identical. In embodiments of the present disclosure, wherein multimers comprising at least two peptide-MHC monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label and each oligonucleotide label may be different.
  • each fluorescent label may be identical and each oligonucleotide label may be different.
  • each fluorescent label may be different and each oligonucleotide label may be identical.
  • the multimer, the first and/or the second ligand according to the preset disclosure comprises one or more fluorescent labels.
  • the multimer, the first and/or the second ligand according to the preset disclosure comprises one or more fluorescent labels selected from the group of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, 2-4'-maleimidylanilino)naphthalene-6- sulfonic acid sodium salt, 5-(((2- iodoacetyl)amino)ethyl)amino), naphthalene-1 -sulfonic acid, Pyrene-1 -butanoic acid, AlexaFluor 350 (7-amino-6-sulfonic acid-4-methyl coumarin-3-acetic acid, AMCA (7- amino-4-methyl coumarin-3-acetic acid), 7
  • the one or more labels are in specific embodiments selected from the group consisting of APC, APC-Cy7, ABC-H7, APC-R700, Alexa FloursTM 488, Alexa FloursTM555, Alexa FloursTM647, Alexa FloursTM700, AmCyan, BB151 , BB700, BUV395, BUV496, BUV563, BUV615, BUV661 , BUV737, BUV805, BV421 , BV480, BV510, BV605, BV71 1 , BV750, BV786, FITC, PE, PE-CF594, PE-Cy5, PE-CY5.5, PE-cy7, Pasific Blue, PERCP, pPerCp-Cy5.5, PE, R718, RY586, V450 and V500 (wherein in BV means Brilliant violet, wherein BLIV means Brilliant ultra violet and PE means R-Phycoerythrin).
  • the one or more labels can be selected from the group consisting of cFluor®B515, cFluor®B532, cFluor®B548, cFluor®B675, cFluor®B690, cFluor®BY575, cFluor®BY610, cFluor®BY667, cFluor®BY710, cFluor®BY750, cFluor®BY781 , cFluor®B250, cFluor®R659, cFluor®R668, cFluor®R685, cFluor®R720, cFluor®R780, cFluor®R840, cFluor®v420, cFluor®v547, cFluor®v450, cFluor®V610 and cFl
  • the multimer, the first and/or the second ligand of the present disclosure comprises one or more chemiluminescent labels, such as one or more labels selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • chemiluminescent labels such as one or more labels selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • the multimer, the first and/or the second ligand of the present disclosure comprises one or more bioluminescent labels, such as one or more labels selected from the group consisting of luciferin, luciferase and aequorin.
  • the multimer, the first and/or the second ligand of the present disclosure comprises one or more enzyme labels, such as one or more enzyme labels selected from the group peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • enzyme labels such as one or more enzyme labels selected from the group peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-
  • the multimer, the first and/or the second ligand of the present disclosure comprises one or more chromophore labels.
  • the multimer, the first and/or the second ligand of the present disclosure comprises one or more metal labels. In one embodiment the multimer, the first and/or the second ligand of the present disclosure comprises one or more radioactive labels such as one or more labels selected from the group consisting of a radionuclide, an isotope, a label comprising a rays, a label comprising rays or a label comprising y rays.
  • said multimer further comprises one or more additional cytokines.
  • said one or more additional cytokines may be different cytokines than said second ligand.
  • one or more additional cytokines are interferons.
  • the interferons are selected from the group consisting of IFN-alpha, IFN- beta, and IFN-gamma.
  • one or more additional cytokines are interleukins.
  • the interleukins are selected from the group consisting of IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL- 21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, -IL-29, IL-30, IL-31 , IL-32, IL-33, IL-34 and IL-35.
  • one or more additional cytokines are tumor necrosis factors.
  • the tumor necrosis factors are TNF-alpha or TNF-beta.
  • one or more additional cytokines are selected from the group consisting of a CSF, c-Kit, EPO, TGF-p, CD40L, CD27L, CD30L, and MIF.
  • nucleic acid encoding the multimer as disclosed elsewhere herein.
  • said nucleic acid comprises or consists of DNA.
  • said nucleic acid comprises or consists of RNA.
  • a vector comprising the nucleic acid as disclosed elsewhere herein.
  • the vector may comprise a promoter operably linked to said nucleic acid.
  • composition comprising the multimer as disclosed elsewhere herein, the nucleic acid as disclosed elsewhere herein and/or the vector as disclosed elsewhere herein.
  • compositions comprising the multimer as disclosed elsewhere herein. In some embodiments of the present disclosure is provided a composition comprising the nucleic acid as disclosed elsewhere herein. In some embodiments of the present disclosure is provided a composition comprising the vector as disclosed elsewhere herein.
  • the technical effects provided by the present invention may additionally be achieved by providing the first and second ligands as disclosed elsewhere herein on separate molecules within the same composition.
  • composition comprising a) a first multimer comprising a first ligand of a first immune cell receptor; and b) a second multimer comprising a second ligand of a second immune cell receptor, wherein said first and second multimers are defined as disclosed elsewhere herein except that said first and second ligands are comprised in separate multimers.
  • a composition for the purposes of the present disclosure may instead comprise at least two different multimers (a first and a second multimer), each comprising either the first ligand or the second ligand as disclosed elsewhere herein.
  • the first multimer may comprise the first ligand as disclosed elsewhere herein.
  • the first multimer and/or the first ligand comprised in said multimer may comprise any modifications as disclosed elsewhere herein.
  • the second multimer may comprise the second ligand as disclosed elsewhere herein.
  • the second multimer and/or the second ligand comprised in said multimer may comprise any modifications as disclosed elsewhere herein.
  • the composition comprises a plurality of the first multimer and a plurality of the second multimer.
  • the composition comprises at least 5, such as at least 10, such as at least 25, such as at least 50 of the first multimer and at least 5, such as at least 10, such as at least 25, such as at least 50 of the second multimer. Provision of the first and second ligands on separate molecules may produce similar effective and robust activation of immune cells as provision of first and second ligands on a single molecule.
  • composition comprising a) a first multimer comprising a plurality of a first ligand of a first immune cell receptor and optionally a plurality of at least one additional ligand; and b) a second multimer comprising a plurality of a second ligand of a second immune cell receptor and optionally a plurality of at least one additional ligand, wherein said first and second multimers are defined as disclosed elsewhere herein except that said first and second ligands are comprised in separate multimers.
  • first and the second multimers are not the same / not identical, and the first and second ligand are not identical but selected from the same group of ligands.
  • first and second ligand are not identical but selected from the same group of ligands.
  • Such an example includes a first multimer comprising a first ligand which may be any of those described under the heading “first ligand” and a second multimer comprising a second ligand which is also selected from the same group but the first and second ligand is different.
  • the additional ligands may be selected from any of the ligands under the heading “second ligand”.
  • composition comprising a) a first multimer comprising a plurality of a first ligand of a first immune cell receptor and optionally a plurality of at least one additional ligand; and b) a second multimer comprising a plurality of a second ligand of a second immune cell receptor and optionally a plurality of at least one additional ligand, wherein the first and second ligands are comprised in separate multimers, and wherein the first and second ligand is selected from the group consisting of; i. MHC-peptide comprising a peptide complexed to an MHC molecule, and ii.
  • an antibody to a second immune cell receptor preferably wherein the antibody is directed to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; and
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and
  • composition comprising a) a first multimer comprising a plurality of a first ligand of a T cell; and b) a second multimer comprising a plurality of ligands selected from the group consisting of; i. MHC-peptide, ii. anti-CD3 antibody, ill.
  • an antibody to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1 ), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; iv. II.
  • LFA-1 LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1 ), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; iv. II.
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and v.
  • composition comprising a) a first multimer comprising a plurality of MHC-peptide; and b) a second multimer comprising a second ligand of a second immune cell receptor, wherein the second multimer comprising a plurality of a second ligands selected from the list consisting of; i.
  • an antibody to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; ii. II.
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and ill.
  • composition comprising c) a first multimer comprising a plurality of anti-CD3 antibody; and d) a second multimer comprising a second ligand of a second immune cell receptor, wherein the second multimer comprising a plurality of a second ligands selected from the list consisting of; i.
  • an antibody to a receptor is selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1 ), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1 ), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D, preferably an anti CD28 antibody; ii. II.
  • ICAM 1 -5 JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5; and ill.
  • the composition comprises a plurality of the first multimer and a plurality of the second multimer.
  • the composition comprises at least 5, such as at least 10, such as at least 25, such as at least 50 of the first multimer and at least 5, such as at least 10, such as at least 25, such as at least 50 of the second multimer.ln some embodiments of the present disclosure, the composition comprises at least 1 , such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or 1000 multimers according to the present disclosure.
  • the composition further comprises one or more cytokines in solution such as one cytokine, two cytokines, three cytokines or more than three cytokines in solution - i.e. not attached to the multimerization domain.
  • one or more cytokines in solution such as one cytokine, two cytokines, three cytokines or more than three cytokines in solution - i.e. not attached to the multimerization domain.
  • the one or more cytokines can in one aspect be selected from the group consisting of IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, -IL-29, IL-30, IL-31 , IL- 32, IL-33, IL-34, IL-35, TGF-p, TNF-alpha or TNF-beta, depending on the target cell to be activated.
  • the composition further comprises one or more pH-stabilizing (buffer) components, such as one or more pH-stabilizing (buffer) components selected from the group consisting of Tris, MES, MOPS, phosphate, carbonate, Bis-tris and HEPES.
  • buffer pH-stabilizing
  • the composition further comprises one or more salts, such as one or more salts selected from the group consisting of NaCI, CaCL, and L-arginine.
  • the composition further comprises one or more stabilizers, such as one or more stabilizers selected from the group consisting of glycerol, PEG and BSA.
  • stabilizers such as one or more stabilizers selected from the group consisting of glycerol, PEG and BSA.
