WO2023147185A1 - Procédés de fabrication et d'utilisation d'échafaudages mimétiques de cellules présentatrices d'antigène pour améliorer des thérapies par lymphocytes t - Google Patents

Procédés de fabrication et d'utilisation d'échafaudages mimétiques de cellules présentatrices d'antigène pour améliorer des thérapies par lymphocytes t Download PDF

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WO2023147185A1
WO2023147185A1 PCT/US2023/012027 US2023012027W WO2023147185A1 WO 2023147185 A1 WO2023147185 A1 WO 2023147185A1 US 2023012027 W US2023012027 W US 2023012027W WO 2023147185 A1 WO2023147185 A1 WO 2023147185A1
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cell
cells
antigen
car
antibody
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WO2023147185A9 (fr
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David J. Mooney
Kongyu ZHANG
Joshua M. BROCKMAN
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
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    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
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    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
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    • A61K39/464412—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
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    • 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)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2510/00—Genetically modified cells

Definitions

  • Immunotherapies involving engineered T cells have shown remarkable clinical outcomes for the treatment of cancer, particularly hematological malignancies, such as leukemia and lymphoma.
  • hematological malignancies such as leukemia and lymphoma.
  • several barriers need to be overcome before these adoptive cellular therapies can be accessed by a broader patient population and be applied to other cancers.
  • the lack of control over the quality of T-cell products limits the cells’ intrinsic ability to proliferate, persist, and effectively kill tumor cells in vivo. This can, in part, be attributed to the synthetic materials used to mediate T-cell activation, a key step in therapeutic T-cell production.
  • Conventional materials fail to recapitulate key features of physiological T-cell activation, resulting in poor control over T-cell quality and yield.
  • the present invention provides compositions and methods for manipulating, e.g., activating, stimulating, expanding, proliferating, and/or energizing T-cells, particularly therapeutic T-cell products including engineered T-cells, such as CAR T-cells.
  • the present invention addresses the limitations of conventional methods and materials used to mediate T-cell activation by providing material scaffolds that mimic physiological antigen presentation and provide precise control over T-cell activation to create and manipulate T cells in vivo.
  • the material scaffolds can be used for in vivo manipulation of T-cells, e.g., activation and expansion of populations of engineered T-cells, such as CAR T-cells.
  • the present invention can be used to improve T-cell immunotherapies, for example, by engineering the materials to tune T cell-intrinsic attributes to enhance therapeutic T-cell products, and by using the materials as an injectable scaffold niche for local T-cell stimulation, to boost systemic antitumor responses of T-cell immunotherapies administered prior to, simultaneous with, or after administration of the scaffold materials. Accordingly, the present invention provides a materials-based approach to manipulate T-cell biology, enabling the controlled enhancement of therapeutic T-cell products including engineered T-cells, such as CAR T-cells and their therapeutic functions in vivo.
  • CAR adoptive chimeric antigen receptor
  • This present invention provides subcutaneously injected materials, which form a scaffold microenvironment for local T-cell simulation, and boosts the systemic anti-tumor activity of administered CAR T-cells.
  • These scaffolds emulate key features of physiological T cell activation and enable the continuous infiltration and stimulation of specific T-cell subpopulations.
  • Presentation of T-cell activating ligands, optionally in combination with costimulatory molecules drives selective T-cell expansion, differentiation, and cytotoxic function, depending on the dose, type, and context in which the ligands are delivered.
  • subjects administered scaffolds following subtherapeutic CAR T-cell dosing promotes improved anti-tumor responses accompanied by increased therapeutic cell output and persistent, memory-like differentiation profiles.
  • the subtherapeutic CAR T-cell dose refers to a dose of CAR T-cells that is too small to produce a therapeutic effect, e.g., a curing effect.
  • the subtherapeutic amounts of CAR T-cells may become therapeutically effective when given with an antigen presenting cell-mimetic scaffold (APC-MS) described herein.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the instant invention provides antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica microrods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a functional molecule selected from the group consisting of a T-cell ligand, a T-cell co-stimulatory molecule, a T-cell homeostatic agent, and a combination thereof.
  • the APC-MS can include one or more T-cell activating ligands.
  • the APC-MS can include one or more T-cell inhibiting ligands.
  • the APC-MS can include one or more T-cell co-stimulatory molecules, optionally, loaded into and/or presented on the scaffold.
  • the APC- MS can include one or more T-cell homeostatic agents, optionally, loaded into and/or presented on the scaffold.