  • the composition comprises one or more pH-stabilizing (buffer) components, one or more salts and one or more stabilizers.
  • the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable diluent, carrier and/or excipient.
  • the composition as disclosed herein is capable of stimulating and activating an immune cell. In some embodiments, the composition as disclosed herein is capable of stimulating, activating and causing expansion of an immune cell.
  • said immune cell is a CD3+ T cell. In some embodiments, said immune cell is a CD8+ T cell. In some embodiments, said immune cell is a CD4+ T cell. In some embodiments, said immune cell is a CD4+CD8+ (double positive) T cell. In some embodiments, said immune cell is a CD4-CD8- (double negative) T cell. In some embodiments, said immune cell is an MAIT cell. In some embodiments, said immune cell is an iNKT cell. In some embodiments, said immune cell is a B cell. In some embodiments, said immune cell is an NK cell.
  • a method of treatment of a disease associated with immune evasion in a subject in need thereof comprising the step(s) of: a) administering the pharmaceutical composition as disclosed elsewhere herein to said subject in a therapeutically effective amount, or a) isolating immune cells, from said subject; b) contacting said immune cells with one or more multimers as disclosed elsewhere herein, or with a composition as disclosed elsewhere herein, thereby obtaining activated and expanded immune cells specific for said disease; and c) transferring said activated and expanded immune cells back into said subject.
  • said immune cells are CD3+ T cells. In some embodiments, said immune cells are CD8+ T cells. In some embodiments, said immune cells are CD4+ T cells. In some embodiments, said immune cells are CD4+CD8+ (double positive) T cells. In some embodiments, said immune cells are CD4-CD8- (double negative) T cells. In some embodiments, said immune cells are MAIT cells. In some embodiments, said immune cells are iNKT cells. In some embodiments, said immune cells are B cells. In some embodiments, said immune cells are NK cells.
  • a pharmaceutical composition as disclosed elsewhere herein for use as a medicament In some aspects of the present disclosure is provided a pharmaceutical composition as disclosed elsewhere herein for use in a method of treatment of a disease associated with immune evasion.
  • said disease associated with immune evasion is cancer.
  • a method for activating one or more immune cells comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; and c) contacting said multimers or said composition, with said sample, thereby activating and optionally expanding said immune cells.
  • each of the two ligands may be comprised separately in two different multimers that are provided either simultaneously or subsequently in any order.
  • a method for activating and expanding one or more immune cells comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; and c) contacting said multimers or said composition, with said sample, thereby activating and expanding said immune cells.
  • each of the two ligands may be comprised separately in two different multimers that are provided either simultaneously or subsequently in any order.
  • One of the advantages of the multimers of the invention is that they may be used to activate a target immune cell, i.e., the immune cell to be activated, such that activation is obtained faster and more specific than the commonly used methods.
  • the activation is specific when a majority of the immune cells activated are the target immune cells e.g., recognizing a particular ligand/antigen.
  • One preferred embodiment relates to a method of activating one or more immune cells, such as expanding a population of immune cells, wherein the multimer comprises a plurality of a first ligand selected from the group consisting of MHC-peptide comprising a peptide complexed to the MHC molecule, an anti-CD3 antibody, a B cell receptor antigen, a B cell receptor antibody, a CAR-T cell antigen and a CAR antibody such as also described under the heading “First ligand”.
  • a first ligand selected from the group consisting of MHC-peptide comprising a peptide complexed to the MHC molecule, an anti-CD3 antibody, a B cell receptor antigen, a B cell receptor antibody, a CAR-T cell antigen and a CAR antibody such as also described under the heading “First ligand”.
  • One preferred embodiment relates to a method of activating one or more immune cells, such as expanding a population of immune cells, wherein the multimer comprises a plurality of a second ligand as described under the heading “Second ligand” and which may be selected from any of the following ligands ICAM 1 -5, JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1A, GITRL, IgA, TRAF2 and TRAF5 and/or the second ligand may be an antibody to a second immune cell receptor, selected from an antibody to any of the following second immune cell receptors LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134
  • the multimer may comprise a plurality of ligands selected from the group of first ligands and a plurality of ligands selected from the group of second ligands, such as plurality of peptide-MHC monomer comprising a peptide complexed to the MHC molecule and a plurality of an anti-CD-28 antibody.
  • the multimers used in the method of the invention may also comprise plurality of two different first ligands such as plurality of peptide-MHC monomer comprising a peptide complexed to the MHC molecule, a plurality of an anti- CD3 antibody and a plurality of a second ligand such as a plurality of an anti-CD-28 antibody.
  • first ligands such as plurality of peptide-MHC monomer comprising a peptide complexed to the MHC molecule, a plurality of an anti- CD3 antibody and a plurality of a second ligand such as a plurality of an anti-CD-28 antibody.
  • the methods are preferably performed at temperature above room temperature such as in a range of 24 - 40°C, or in the range 30-40°C or in the range 35-40°C, such as around 24°C, around 25°C, around 26°C, around 27°C, around 28°C, around 29 °C, around 30°C, around 31 °C, around 32°C, around 33°C, around 34°C, around 35°C, around 36°C, around 37°C, around 38°C, around 39°C or such as around 40 °C.
  • the method may be performed in the range of a few hours to several weeks depending on the target immune cells and the result to be achieved, e.g., if activation is measured by expression of cytokines this may be achieved within a few days e.g., 1 to 10 days or 5 to 10 days, wherein proliferation of target immune cell may take longer such as 5 to 30 days or 10 to 20 days.
  • One specific embodiment includes a method for regulating target immune cells, where the target immune cells are not necessarily activated or stimulated.
  • Regulating the target immune cells includes in the context of the present invention any change in the target cells, such as up or down regulating certain surface markers and changes in the level of cytokine production.
  • one embodiment of the invention relates to a method for regulating one or more immune cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; and c) contacting said multimers or said composition, with said sample, thereby regulating said immune cells.
  • each of the two ligands may be comprised separately in two different multimers that are provided either simultaneously or subsequently in any order.
  • One preferred embodiment relates to a method of regulating one or more immune cells, wherein the multimer comprises a plurality of a first ligand selected from the group consisting of MHC-peptide comprising a peptide complexed to the MHC molecule, an anti-CD3 antibody, a B cell receptor antigen, a B cell receptor antibody, a CAR-T cell antigen and a CAR antibody such as also described under the heading “First ligand”.
  • a first ligand selected from the group consisting of MHC-peptide comprising a peptide complexed to the MHC molecule, an anti-CD3 antibody, a B cell receptor antigen, a B cell receptor antibody, a CAR-T cell antigen and a CAR antibody such as also described under the heading “First ligand”.
  • One preferred embodiment relates to a method of regulating one or more immune cells, wherein the multimer comprises a plurality of a second ligand as described under the heading “Second ligand” and which may be selected from any of the following ligands ICAM 1 -5, JAM-A, NKG2DL, ICOS-L, BTLA, CD160, CSF, LFA-3 (CD58), CD40L, CD30L, CD48, CD70, CD80, CD86, CD112, CD155, CD137L, OX40L, TL1 A, GITRL, IgA, TRAF2, TRAF5, PD-L1 , HVEM, CECAM-1 , PTPN6, CD28, PD1 , EPO, SCF and EPO and/or the second ligand may be an antibody to a second immune cell receptor, selected from an antibody to any of the following second immune cell receptors LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD
  • One specific embodiment includes a method for inhibiting target immune cells.
  • One embodiment of the invention relates to a method for inhibiting one or more immune cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; and c) contacting said multimers or said composition, with said sample, thereby inhibiting said immune cells.
  • each of the two ligands may be comprised separately in two different multimers that are provided either simultaneously or subsequently in any order.
  • One preferred embodiment relates to a method of inhibiting one or more immune cells, wherein the multimer comprises a plurality of a first ligand selected from the group consisting of MHC-peptide comprising a peptide complexed to the MHC molecule, an anti-CD3 antibody, a B cell receptor antigen, a B cell receptor antibody, a CAR-T cell antigen and a CAR antibody such as also described under the heading “First ligand”.
  • a first ligand selected from the group consisting of MHC-peptide comprising a peptide complexed to the MHC molecule, an anti-CD3 antibody, a B cell receptor antigen, a B cell receptor antibody, a CAR-T cell antigen and a CAR antibody such as also described under the heading “First ligand”.
  • One preferred embodiment relates to a method of inhibiting one or more immune cells, wherein the multimer comprises a plurality of a second ligands selected from the group consisting of CD80, CD86, PD-L1 , HVEM, CECAM-1 (phosphatidylserine), CD155, HVEM, PTPN6, CD28, CTLA4, CD28, CTLA4 and PD1 and/or the second ligand may be an antibody to a second immune cell receptor, selected from an antibody to any of the following second immune cell receptors CTLA4 (CD152), PD1 , BTLA, TIM3, TIGIT, CD160, LAG3, LAIR1 (CD305), CD80, CD86, PD-L1 and TIGIT and/or the second ligand may be any of the cytokines selected from IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 10, IL-1 1 , IL-12, IL-15
  • a method for isolating one or more immune cells of low quantity in a sample comprising the steps of: a) providing a sample comprising a population of immune cells in low quantity; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; c) contacting said multimers or said composition, with said sample, thereby activating said immune cells; and d) isolating said activated immune cells, such as by FACS, thereby isolating said one or more immune cells of low quantity in said sample.
  • a method for isolating one or more immune cells of low quantity in a sample comprising the steps of: a) providing a sample comprising a population of immune cells in low quantity; b) providing one or more multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; c) contacting said multimers or said composition, with said sample, thereby activating and expanding said immune cells; and d) isolating said activated and expanded immune cells, such as by FACS, thereby isolating said one or more immune cells of low quantity in said sample.
  • a method for assaying the ability of one or more immune cells in a sample to activate in response to a multimer as disclosed elsewhere herein comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more of said multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; c) contacting said multimers or said composition, with said sample; and d) measuring the activation of said immune cells, such as by FACS, thereby assaying the ability of said one or more immune cells to activate in response to said multimer.
  • a method for assaying the ability of one or more immune cells in a sample to activate in response to a multimer as disclosed elsewhere herein comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more of said multimers as disclosed elsewhere herein, or a composition as disclosed elsewhere herein; c) contacting said multimers or said composition, with said sample; and d) measuring the activation and expansion of said immune cells, such as by FACS, thereby assaying the ability of said one or more immune cells to activate in response to said multimer.
  • said one or more immune cells each comprise said first and second immune cell receptors as disclosed elsewhere herein.
  • said immune cells are CD3+ T cells. In some embodiments, said immune cells are CD8+ T cells. In some embodiments, said immune cells are CD4+ T cells. In some embodiments, said immune cells are CD4+CD8+ (double positive) T cells. In some embodiments, said immune cells are CD4-CD8- (double negative) T cells. In some embodiments, said immune cells are MAIT cells. In some embodiments, said immune cells are iNKT cells. In some embodiments, said immune cells are B cells. In some embodiments, said immune cells are NK cells. In some embodiments, said immune cells are gamma-delta T cells. In some embodiments, said immune cells are innate lymphoid cells.
  • said immune cells are granulocytes. In some embodiments, said immune cells are macrophages. In some embodiments, said immune cells are dendritic cells. In some embodiments, the immune cell is an engineered T cell, such as TCR-T. In some embodiments, the immune cell is a CAR T cell. In some embodiments, the immune cell is a MART-1 specific T cell. In some embodiments, the immune cell is a CMV specific T cell.
  • sample refers to e.g., a liquid sample or a solid sample.
  • a liquid sample include, but is not limited to, blood, lymph, cerebrospinal fluid, synovial fluid, sputum, lymph, semen, fluid cultures of cells, suspensions of solid tissue.
  • Examples of a solid sample include, but is not limited to, solid tissue, tissue sections, organ, part of organ or tissue, human bodies or any part thereof, animal bodies of any part thereof and cells embedded in a solid matrix, e.g., paraffin.
  • the sample can be derived from a human or an animal.
  • the sample is preferably a biological sample.
  • the sample is blood. In some embodiments of the present disclosure, the sample is lymph. In some embodiments of the present disclosure, the sample is cerebrospinal fluid. In some embodiments of the present disclosure, the sample is synovial fluid. In some embodiments of the present disclosure, the sample is sputum. In some embodiments of the present disclosure, the sample is lymph. In some embodiments of the present disclosure, the sample is semen. In some embodiments of the present disclosure, the sample is fluid cultures of cells.
  • the sample is suspensions of solid tissue, such as suspensions of tissue sections, organs, part of organs or tissues, human bodies or any part thereof, animal bodies of any part thereof, and cells embedded in a solid matrix, e.g., paraffin.
  • solid tissue such as suspensions of tissue sections, organs, part of organs or tissues, human bodies or any part thereof, animal bodies of any part thereof, and cells embedded in a solid matrix, e.g., paraffin.
  • the subject is a mammal. In some embodiments, the subject is a human being. In some embodiments, the subject is a non-human animal.
  • the invention may further be described by one or more of the following items.
  • An immune cell activating multimer comprising a plurality of a) a first ligand of a first immune cell receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell.
  • MHC molecule is from an organism selected from the group consisting of human, gorilla, chimpanzee, rhesus macaque and rodent, such as mouse or rat.
  • said anti-CD3 antibody is an IgG antibody, such as a mouse monoclonal lgG2a antibody.
  • said multimer according to any one of item 12 to 13, wherein said anti-CD3 antibody comprises a) a heavy chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 1 , a CDR2 region as set forth in SEQ ID NO: 2, and a CDR3 region as set forth in SEQ ID NO: 3; and b) a light chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 4, a CDR2 region as set forth in SEQ ID NO: 5, and a CDR3 region as set forth in SEQ ID NO: 6.
  • the multimer according to any one of the preceding items, wherein said second ligand is an antibody.
  • the multimer according to any one of the preceding items, wherein said second ligand is an IgG antibody.
  • the multimer according to any one of the preceding items, wherein said second ligand is an antibody to an immune cell receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS-1 , CD2, CD28, CD40, CD134, CD152 (CTLA-4), CD40L, an ITAM, an ITIM, PD-1 , TIGIT, LAG-3, and TIM-4.
  • said second ligand is an anti-CD28 antibody.
  • said anti-CD28 antibody comprises a) a heavy chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 7, a CDR2 region as set forth in SEQ ID NO: 8, and a CDR3 region as set forth in SEQ ID NO: 9; and b) a light chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 10, a CDR2 region as set forth in SEQ ID NO: 11 , and a CDR3 region as set forth in SEQ ID NO: 12.
  • each of said first and/or second ligands such as each of said peptide-MHC monomers, is associated with one or more multimerization domains, such as a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable of binding said peptide- MHC monomers.
  • a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying
  • the multimer according to any one of the preceding items wherein said multimer comprises at least one DNA barcode oligonucleotide, said at least one DNA barcode oligonucleotide comprising a primer region, a DNA barcode sequence that specifies the MHC-peptide specificity, and a capture region.
  • said first and/or second ligands, such as said peptide-MHC monomers are linked to a backbone molecule.
  • the multimer according to item 33, wherein the backbone molecule comprises polysaccharides, including glucans such as dextran. 27.
  • any one of items 38 to 43 wherein one or more labels comprise at least one oligonucleotide label, such as a nucleic acid molecule comprising or consisting of DNA, RNA, and/or artificial nucleotides, such as PLA or LNA.
  • the multimer according to any one of items 38 to 45, wherein one or more labels are oligonucleotides comprising one or more component selected from the group consisting of: a. a barcode region; b. a 5’ first primer region (forward); c.
  • a 3’ second primer region reverse
  • d a random nucleotide region
  • e a connector molecule
  • f a stability-increasing component
  • g a short nucleotide linker in between any of the above-mentioned components
  • h an adaptors for sequencing
  • i an annealing region.
  • one or more labels comprise at least one fluorescent label selected from the group consisting of PE, 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6- (fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine, Cy2, Cy3, Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeston Red and Green fluorescent protein (GFP).
  • fluorescent label selected from the group consisting of PE, 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6- (fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhod
  • one or more labels are bioluminescent labels, such as a label selected from the group consisting of luciferin, luciferase and aequorin.
  • one or more labels are enzyme labels, such as an enzyme label selected from the group consisting of peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • enzyme labels such as an enzyme label selected from the group consisting of peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate, dehydrogenase, triose phosphate
  • one or more labels are radioactive labels, such as a label selected from the group consisting of a radionuclide, an isotope, a label comprising a rays, a label comprising rays or a label comprising y rays.
  • interferons are selected from the group consisting of IFN-alpha, IFN-beta, and IFN-gamma.
  • interleukins are selected from the group consisting of IL-1 , IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL- 27, IL-28, -IL-29, IL-30, IL-31 , IL-32, IL-33, IL-34 and IL-35.
  • said additional cytokines are selected from the group consisting of a CSF, c-Kit, EPO, TGF-p, CD40L, CD27L, CD30L, and MIF.
  • said first ligand is said peptide-MHC monomer and said second ligand is said anti-CD28 antibody.
  • the multimer according to any one of the preceding items wherein said multimer comprises at least 2, such as at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30 molecules of said first ligand, such as of said peptide-MHC monomer and/or said anti-CD3 antibody.
  • the multimer according to any one of the preceding items wherein the multimer comprises no more than 30, such as no more than 25, such as no more than 20, such as no more than 15, or such as no more than 10 molecules of said first ligand, such as of said peptide-MHC monomer and/or said anti-CD3 antibody.
  • said multimer comprises 2 to 30, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, or such as 10 to 20 molecules of said first ligand, such as of said peptide-MHC monomer and/or said anti-CD3 antibody, or any combination of these intervals.
  • the multimer comprises no more than 30, such as no more than 25, such as no more than 20, such as no more than 15, or such as no more than 10 molecules of said second ligand, such as of said anti-CD28 antibody.
  • the multimer according to any one of the preceding items wherein said multimer comprises 1 to 30, such as 1 to 2, such as 2 to 3, such as 3 to 4, such as 4 to 5, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, or such as 10 to 20 molecules of said second ligand, such as of said anti-CD28 antibody, or any combination of these intervals.
  • said multimer comprises 1 to 30, such as 1 to 2, such as 2 to 3, such as 3 to 4, such as 4 to 5, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, or such as 10 to 20 molecules of said second ligand, such as of said anti-CD28 antibody, or any combination of these intervals.
  • a vector comprising the nucleic acid according to item 78.
  • a composition comprising the multimer according to any one of items 1 to 77, the nucleic acid according to item 78 and/or the vector according to item 78.
  • a composition comprising a) a first multimer comprising a first ligand of a first immune cell receptor; and b) a second multimer comprising a second ligand of a second immune cell receptor, wherein said first and second multimers are defined according to any one of items 1 to 77 except that said first and second ligands are comprised in separate multimers.
  • the composition according to any one items 79 to 16 wherein said composition is capable of stimulating and activating an immune cell.
  • composition according to any one items 79 to 82, wherein said composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable diluent, carrier and/or excipient.
  • a method for activating and optionally expanding one or more immune cells comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more multimers according to any one of items 1 to 77, or a composition according to any of items 79 to 83; and c) contacting said multimers or said composition, with said sample, thereby activating and optionally expanding said immune cells.
  • a method for isolating one or more immune cells of low quantity in a sample comprising the steps of: a) providing a sample comprising a population of immune cells in low quantity; b) providing one or more multimers according to any one of items 1 to 77, or a composition according to any of items 79 to 83; c) contacting said multimers or said composition, with said sample, thereby activating and optionally expanding said immune cells; and d) isolating said activated, or said activated and expanded, immune cells, such as by FACS, thereby isolating said one or more immune cells of low quantity in said sample.