  • the term "loaded” refers to the functional molecule being incorporated into the scaffold, e.g., encapsulated within the scaffold material.
  • the term "presented” refers to the functional molecule being present in and/or onto the scaffold surface, e.g., attached to the scaffold surface.
  • the functional molecule is presented on the scaffold surface such that the scaffold mimics the natural antigen-presenting cells (APCs) ability to present the functional molecule.
  • APCs natural antigen-presenting cells
  • the antigen presenting cell-mimetic scaffolds is formulated for treating a cancer in a subject, wherein the subject is administered the population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS.
  • the antigen presenting cell-mimetic scaffolds is formulated to enhance the systemic anti-tumor activity of a population of engineered T-cells in the subject, wherein the subject is administered the population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS.
  • the antigen presenting cell-mimetic scaffolds is formulated for enhancing the systemic anti-tumor activity of an engineered T-cell therapy in a subject, wherein the subject is administered the population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS.
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) that sequester T-cells, such as engineered T-cells, selected from the group consisting of natural killer T-cells (NKT), CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combination thereof.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • engineered T-cells selected from the group consisting of natural killer T-cells (NKT), CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combination thereof.
  • the engineered T-cells are CAR T-cells.
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing one or more T-cell homeostatic agents which is loaded into and/or presented on the SLB layer.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing one or more T-cell homeostatic agents which are loaded into the base layer.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing the one or more T-cell homeostatic agents which is released from the scaffold in a controlled-release manner.
  • the one or more T-cell homeostatic agents is released from the scaffold in a controlled release manner over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or more.
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing the one or more T-cell homeostatic agents which are released from the scaffold in a sustained manner for up to 7 days. In one embodiment, the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing the one or more T-cell homeostatic agents which are released from the scaffold in a sustained manner for up to 15 days. In one embodiment, the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing the T-cell homeostatic agent which is released from the scaffold in a sustained manner for up to 30 days.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the T-cell homeostatic agent is released from the scaffold in a sustained manner for up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or more.
  • the T-cell homeostatic agent is released from the scaffold in a sustained manner for at least 7 days.
  • the T-cell homeostatic agent is released from the scaffold in a sustained manner for at least 15 days. In some embodiments, the T-cell homeostatic agent is released from the scaffold in a sustained manner for at least 30 days. In some embodiments, the T-cell homeostatic agent is released from the scaffold in a sustained manner for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or more.
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing the one or more T-cell homeostatic agents which is selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, and transforming growth factor beta (TGF-P), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • TGF-P transforming growth factor beta
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing a one or more T-cell homeostatic agents which is IL -2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof with at least one additional homeostatic agent selected from the group consisting of IL-7, IL-21, IL-15, and IL-15 superagonist.
  • the T-cell homeostatic agent may be selected from the group consisting of an N-terminal IL -2 fragment comprising the first 30 amino acids of IL-2 (pl-30), an IL -2 superkine peptide, and an IL -2 partial agonist peptide, or a combination thereof.
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell ligand, wherein the one or more T-cell ligands is a T-cell activating ligand or a T-cell inhibiting ligand.
  • the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is adsorbed onto the fluid supported lipid bilayer (SLB).
  • the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is adsorbed via affinity pairing or chemical coupling.
  • the chemical coupling comprises a click chemistry reagent (e.g., DBCO or azide).
  • a click chemistry reagent e.g., DBCO or azide.
  • the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is adsorbed via affinity pairing comprising a biotin-streptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an aptamer affinity pair, a capture protein pair, an Fc receptor-IgG pair, a metal-chelating lipid pair, a metalchelating lipid-histidine (HlS)-tagged protein pair, or a combination thereof.
  • affinity pairing comprising a biotin-streptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an aptamer affinity pair, a capture protein pair, an Fc receptor-IgG pair, a metal-chelating lipid pair, a metalchelating lipid-histidine (
  • the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is adsorbed via chemical coupling comprising azide-alkyne chemical (AAC) reaction, dibenzo- cyclooctyne ligation (DCL), or tetrazine-alkene ligation (TAL).
  • AAC azide-alkyne chemical
  • DCL dibenzo- cyclooctyne ligation
  • TAL tetrazine-alkene ligation
  • the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is coated onto the fluid supported lipid bilayer (SLB). In some embodiments, the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is partly embedded onto the fluid supported lipid bilayer (SLB).
  • the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is loaded into and/or presented on the fluid supported lipid bilayer (SLB).
  • SLB fluid supported lipid bilayer
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell ligand and/or one or more T-cell co-stimulatory molecules coated onto the fluid supported lipid bilayer (SLB).