  • a method for assaying the ability of one or more immune cells in a sample to activate in response to a multimer according to any one of items 1 to 77 comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more of said multimers according to any one of items 1 to 77, or a composition according to any of items 79 to 83; c) contacting said multimers or said composition, with said sample; and d) measuring the activation and expansion of said immune cells, such as by FACS, thereby assaying the ability of said one or more immune cells to activate in response to said multimer.
  • the immune cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, MAIT cells, gamma-delta T cells, NK cells, iNKT cells, B cells, innate lymphoid cells, granulocytes, macrophages, and dendritic cells.
  • a pharmaceutical composition according to item 83 for use as a medicament for use as a medicament.
  • a method of treatment of a disease associated with immune evasion, such as cancer, in a subject in need thereof comprising the step(s) of: a) administering the pharmaceutical composition according to item 83 to said subject in a therapeutically effective amount, or a) isolating immune cells, such as T cells, from said subject; b) contacting said immune cells with one or more multimers according to any one of items 1 to 77, or with a composition according to any of items 79 to 83, thereby obtaining activated and expanded immune cells specific for said disease, such as specific for cells of said cancer; and c) transferring said activated and expanded immune cells back into said subject.
  • Additional items include the following.
  • An immune cell activating multimer comprising a plurality of a) a first ligand of a first immune receptor; and b) a second ligand of a second immune cell receptor, wherein i. said first and second ligands are different; ii. said first and second immune cell receptors are different; and ill. said first and second immune cell receptors are present on the same immune cell.
  • MHC molecule is from an organism selected from the group consisting of human, gorilla, chimpanzee, rhesus macaque and rodent, such as mouse or rat.
  • the multimer according to item 11 wherein said first ligand is an antibody.
  • the multimer according to item 11 wherein said first ligand is an anti-CD3 antibody.
  • the multimer according to item 12, wherein said anti-CD3 antibody is an IgG antibody, such as a mouse monoclonal lgG2a antibody.
  • said anti-CD3 antibody comprises a) a heavy chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 1 , a CDR2 region as set forth in SEQ ID NO: 2, and a CDR3 region as set forth in SEQ ID NO: 3; and b) a light chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 4, a CDR2 region as set forth in SEQ ID NO: 5, and a CDR3 region as set forth in SEQ ID NO: 6.
  • the first ligand is a B-cell receptor antigen or a B-cell receptor antibody.
  • the multimer according to item 1 wherein the first ligand is selected from CAR T cells receptor antigen and a CAR antibody.
  • the multimer according to item 16 wherein the first ligand is selected from the group consisting of CD19, CD20, CD22, CD39, BCM (B-cell maturation antigen), PSMA (Prostate Specific Membrane Antigen), HER2, EGFR (epidermal growth factor receptor) and ENTPD (ectonucleoside triphosphate diphosphohydrolase).
  • the multimer according to item 16 wherein the first ligand is selected from the group consisting of an antibody to a CAR specific for any of CD19, CD20, CD22, CD39, BCM (B-cell maturation antigen), PSMA (Prostate Specific Membrane Antigen), HER2, EGFR (epidermal growth factor receptor) and ENTPD (ectonucleoside triphosphate diphosphohydrolase).
  • BCM B-cell maturation antigen
  • PSMA Prostate Specific Membrane Antigen
  • HER2 Prostate Specific Membrane Antigen
  • HER2 epidermal growth factor receptor
  • ENTPD ectonucleoside triphosphate diphosphohydrolase
  • a second immune receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18) a HVEM, SLAM family, TIM1 and NKG2D
  • the second ligand is an
  • said second ligand is an anti-CD28 antibody.
  • said anti-CD28 antibody comprises a) a heavy chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 7, a CDR2 region as set forth in SEQ ID NO: 8, and a CDR3 region as set forth in SEQ ID NO: 9; and b) a light chain variable domain comprising a CDR1 region as set forth in SEQ ID NO: 10, a CDR2 region as set forth in SEQ ID NO: 11 , and a CDR3 region as set forth in SEQ ID NO: 12.
  • said second ligand is a cytokine.
  • said second ligand is selected from the group consisting of IFN-alpha, IFN-beta, IFN-gamma, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, -IL-29, IL-30, IL-31 , IL-32, IL- 33, IL-34, IL-35, TGF-p , TNF-alpha and TNF-beta.
  • the multimer according to any one of the preceding items wherein said multimer comprises at least one DNA barcode oligonucleotide, said at least one DNA barcode oligonucleotide comprising a primer region, a DNA barcode sequence that specifies the MHC-peptide specificity, and a capture region.
  • the multimer according to any one of the preceding items, wherein the first and/or the second ligand, is linked to a backbone molecule.
  • the multimer according to item 31 , wherein the backbone molecule is a protein, such as a streptavidin, a coiled-coil polypeptide or a streptactin.
  • one or more labels comprise at least one oligonucleotide label, such as a nucleic acid molecule comprising or consisting of DNA, RNA, and/or artificial nucleotides, such as PLA or LNA.
  • oligonucleotide label such as a nucleic acid molecule comprising or consisting of DNA, RNA, and/or artificial nucleotides, such as PLA or LNA.
  • one or more labels are oligonucleotides comprising one or more component selected from the group consisting of: a. a barcode region; b. a 5’ first primer region (forward); c. a 3’ second primer region (reverse); d. a random nucleotide region; e. a connector molecule; f. a stability-increasing component; g. a short nucleotide linker in between any of the above-mentioned components; h. an adaptors for sequencing; and i. an annealing region.
  • one or more labels are oligonucleotides comprising one or more component selected from the group consisting of: a. a barcode region; b. a 5’ first primer region (forward); c. a 3’ second primer region (reverse); d. a random nucleotide region; e. a connector molecule; f. a stability-increasing component; g. a short nu
  • one or more labels comprise at least one fluorescent label selected from the group consisting of PE, 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6- (fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine, Cy2, Cy3, Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeston Red and Green fluorescent protein (GFP).
  • fluorescent label selected from the group consisting of PE, 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6- (fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhod
  • chemiluminescent labels such as a label selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • one or more labels are enzyme labels, such as an enzyme label selected from the group consisting of peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • enzyme labels such as an enzyme label selected from the group consisting of peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate, dehydrogenase, triose phosphate
  • said multimer further comprises one or more ligand(s) in addition to, and different from, the first and second ligand.
  • the multimer according to any one of the proceeding items wherein the multimer comprises at least two different ligands selected from the group consisting of first ligands of items 2 to 18.
  • the multimer according to any one of the proceeding items wherein the multimer comprises at least two different ligands selected from the group consisting of second ligands of items 20 to 30.
  • the multimer according to any one of items 54 to 56, wherein said multimer further comprises one or more additional cytokines.
  • said additional cytokines are selected from the group consisting of interferons, interleukins, and tumor necrosis factors.
  • interferons are selected from the group consisting of IFN-alpha, IFN-beta, and IFN-gamma.
  • cytokines are interleukins selected from the group consisting of IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11 , IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, -IL-29, IL-30, IL-31 , IL-32, IL-33, IL-34 and IL-35.
  • tumor necrosis factors are TNF- alpha or TNF-beta.
  • the multimer comprises one or more ligand(s) in addition the first and second ligand, wherein the one or more additional ligand(s) is selected from the group consisting of a an antibody to an immune cell receptor wherein the antibody is specific to the immune receptor selected from the group consisting of LFA-1 , 4-1 BB (CD137), ICOS (e.g., ICOS-1), CD2, CD27, CD28, CD30 (TNFRSF8), CD40, 0X40 (CD134), CD226 (DNAM-1), CD244 (2B4), DR3, GITR (TNFRSF18), an ITAM, an ITIM, a HVEM, SLAM family, MIF, TIM1 , TIM2, TIM-4 and NKG2D.
  • the multimer comprises one or more ligand(s) in addition the first and second ligand, wherein the one or more additional ligand(s) is selected from the group
  • the multimer comprises one or more ligand(s) in addition the first and second ligand, wherein the one or more ligand(s) is selected from the group consisting of ICOS-L, BTLA, CSF, c-Kit, EPO, TGF- , CD40L, CD30L, CD70, CD80, CD86, CD137L, OX40L, TL1 A, GITRL, MIF or ligands to any of the second immune cell receptors according to item 62.
  • said first ligand is said peptide-MHC monomer and said second ligand is said anti-CD28 antibody.
  • the multimer comprises no more than 30, such as no more than 25, such as no more than 20, such as no more than 15, or such as no more than 10 molecules of said first ligand, such as of said peptide-MHC monomer and/or said anti-CD3 antibody.
  • said multimer comprises 2 to 30, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, or such as 10 to 20 molecules or any combination of these intervals of said first ligand, such as of said peptide-MHC monomer, anti- CD3 antibody, BCR antigen or an antibody hereto or CAR T cell antigen.
  • the multimer comprises no more than 30, such as no more than 25, such as no more than 20, such as no more than 15, or such as no more than 10 molecules of said second ligand, such as of said anti-CD28 antibody.
  • said multimer comprises 1 to 30, such as 1 to 2, such as 2 to 3, such as 3 to 4, such as 4 to 5, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, or such as 10 to 20 molecules of said second ligand, such as of said anti-CD28 antibody, or any combination of these intervals.
  • said immune cell is selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, CD4+CD8+ T cells, CD4-CD8- T cells, MAIT cells, iNKT cells, B cells, NK cells, CAR T cells and engineered T cells, such as and TCR-T cells.
  • a vector comprising the nucleic acid according to item 78.
  • a composition comprising the multimer according to any one of items 1 to 77, the nucleic acid according to item 78 and/or the vector according to item 79.
  • a composition comprising; a) a first multimer comprising a first ligand of a first immune cell receptor; and b) a second multimer comprising a second ligand of a second immune cell receptor, wherein said first and second multimers are defined according to any one of items 1 to 77 except that said first and second ligands are comprised in separate multimers.