  • APC-MS antigen presenting cell-mimetic scaffolds
  • SLB fluid supported lipid bilayer
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell ligand and/or one or more T-cell co-stimulatory molecules partly embedded onto the fluid supported lipid bilayer (SLB).
  • APC-MS antigen presenting cell-mimetic scaffolds
  • SLB fluid supported lipid bilayer
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell ligand and/or one or more T-cell co-stimulatory molecules loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • APC-MS antigen presenting cell-mimetic scaffolds
  • MSR mesoporous silica micro-rods
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell ligand and/or one or more T-cell co-stimulatory molecules loaded into and/or presented on the fluid supported bilayer (SLB) or the mesoporous silica micro-rods (MSR) via affinity pairing or chemical coupling.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • SLB fluid supported bilayer
  • MSR mesoporous silica micro-rods
  • the affinity coupling comprises a biotinstreptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an affinity pair, a capture protein pair, an Fc receptor-IgG pair, a metal-chelating lipid pair, or a combination thereof, optionally, wherein the chemical coupling comprises azide-alkyne chemical (AAC) reaction, dibenzocyclooctyne ligation (DCL), or tetrazine-alkene ligation (TAL).
  • AAC azide-alkyne chemical
  • DCL dibenzocyclooctyne ligation
  • TAL tetrazine-alkene ligation
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell ligand and/or a T-cell co-stimulatory, wherein the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell ligand and/or a T-cell co-stimulatory, wherein the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is an antibody molecule or an antigen-binding fragment thereof.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the one or more T-cell ligands and/or the T-cell co-stimulatory molecule is an antibody molecule or an antigen-binding fragment thereof.
  • the T-cell activating ligands and/or the T-cell inhibiting ligands are anti-idiotype antibodies or derivatives thereof.
  • the anti-idiotype antibodies or derivatives thereof may bind to the antigen-binding domain of a CAR molecule of the administered population of engineered T-cells in the subject.
  • the T-cell activating ligands and/or the T-cell inhibiting ligands are selected from the group consisting of an anti-idiotype CD3 antibody (aCD3) or an antigen-binding fragment thereof; an anti-idiotype CD5 antibody (aCD5) or an antigen-binding fragment thereof; an anti-idiotype CD7 antibody (aCD7) or an antigen-binding fragment thereof; an anti-idiotype CD28 antibody (aCD28) or an antigen-binding fragment thereof; an anti-idiotype CD19 antibody (aCD19) or an antigen-binding fragment thereof; an anti-idiotype CD20 antibody (aCD20) or an antigen-binding fragment thereof; an anti-idiotype CD22 antibody (aCD22) or an antigenbinding fragment thereof; an anti-idiotype CD70 antibody (aCD70) or an antigen-binding fragment thereof; an anti-idiotype CD123 antibody (aCD123) or an antigen-binding fragment thereof; an antiidiotype CS1 antibody (aCSl) or an anti
  • the T-cell activating ligands and/or the T-cell inhibiting ligands are T-cell ligands or derivatives thereof that bind to a CAR molecule of the administered population of engineered T-cells in the subject.
  • the T-cell activating ligands and/or the T- cell inhibiting ligands are selected from the group consisting of a CD3 molecule or a fragment thereof; a CD5 molecule or a fragment thereof; a CD7 molecule or a fragment thereof; a CD28 molecule or a fragment thereof; a CD 19 molecule or a fragment thereof; a CD20 molecule or a fragment thereof; a CD22 molecule or a fragment thereof; a CD70 molecule or a fragment thereof; a CD123 molecule or a fragment thereof; a CS1 molecule or a fragment thereof; a BCMA molecule or a fragment thereof; a SLAMF7 molecule or a fragment thereof; a Claudin-6 molecule or a fragment thereof; a NKG2D molecule or a fragment thereof; a NKG2DL molecule or a fragment thereof; a GD2 molecule or a fragment thereof; a Her2 molecule or a fragment thereof; and a mes
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell activating ligand and/or a T-cell inhibiting ligand and, optionally, a co-stimulatory molecule, wherein the T -cell activating ligand and/or the T -cell inhibiting ligand are selected from the group consisting of an anti-CD3 antibody or an antigenbinding fragment thereof, an anti-CD2 antibody or an antigen-binding fragment thereof, an anti-CD47 antibody or an antigen-binding fragment thereof, anti-macrophage scavenger receptor (MSR1) antibody or an antigen-binding fragment thereof, an anti-T-cell receptor (TCR) antibody or an antigen-binding fragment thereof, a major histocompatibility complex (MHC) molecule or a multimer thereof loaded with an MHC peptide, and an MHC-immunoglobulin (Ig) conjugate or a multimer thereof, ICAM-1