  • composition according to any one of items 80 to 81 wherein said composition is capable of stimulating and activating an immune cell.
  • a method for activating and optionally expanding one or more immune cells comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more multimers according to any one of items 1 to 77, or a composition according to any of items 80 to 84; and c) contacting said multimers or said composition, with said sample, thereby activating and optionally expanding said immune cells.
  • a method for isolating one or more immune cells of low quantity in a sample comprising the steps of: a) providing a sample comprising a population of immune cells in low quantity; b) providing one or more multimers according to any one of items 1 to 77, or a composition according to any of items 80 to 84; c) contacting said multimers or said composition, with said sample, thereby activating and optionally expanding said immune cells; and d) isolating said activated, or said activated and expanded, immune cells, such as by FACS, thereby isolating said one or more immune cells of low quantity in said sample.
  • a method for assaying the ability of one or more immune cells in a sample to activate in response to a multimer according to any one of items 1 to 77 comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more of said multimers according to any one of items 1 to 77, or a composition according to any of items 80 to 84; c) contacting said multimers or said composition, with said sample; and d) measuring the activation and expansion of said immune cells, such as by FACS, thereby assaying the ability of said one or more immune cells to activate in response to said multimer.
  • the immune cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, MAIT cells, gamma-delta T cells, NK cells, iNKT cells, B cells, innate lymphoid cells, granulocytes, macrophages, and dendritic cells.
  • a pharmaceutical composition according to item 84 for use as a medicament for use as a medicament.
  • a method of treatment of a disease associated with immune evasion, such as cancer, in a subject in need thereof comprising the step(s) of: a) administering the pharmaceutical composition according to item 84 to said subject in a therapeutically effective amount, or d) isolating immune cells, such as T cells, from said subject; e) contacting said immune cells with one or more multimers according to any one of items 1 to 77, or with a composition according to any of items 80 to 84, thereby obtaining activated and expanded immune cells specific for said disease, such as specific for cells of said cancer; and f) transferring said activated and expanded immune cells back into said subject.
  • a disease associated with immune evasion such as cancer
  • Example 1 Stimulation of HLA-A*0201/EBV peptide antigen specific CD8+ T cells specific by Dextramer reagents comprising MHC monomer and varying numbers of anti- CD28 Abs
  • Dextramer reagents Dextramer reagents conjugated with a fixed number of MHC monomers and various numbers of anti-CD28 per Dextramer backbone were manufactured in the following MHC/aCD28/Dextramer ratios: 12/0/1 ; 12/1/1 ; 12/6/1 .
  • the monomers were either HLA-A*0201/EBV or HLA-A*0201/ALI (Negative control).
  • the anti-CD28 were of clone CD28.2.
  • the Dextramer backbone was dextran-NONE.
  • PBMC donor cells were washed on resuspended in growth medium (XVIVO 15(Lonza) supplemented with 5% Human serum) to a cell concentration of 2x10e7/ml.
  • 1 x10e6 cells were distributed in a 96U microtiter culture plate (Sartorius), and MHC/aCD28/Dextramer reagents were added in a concentration of 5,3 nM. After 30 minutes binding phase 100 pl growth medium was added per well. After 24 h of incubation cells were washed and stained with fluorophore labelled antibodies and Dextramer-PE reagents HLA- A2*0201/EBV/Dextramer-PE. The incubation of PBMCs with HLA- A*0201/EBV/aCD28/Dextramer but not the HLA-
  • A*0201/ALI/aCD28/Dextramer resulted in an antigen specific stimulation of HLA- A*0201/EBV specific CD8+ T cells, as shown by a specific TCR down regulation (see Figure 1 ), and by the specific upregulation of activation markers, CD69 and CD137, on the cell surface (see Figure 2).
  • aCD28Ab was not essential to obtain stimulation the HLA-A*0201/EBV specific CD8+ T cells, as the stimulation was similar whether aCD28 was present or not on the stimulating Dextramer reagent, see the stimulation induced by MHC/aCD28/Dextramer containg various ratios of aCD28 ( Figure 2).
  • Example 2 Generation of nano matrices comprising MHC monomer, anti-CD28 Fabs, and parental 15E8 antibody.
  • MHC monomer and Fab variants described in example 6 of WO2016/180721 and purified parental murine antibodies specific for CD28, respectively, are used for the generation of nanomatrices.
  • Dextran is used as a mobile polymer chain.
  • magnetic nanomatrices is produced, and therefore a modified procedure of Molday and MacKenzie is used.
  • Ten grams of Dextran T40 (GE Healthcare), 1.5 g FeCh 6 H 2 0 and 0.64 g FeCI 2 4 H 2 0 are dissolved in 20 ml H 2 0, and heated to 40° C. While stirring, 10 ml 4 N NaOH are added slowly and the solution is heated to 70° C for 5 min. The particle suspension is neutralized with acetic acid.
  • HGMF high-gradient magnetic field
  • the magnetic nanomatrices are eluted with 0.05 M sodium acetate.
  • the nanomatrices will form a brown suspension.
  • the relative particle concentration is given as optical density at 450 nm.
  • the size of the nanomatrices is determined by electron microscopy and dynamic light scattering.
  • the nanomatrices should show superparamagnetic behavior, as determined by susceptibility measurements.
  • pMHC and antibodies and Fabs are conjugated to separate nanomatrices by standard bioconjugation chemistry (Bioconjugate Techniques, 2nd Edition, By Greg T. Hermanson, Published by Academic Press, Inc., 2008). After sterile filtration, the resulting nanomatrices are stored in PBS buffer/0.3% Poloxamer 188 at -70°C.
  • nanomatrices are generated: A nanomatrix comprising MHC monomer, a nanomatrix comprising parental anti-CD28 antibody 15E8, a nanomatrix comprising anti-CD28 Fab variant huCD_v3, and a nanomatrix comprising anti-CD28 Fab variant huCD3 v4.
  • Example 3 Generation of nano matrices comprising anti-CD3 Fabs, parental OKT3 antibody, anti-CD28 Fabs, and parental 15E8 antibody
  • Fab variants of example 6 of WO2016/180721 and purified parental murine antibodies specific for CD3 and specific for CD28, respectively, are used for the generation of nanomatrices.
  • Dextran is used as a mobile polymer chain.
  • magnetic nanomatrices is produced, and therefore a modified procedure of Molday and MacKenzie, 1982 is used.
  • Ten grams of Dextran T40 (GE Healthcare), 1.5 g FeCh 6 H 2 0 and 0.64 g FeCI 2 4 H 2 0 are dissolved in 20 ml H 2 0, and heated to 40° C. While stirring, 10 ml 4 N NaOH are added slowly and the solution is heated to 70° C for 5 min. The particle suspension is neutralized with acetic acid.
  • HGMF high-gradient magnetic field
  • the magnetic nanomatrices After removing the column from the external magnetic field, the magnetic nanomatrices are eluted with 0.05 M sodium acetate. The nanomatrices will form a brown suspension. The relative particle concentration is given as optical density at 450 nm. The size of the nanomatrices is determined by electron microscopy and dynamic light scattering. The nanomatrices should show superparamagnetic behavior, as determined by susceptibility measurements. Antibodies and Fabs are conjugated to separate nanomatrices by standard bioconjugation chemistry (Bioconjugate Techniques, 2nd Edition, By Greg T. Hermanson, Published by Academic Press, Inc., 2008). After sterile filtration, the resulting nanomatrices are stored in PBS buffer/0.3% Poloxamer 188 at -70°C.
  • nanomatrix comprising parental anti-CD3 antibody OKT3, a nanomatrix comprising parental anti-CD28 antibody 15E8, a nanomatrix comprising anti-CD3 Fab variant chCD3_mut, a nanomatrix comprising anti- CDS Fab variant huCD3_mut, a nanomatrix comprising anti-CD28 Fab variant huCD_v3, and a nanomatrix comprising anti-CD28 Fab variant huCD3 v4.
  • Nanomatrices comprising MHC monomer and antibody and Fab variants of herein disclosed Example 2 are used for the stimulation of T cells. Therefore, Pan T cells are isolated from PBMC using the Pan T Cell Isolation Kit, human (Miltenyi Biotec). Afterwards, the cells are labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured at a density of 1 E+06 cells per cm 2 in TexMACS GMP Medium (Miltenyi Biotec) supplemented with 200 ILI/mL MACS GMP IL-2 (Miltenyi Biotec).
  • CFSE carboxyfluorescein succinimidyl ester
  • a nanomatrix comprising a MHC monomer is used alone or in different combinations with a nanomatrix comprising an anti-CD28 Fab variant or the full-length antibody (15E8), respectively.
  • the final concentration during T cell stimulation is 500 ng protein/mL for anti-CD28 full length antibodies and 333 ng protein/mL for MHC monomer and anti-CD28 Fab variants, respectively.
  • the stimulation capacity of the nanomatrices is determined after 48 hours of cell culture using flow-cytometry for the detection of the early activation markers CD25 and CD69 (among viable T cells). Cells are afterwards cultured for further 5 days, and the proliferation rate (among viable cells) is determined via flow-cytometric acquisition.
  • Example 5 Stimulation of T cells by nanomatrices comprising an anti-CD3 protein alone or in combination with nanomatrices comprising an anti-CD28 protein
  • Nanomatrices comprising antibody and Fab variants of herein disclosed Example 3 are used for the stimulation of T cells. Therefore, Pan T cells are isolated from PBMC using the Pan T Cell Isolation Kit, human (Miltenyi Biotec). Afterwards, the cells are labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured at a density of 1 E+06 cells per cm 2 in TexMACS GMP Medium (Miltenyi Biotec) supplemented with 200 ILI/mL MACS GMP IL-2 (Miltenyi Biotec).
  • CFSE carboxyfluorescein succinimidyl ester
  • a nanomatrix comprising either an anti-CD3 Fab variant or the full-length antibody (OKT3) is used alone or in different combinations with a nanomatrix comprising an anti-CD28 Fab variant or the full-length antibody (15E8), respectively.
  • the final concentration during T cell stimulation is 500 ng protein/mL for each anti-CD3 and anti-CD28 full length antibodies and 333 ng protein/mL for each anti- CD3 and anti-CD28 Fab variants, respectively.
  • the stimulation capacity of the nanomatrices is determined after 48 hours of cell culture using flow-cytometry for the detection of the early activation markers CD25 and CD69 (among viable T cells). Cells are afterwards cultured for further 5 days, and the proliferation rate (among viable cells) is determined via flow-cytometric acquisition.
  • Example 6 Generation of an immune cell activatino multimer bv first couolino oeotide- MHC (first liqand) and subsequently Anti-CD28 antibody (second liqand) to a dextran backbone via streotavidin-biotin interactions
  • This example describes how an immune cell activating multimer was assembled.
  • First peptide-MHC (first ligand) and subsequently an Anti-CD28 antibody (second ligand) was coupled to a dextran backbone.
  • the ratio of peptide-MHC:Anti-CD28 Ab:dextran was 5:6:1.
  • Streptavidin conjugated dextran was manufactured and subsequently sterile filtered by using a 0.22 pm sterile filter
  • Assembly buffer (0.05M Tris-hydrochloric acid, 1% BSA, pH 7.2) was sterile filtered by using a 0.22 pm sterile filter.
  • Mono-biotinylated peptide-MHC was manufactured in the form of HLA-A*0201/ NLVPMVATV (SEQ ID NO: 13) complex.
  • the peptide-MHC and Anti-CD28 antibody was attached to the dextran by a non- covalent Biotin-Streptavidin interaction between the streptavidin conjugated to dextran and the biotinylated Heavy Chain part of the peptide-MHC and the biotinylated Fc domain of the antibody, respectively.
  • the reagent was assembled under a sterile condition in the following order: a. Streptavidin conjugated dextran. b. peptide-MHC c. Anti-CD28 antibody d. assembly buffer
  • the assembled multimer was sterile filtered and stored at 2-8°C until further use.
  • the above method can be used for generation of immune cell activating multimer comprising any of the first ligands and second ligands mentioned in the present disclosure.
  • Streptavidin conjugated dextran was manufactured and subsequently sterile filtered by using a 0.22 pm sterile filter.
  • Assembly buffer (0.05M Tris-hydrochloric acid, 1% BSA, pH 7.2) was sterile filtered by using a 0.22 pm sterile filter.
  • Mono-biotinylated peptide-MHC was manufactured in the form of HLA-A*0201/ NLVPMVATV (SEQ ID NO: 13).
  • the MHC-complex and Anti-CD28 antibody was attached to the dextran by a non-covalent Biotin-Streptavidin interaction between the streptavidin conjugated to dextran and the biotinylated Heavy Chain part of the peptide-MHC and the biotinylated Fc domain of the antibody, respectively.
  • the reagent was assembled under a sterile condition in the following order: a. Streptavidin conjugated dextran. b. Anti-CD28 antibody c. peptide-MHC d. assembly buffer
  • the assembled and sterile filtered reagent was kept at 2-8°C until further use.
  • the above method can be used for generation of immune cell activating multimer comprising any of the first ligands and second ligands mentioned in the present disclosure.
  • Example 8 Generation of an immune cell activation multimer by coupling peptide-MHC (first ligand) and Anti-CD28 antibody (second ligand) to a dextran backbone in a one-pot fashion via streptavidin-biotin interactions.
  • This example describes how an immune cell activation multimer was assembled by coupling peptide-MHC (first ligand) and an Anti-CD28 antibody (second ligand) to a dextran backbone in a one-pot fashion - i.e. in a simultaneous coupling reaction.
  • the ratio of peptide-MHC:Anti-CD28 Ab:dextran was 5:6:1 .
  • Streptavidin conjugated dextran was manufactured and subsequently sterile filtered by using a 0.22 pm sterile filter.
  • Assembly buffer (0.05M Tris-hydrochloric acid, 1% BSA, pH 7.2) was sterile filtered by using a 0.22 pm sterile filter.
  • Mono-biotinylated peptide-MHC was manufactured in the form of HLA-A*0201/ NLVPMVATV (SEQ ID NO: 13) complex.
  • the peptide-MHC and Anti-CD28 antibody was attached to the dextran by a non- covalent Biotin-Streptavidin interaction between the streptavidin conjugated to dextran and the biotinylated Heavy Chain part of the peptide-MHC and the biotinylated Fc domain of the antibody, respectively.
  • the reagent was assembled under a sterile condition in the following order: a. Mix peptide-MHC and Anti-CD28 antibody b. Add the mixture into Streptavidin conjugated dextran c. Add assembly buffer
  • the assembled and sterile filtered multimer was kept at 2-8°C until further use.
  • the above method can be used for generation of immune cell activating multimer comprising any of the first ligands and second ligands mentioned in the present disclosure.
  • Example 9 Generation of an immune cell activation multimer by coupling peptide-MHC (first ligand) and Anti-CD28 Fab (second ligand) to a dextran backbone via streptavidinbiotin interactions.
  • This example describes how an immune cell activation multimer was assembled in a similar way as described in Example 6, 7 and 8, but instead by using an Anti-CD28 antibody, an Anti-CD28 Fab was used.
  • the ratio of peptide-MHC:Anti-CD28 Fab:dextran was 5:6:1 .
  • the Mono-biotinylated Anti-CD28 Fab (second ligand) was commercially purchased.
  • Example 10 Generation of an immune cell activation multimer by coupling peptide- MHC (first ligand) and Anti-CD28 antibody / Anti-CD28 Fab (second ligand) to a dextran backbone via covalent interactions
  • first ligand Anti-CD28 antibody / Anti-CD28 Fab
  • second ligand Anti-CD28 Fab
  • covalent interactions such as di-sulphide bonds are used for the attachment.
  • the above method can be used for generation of immune cell activating multimer comprising any of the first ligands and second ligands mentioned in the present disclosure.
  • Example 11 Generation of an immune cell activation multimer by coupling different amount of peptide MHC (first ligand) and Anti-CD28 antibody / Anti-CD28 Fab (second ligand) to a dextran backbone.
  • This example describes use of different amounts of peptide-MHC (first ligand) and Anti- CD28 antibody / Anti-CD28 Fab (second ligand) to assemble the immune cell activation multimer made in a similar way as described in Example 6, 7, 8, 9 and 10.
  • the amounts used are summarized the Table 11.1.
  • Table 11 .1 the ratio of first and second ligand per dextran.
  • Example 12 MART-1 cells - Stimulation of HLA-A*0201/MART-1 peptide specific CD8+ T cells in PBMCs from healthy donors by dextran backbone multimers comprising peptide-MHC and anti-CD28 Fab
  • an immune cell activation multimers comprising a dextran backbone conjugated with peptide-MHC monomers and anti-CD28 Fab were manufactured in the peptide-MHC/anti-CD28 Fab/dextran backbone ratio of 8/16/1 .
  • the peptide-MHC utilized were either HLA-A0201 presenting the ELAGIGILTV peptide (derived from the MART-1 epitope) (SEQ ID NO: 14) or HLA-A0201 presenting the ALIAPVHAV peptide (serving as a negative control) (SEQ ID NO: 15).
  • the anti-CD28 component consisted of a Fab fragment derived from the 15E8 antibody clone.
  • PBMCs Peripheral blood mononuclear cells
  • a growth medium such as XVIVO 15 (Lonza) supplemented with 5% human serum, to achieve a cell concentration of 2 x 10 A 7 cells/mL.
  • Approximately 2 x 10 A 6 cells were distributed per well in a 48-well microtiter culture plate (Sartorius), and the peptide-MHC/anti-CD28 Fab/dextran stimulating multimers were added at a concentration of 0.64 nM. Following a 30-minute binding phase, 400 pL of growth medium was added to each well.
  • the culture medium was refreshed every other day, with IL-2 supplementation starting on day 3, and IL-2, IL-7, and IL-15 supplementation commencing on day 5.
  • cultured cells were restimulated with the peptide- MHC/anti-CD28 Fab/dextran multimers.
  • HLA-A0201 /MART-1 -specific CD8+ T cells assessed the stimulation and expansion of HLA- A0201 /MART-1 -specific CD8+ T cells using HLA-A0201 /ELAGIGILTV (SEQ ID NO: 14) and HLA-A*0201/ALIAPVHAV (SEQ ID NO: 15) multimers, along with an antibody panel specific for cell surface markers including CD3, CD8, and CD4.
  • HLA-A0201/MART-1 -specific T cells were undetectable in PBMC samples at day 0 for all five donors, Dextramer staining demonstrated significant stimulation and proliferation of HLA-A0201 /MART- 1 -specific T cells by day 13, with further expansion noted by day 33 in three of the five donors.
  • expansion of HLA-A0201/MART-1 - specific CD8+ T cells was observed exclusively with the specific stimulating reagent comprising HLA-A0201/MART-1/aCD28 Fab/dextran, and not with the control reagent, HLA-A*0201/ALIAPVHAV/aCD28 Fab/dextran (SEQ ID NO: 14).
  • Example 13 CMV cells - Stimulation of HLA-B*0702/CMV peptide antigen specific CD8+ T cells in PBMCs from healthy donors by dextran backbone multimers comprising peptide-MHC and anti-CD28 Ab
  • Immune cell activation multimers comprising a dextran backbone conjugated with a defined number of peptide-MHC and anti-CD28 Ab molecules were manufactured with specific peptide-MHC/anti-CD28 Ab/dextran backbone ratios, including 12/0/1 , 12/6/1 , 6/6/1 , 3/6/1 , and 6/12/1.
  • the peptide-MHC used included HLA-B*0702 molecules presenting the TPRVTGGGAM peptide (a cytomegalovirus (CMV) epitope) (SEQ ID NO: 16), while the anti-CD28 Ab component was an antibody fragment derived from the CD28.2 antibody clone.
  • PBMCs Peripheral blood mononuclear cells from a donor source were prepared by washing and resuspending in a growth medium, specifically XVIVO 15 (Lonza) supplemented with 5% human serum, at a concentration of 6 x 10 A 6 cells/mL. Approximately 3 x 10 A 5 cells per well were dispensed into a 96 U-bottom microtiter culture plate (Sartorius). The peptide-MHC/anti-CD28 Ab/dextran multimers were added at concentrations of 0.15 nM, 0.5 nM, or 5 nM, and following a 30-minute incubation for binding, 50 pL of growth medium was added to each well. The culture medium was refreshed every other day, with IL-2 supplementation beginning on day 3.
  • a growth medium specifically XVIVO 15 (Lonza) supplemented with 5% human serum
  • HLA-B0702/TPRVTGGGAM epitope SEQ ID NO: 16
  • HLA-B0702/TPRVTGGGAM SEQ ID NO: 16
  • HLA-B*0702/control peptide Dextramer multimers employed HLA-B*0702/control peptide Dextramer multimers alongside an antibody panel specific for surface markers, including CD3, CD8, and CD4.
  • Example 14 iNKT cells - Stimulation and expansion of PBMC derived iNKT cells using dextran backbone multimers comorisino HLA-CD1d monomer and anti-CD28 Ab
  • Immune cell activation multimers comprising a dextran backbone are conjugated with an optimized number of HLA-CD1 d monomers and anti-CD28 antibody (Ab) or Fab molecules per dextran backbone, facilitating targeted activation of invariant natural killer T (iNKT) cells.
  • Abs anti-CD28 antibody
  • Fab molecules per dextran backbone
  • PBMCs Peripheral blood mononuclear cells
  • a growth medium such as XVIVO 15 (Lonza) supplemented with 5% human serum
  • a cell density of approximately 5 x 10 A 6 cells/mL.
  • Aliquots of 0.2-1 mL are distributed into cell culture plates, such as 96U, 48-well, or 24-well plates.
  • the stimulating multimer comprising HLA-CD1d monomers loaded with a- Galactosylceramide (a-GC) or other CD Id-presented glycolipid antigens, is added to the culture at a final concentration of approximately 0.05-0.5 nM dextran backbone.
  • a-GC Galactosylceramide
  • CD Id-presented glycolipid antigens is added to the culture at a final concentration of approximately 0.05-0.5 nM dextran backbone.
  • Cells are cultured in this medium, with IL-2 supplementation added from day 3 onward.
  • iNKT cell TCR clonotypes are evaluated using single-cell genomic platforms, such as 10x Genomics or BD Rhapsody, in conjunction with CD1 d dCODE Dextramer multimers for clonotype identification and characterization.
  • Immune cell activation multimers comprising a dextran backbone are conjugated with an optimized number of HLA-MR1 monomers and anti-CD28 antibody (Ab) or Fab molecules per dextran backbone, specifically designed to enhance activation and expansion of mucosal-associated invariant T (MAIT) cells.
  • PBMCs Peripheral blood mononuclear cells
  • a growth medium such as XVIVO 15 (Lonza) supplemented with 5% human serum, to achieve a cell density of approximately 5 x 10 A 6 cells/mL.
  • Aliquots of 0.2-1 mL are distributed into cell culture plates, including but not limited to 96U, 48-well, or 24-well plates.
  • the stimulating reagent comprising HLA-MR1 monomers loaded with 5-(2- oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) or other MR1 -binding metabolites, is added at a final concentration of approximately 0.05-0.5 nM dextran backbone.
  • Cells are cultured in this medium, with IL-2 supplementation beginning on day 3.
  • Example 16 B cells - Stimulation and expansion of PBMC derived B cells using dextran backbone multimers comprising BCR antigen and CD40 ligand or anti-CD40 Ab
  • Immune cell activation multimers comprising a dextran backbone are conjugated with an optimized number of B cell receptor (BCR) antigens and CD40 ligand (CD40L) or anti- CD40 antibody (Ab) molecules per dextran backbone, designed to promote specific activation of B cells.
  • BCR B cell receptor
  • CD40L CD40 ligand
  • Ab anti- CD40 antibody
  • PBMCs Peripheral blood mononuclear cells
  • a growth medium such as XVIVO 15 (Lonza) supplemented with 5% human serum, to achieve a target cell density of approximately 5 x 10 A 6 cells/mL. Aliquots of 0.2-1 mL are then distributed into cell culture plates, including but not limited to 96U, 48-well, or 24-well plates.
  • the stimulating reagent comprising BCR-recognized antigens conjugated to the dextran backbone, is introduced at a final concentration of approximately 0.05-0.5 nM dextran backbone.
  • Cultured cells are maintained in growth medium supplemented with cytokines to support cell growth. Between days 5 and 14, cell cultures are analyzed to assess the activation and expansion of B cells, as well as cellular phenotypes, by staining with Longmer multimers conjugated with the B cell antigen and a mixture of antibodies specific for surface markers, including CD19 and CD40.
  • Enriched B cells will be further analyzed to determine BCR specificity for the targeted BCR antigen. Additionally, the B cell clonotype will be evaluated using single-cell genomic platforms, such as 10x Genomics or BD Rhapsody, in conjunction with dCODE Lanemer multimers conjugated to the BCR antigen to facilitate clonotype identification and characterization.
  • Example 17 CAR T cells - Stimulation and expansion of CAR T cells using dextran backbone multimers comprising CAR antigen (CD19) and anti-CD28 Ab.
  • Immune cell activation multimers comprising a dextran backbone are conjugated with an optimized number of chimeric antigen receptor (CAR) antigens, such as CD19, along with anti-CD28 antibody (Ab) or Fab fragments per dextran backbone, to enhance cellular activation.
  • CAR chimeric antigen receptor
  • Abs anti-CD28 antibody
  • Fab fragments per dextran backbone to enhance cellular activation.
  • Engineered CAR T cells within a peripheral blood mononuclear cell (PBMC) sample may be prepared by washing and resuspending the cells in a growth medium, for example, XVIVO 15 (Lonza) supplemented with 5% human serum, to achieve a cell density of approximately 5 x 10 A 6 cells/mL. Aliquots of 0.2-1 mL may then be distributed into cell culture plates, such as 96U, 48-well, or 24-well plates.
  • a growth medium for example, XVIVO 15 (Lonza) supplemented with 5% human serum
  • the stimulating multimer containing CAR antigens conjugated to the dextran backbone will be introduced at a final concentration of approximately 0.05-0.5 nM dextran backbone.
  • CAR T cells will be cultured in the growth medium with supplemental IL-2 added on day 3. Between days 5 and 14, cell cultures will undergo analysis to assess CAR T cell activation, expansion, and phenotypic characteristics by staining with CAR Dextramer multimers and a mixture of antibodies targeting specific cell surface markers, including but not limited to CD3, CD8, and CD4.
  • enriched CAR T cells will undergo potency analysis, assessing their ability to secrete interferon-gamma (IFN-y), and cytotoxicity assays will be conducted to evaluate the CAR T cells' cytotoxic effects against CD19-expressing B cells.
  • IFN-y interferon-gamma
  • a specialized NF-KB Luciferase Reporter Jurkat Cell Line is provided. This cell line is designed for monitoring nuclear factor Kappa B (NF-KB) signal transduction pathways, with utility in measuring T cell receptor (TCR)-mediated T cell activation through costimulation with anti-CD3 and anti-CD28 antibodies, leading to the transcription of a luciferase reporter gene.
  • NF-KB nuclear factor Kappa B
  • Reporter cells were adjusted to a concentration of approximately 1 million cells per milliliter in RPMI1640 medium supplemented with 10% fetal calf serum (FCS). Aliquots of 50 pL were plated into wells of a cell culture plate with white walls and a clear bottom. An anti-CD3 antibody was added to the wells containing cells at a concentration of 1 pg/mL, along with either (1 ) varying commercial anti-CD28 antibodies or an anti-CD28 Fab fragment at concentrations ranging from 0 to 1 pg/mL or (2) a dextran backbone conjugated with the same antibodies. After a 6-hour incubation period, luciferase induction was evaluated by adding a substrate containing D-luciferin, and luminescence was subsequently measured using a luminometer.
  • FCS fetal calf serum
  • the anti-CD28 Fab fragment generated luciferase signals of similar magnitude to those of other anti-CD28 antibodies. This result indicates that conjugation of the anti- CD28 Fab fragment to a dextran backbone facilitates crosslinked co-engagement of CD28, thereby enabling effective signaling through the NF-KB pathway.
  • Example 19 The phenotype of TCR engineered T cells stimulated with Dextramer multimers comprising two different ligands closely mimics that of cells exposed to antioen-presentino cells (APCs)
  • APCs antioen-presentino cells
  • Dextramer multimers comprising the two ligands, peptide-MHC (HLA-A*1 1 :01 / KRas G12V ) (VVGAVGVGK; SEQ ID NO:18) and anti-CD28 antibody, with the following stoichiometry 2/12 peptide-MHC/ 6 anti-CD28 Ab were generated according to Example 2.
  • Control Dextramer multimers comprising only peptide-MHC or a negative control peptide-MHC and anti-CD28 antibody were included for comparison.
  • the commercially available T cell stimulation reagent TransAct Miltenyi
  • B-Lymphoblastoid cell line B-LCL/antigen
  • Dextramer construct 1 Dextramer multimers comprising 2/12 peptide-MHC and 6 anti-CD28 antibody molecules (data only shown for 12 peptide-MHC)
  • Dextramer construct 2 Dextramer multimers comprising only 2/12 peptide-MHC (control)
  • ⁇ Dextramer Construct 3 (Negative Control): Dextramer with a non-specific peptide-MHC and anti-CD28 antibody (control for non-specific activation).
  • T Cell Stimulation Reagent TransAct (Miltenyi) - A commercially available reagent to stimulate T cells
  • B-LCL B-Lymphoblastoid Cell Line
  • APCs Antigen-presenting cells
  • TCR-engineered T cells were plated in tissue culture plates and stimulated with the constructs described above. Each construct was applied at the following dilution 1 :100 to the engineered T cells to achieve a final concentration at 0.65 nM. Cells were cultured for 9-12 days, during which time they were monitored for antigen-specific expansion using Dextramer staining as described in Example 15 in W02009003492 as well as cytokine production, and surface marker expression.
  • the test Dextramer reagent comprising peptide-MHC/anti-CD28 Ab induced significant activation in TCR-engineered T cells, demonstrated by cytokine production (Table 19.1 ) and upregulation of CD69 and CD137 markers (data not shown). This activation was shown to be both peptide-MHC specific and dependent on CD28 co-stimulation.
  • TCR-engineered T cells stimulated with the test Dextramer multimer displayed a phenotype similar to that observed in cells stimulated with APCs (B-LCL/antigen) (Figure 3), suggesting effective mimicry of natural antigen presentation.
  • the Dextramer multimer demonstrated efficient enrichment of TCR-engineered T cells, comparable to the enrichment observed with APCs, with expansion shown to be peptide-MHC specific (Table 19.2).
  • the Dextramer multimer comprising peptide-MHC and anti-CD28 antibody provided efficient, specific stimulation of TCR-engineered T cells, with functionality closely matching that achieved with APCs, making it a promising tool for targeted T cell activation and enrichment in immunotherapy applications.
  • Table 19.1 The test Dextramer multimer comprising peptide-MHC/anti-CD28 Ab was observed to induce significant activation in TCR-engineered T cells, as evidenced by the secretion of IFN-y. In contrast, wild-type T cells exposed to the same reagent did not demonstrate IFN-y secretion.
  • Example 20 Comparison of potency of TCR engineered T cells expanded using Dextramer multimers comprising peptide-MHC and anti-CD28 antibody molecules compared to control process derived TCR engineered T cells using the Repeated Antigen Stimulation Assay (RASA)
  • This experiment will compare the potency of TCR-engineered T cells expanded with Dextramer multimers, comprising peptide-MHC and anti-CD28 antibody molecules, against a control process for generating TCR-engineered T cells.
  • the experiment will utilize the Repeated Antigen Stimulation Assay (RASA).
  • Dextramer multimers containing two ligands, peptide-MHC (HLA-A*02:01/ SLLQHLIGL peptide) (SEQ ID NO: 17) and anti-CD28 antibody will be prepared with the following stoichiometric ratio: 10 peptide-MHC to 6 anti-CD28 Ab molecules, as detailed in Example 2.
  • a negative control Dextramer comprising 10 peptide-MHC with a nonsense peptide and 6 anti-CD28 molecules will be included as negative control reagent.
  • TCR- engineered T cells will be plated in tissue culture plates at a density of 300,000 cells per well and stimulated with the peptide-MHC/anti-CD28 Dextramer multimers. Each construct will be applied at two different dilutions, 1 :100 and 1 :1000, to achieve a final concentration of 0.65 nM or 0.065 nM, respectively.
  • the cells will be cultured for 9 to 12 days, during which they will be monitored for antigenspecific expansion by Dextramer staining, as described in Example 15 of W02009003492, along with assessment of cytokine production and surface marker expression.
  • the TCR-engineered T cells will be tested for their cytotoxicity potential using the RASA protocol as follows: The peptide-MHC/anti-CD28 Ab Dextramer-expanded TCR-engineered T cells will be co-cultured with cancer cells, with repeated exposure to the cancer cells every third day. At day 14, the levels of IFN-y secretion and cytotoxic activity will be evaluated.
  • IFN-y Secretion The IFN-y secretion will be measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatant after a 24-hour co-culture period and after reexposures.
  • ELISA enzyme-linked immunosorbent assay
  • Cytotoxicity Assessment The cytotoxicity of the TCR-engineered T cells will be evaluated by a standard chromium-51 release assay or a flow cytometry-based dead cell detection assay. Target cancer cells will be labeled with a radioactive isotope or viability dye, and the percentage of target cell lysis will be determined after each round of coculture.
  • Dextramer multimers comprising two ligands, a peptide-MHC (HLA-A*02:01/ SLLQHLIGL) (SEQ ID NO: 17) and an anti-CD28 antibody, are prepared in a defined stoichiometric ratio of 10 peptide-MHC molecules to 6 anti-CD28 molecules, as outlined in Example 2.
  • a negative control Dextramer reagent incorporating 10 peptide-MHC molecules bound to a nonsense peptide along with 6 anti-CD28 Ab molecules, is prepared as a control reagent.
  • TCR-engineered T cells are plated in tissue culture plates at a density of 300,000 cells per well and stimulated with the prepared peptide-MHC/anti-CD28 Ab Dextramer multimers. Each Dextramer construct is tested at two dilutions, 1 :100 and 1 :1000, resulting in final concentrations of 0.65 nM and 0.065 nM, respectively.
  • TCR-engineered T cells are plated at the same density of 300,000 cells per well and co-cultured with antigen-presenting cells (APCs) expressing the HLA-A*02:01/ SLLQHLIGL complex (SEQ ID NO: 17).
  • APCs antigen-presenting cells
  • This cell line-based setup follows standard potency assay protocols, providing a traditional antigen presentation environment.
  • Both experimental groups (peptide-MHC/anti-CD28 Ab Dextramer multimer-stimulated and cell line-stimulated TCR-engineered T cells) are incubated at 37°C for 24 hours. Following incubation, the cell culture supernatant is harvested from each well, and the concentration of IFN-y in the supernatant is quantified using enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • This experiment aims to demonstrate that the peptide-MHC/anti-CD28 Ab Dextramer multimers can effectively stimulate TCR-engineered T cells, producing IFN-y levels comparable to or greater than those generated using traditional APC cell lines. Such a result would indicate that the Dextramer multimers serve as reliable, standardized, and reproducible replacements for cell line-based potency assays.
  • peptide-MHC/anti-CD28 Ab Dextramer multimers addresses several limitations associated with cell-based potency assays, such as variability in antigen presentation due to differences in APC quality and consistency across assay runs. Additionally, the artificial scaffold nature of the Dextramer multimers allows for enhanced standardization and simplifies assay setup, reducing the need for complex cell culture requirements and potentially improving the reproducibility of potency assessments for TCR-engineered T cell therapies.
  • Example 22 Evaluation of peptide-MHC/anti-CD28 Dextramer reagents comprising different number of peptide-MHC and anti-CD28 antibody molecules
  • Dextramer multimers comprising the two ligands, peptide-MHC (HLA-A*11 :01/ KRasG12V) and anti-CD28 antibody, with different combinations of the following stoichiometries 2/12 peptide-MHC and 0/0.5/6/24 anti-CD28 antibody were generated according to Example 2.
  • Control Dextramer reagents comprising only peptide-MHC or a negative control peptide-MHC and anti-CD28 antibody were included for comparison.
  • the commercially available ? cell stimulation reagent TransAct Miltenyi
  • B-Lymphoblastoid cell line B-LCL/antigen
  • ⁇ Dextramer construct 1 Dextramer multimer comprising 12 peptide-MHC and 0 anti-CD28 antibody molecules
  • ⁇ Dextramer construct 2 Dextramer multimer comprising 12 peptide-MHC and 0.5 anti-CD28 antibody molecules
  • ⁇ Dextramer construct 3 Dextramer multimer comprising 12 peptide-MHC and 6 anti-CD28 antibody molecules
  • ⁇ Dextramer construct 4 Dextramer multimer comprising 12 peptide-MHC and 24 anti-CD28 antibody molecules
  • ⁇ Dextramer construct 5 Dextramer multimer comprising 2 peptide-MHC and 6 anti-CD28 antibody molecules
  • Dextramer Construct 6 (Negative Control): Dextramer with 12 nonspecific peptide-MHC and 6 anti-CD28 antibody molecules (control for non-specific activation).
  • T Cell Stimulation Reagent TransAct (Miltenyi) - A commercially available reagent to stimulate T cells
  • B-LCL B-Lymphoblastoid Cell Line
  • APCs Antigen-presenting cells
  • TCR-engineered T cells were plated in tissue culture plates and stimulated with the constructs described above. Each construct was applied at the following dilution 1 :100 to the engineered T cells to achieve a final concentration at 0.65 nM. Cells were cultured for 9 days, during which time they were monitored for the expansion of the TCR- engineered T cells (CD4 and CD8 T cells) by flow cytometry. All peptide-MHC/anti-CD28 antibody constructs demonstrated an ability to induce expansion of the TCR-engineered T cell population as compared to both the unstimulated control and Dextramer control reagent (Table 22.1).
  • the 12 peptide-MHC/6 anti-CD28 Ab, 12 Peptide-MHC/24 anti-CD28 Ab, and 2 peptide-MHC/6 anti-CD28 Ab constructs induced the highest levels of expansion. These constructs also outperformed both TransAct and B-LCL/antigen in promoting T cell proliferation.

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Abstract

La présente invention concerne des multimères capables d'activer efficacement des cellules immunitaires, telles que des lymphocytes T. En particulier, l'invention concerne des multimères comprenant au moins deux ligands différents, tels qu'un peptide CMH et un anticorps anti-CD28, capables de se lier à et d'activer ensemble ladite cellule immunitaire. L'invention concerne en outre un acide nucléique, des vecteurs et des compositions codant pour lesdits multimères ou comprenant ceux-ci, et divers procédés pour leur utilisation.
PCT/EP2024/083183 2023-11-22 2024-11-21 Multimères d'activation de cellules immunitaires Pending WO2025109111A1 (fr)

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