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell activating ligand and/or a T-cell inhibiting ligand and, optionally, a co-stimulatory molecule, wherein the T-cell co-stimulatory molecule is an antibody, or an antigen-binding fragment thereof, which specifically binds to a co-stimulatory antigen selected from the group consisting of CD28, 4.1BB (CD137), 0X40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LT(3R (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRT AM (CD355),TIM1 (HAVCR1, KIMI), CD2 (LFA2, 0X34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4),
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, a co-stimulatory molecule, wherein the T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, the T-cell co-stimulatory molecule, comprise bispecific antibodies or antigen binding fragments thereof.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) containing a T-cell activating ligand and/or a T-cell inhibiting ligand and, optionally, a co-stimulatory molecule, wherein the T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, the T-cell co-stimulatory molecule, comprise a pair selected from the group consisting of CD3/CD28, CD3/ICOS optionally together with CD28, CD3/CD27 optionally together with CD28, and CD3/CD137 optionally together with CD28, or a combination thereof.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the T-cell activating ligand comprises activating clones of antibodies against CD3/CD28, CD3/ICOS, CD3/CD27, and/or CD3/CD137; and/or (ii) the one or more T-cell ligands comprises an activating clone of antibodies against CD28.
  • the T-cell inhibiting ligands comprise inhibiting clones of antibodies against CD3/CD28, CD3/ICOS, CD3/CD27, and/or CD3/CD137; and/or (ii) the one or more T-cell ligands comprises an activating clone of antibodies against CD28.
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) which further comprise an immunoglobulin molecule that binds specifically to an Fc-fusion protein.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) which further comprise a recruitment compound selected from the group consisting of granulocyte macrophage -colony stimulating factor (GM-CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), a C-X-C motif chemokine ligand 12 (CXCL12), Interferon gamma (IFNy), or a FMS-like tyrosine kinase 3 (Flt-3) ligand, or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof.
  • the scaffolds further comprise a recruitment compound which is granulocyte macrophage colony stimulating factor (GM-CSF), or an agonist thereof, a mimetic thereof, a variant thereof, or a functional fragment thereof.
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS) which further comprise an antigen.
  • the antigen comprises a tumor antigen.
  • the tumor antigen comprises a protein, a glycoprotein, a glycolipid, or a carbohydrate expressed on the surface of a tumor cell.
  • the tumor antigen is selected from the group consisting of MAGE- 1, MAGE-2, MAGE- 3, CEA, Tyrosinase, midkin, BAGE, CASP-8, P-catenin, - catenin, y-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gplOO, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf 112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L, CYNL2, TPR, SOX
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica microrods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, one or more T-cell co-stimulatory molecules, loaded into and/or presented on the scaffold; and, optionally, one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the weight ratio of the supported lipid bilayer (SLB) to the mesoporous silica micro-rods (MSR) is between about 10:1 and about 1:20.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the weight ratio reflects the ratio of SLB to MSR prior to loading. In another embodiment, the weight ratio is adjusted to achieve the desired scaffold composition. In one embodiment, the weight ratio of the SLB to the MSR may be between about 9:1 and about 1:15, between about 5:1 and about 1:10, between about 3:1 and about 1:5, including all ratios in between, e.g., about 3;1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10. In one embodiment, the fluid supported lipid bilayer (SLB) comprises a lipid within melting temperature (Tm) range about -2°C to about 100°C.
  • Tm melting temperature
  • the fluid supported lipid bilayer comprises a lipid within Tm range about -2°C to about 75°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 55°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about - 2°C to about 25°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 15°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 10°C.
  • the fluid supported lipid bilayer comprises a lipid within Tm range about -2°C to about 5°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about 27°C to about 35°C.
  • the fluid supported lipid bilayer comprises a lipid within melting temperature (Tm) of about 1°C to about 50°C, about 25°C to about 45°C, about 25°C to about 75°C, about 50°C to about 75°C.
  • the fluid supported lipid bilayer comprises a lipid within Tm range of about -2°C, about -1°C, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C
  • the fluid supported lipid bilayer comprises a phospholipid. In one embodiments, the fluid supported lipid bilayer (SLB) comprises a phosphatidylcholine (PC). In one embodiment, the fluid supported lipid bilayer (SLB) comprises a lipid selected from the group consisting of cholesterol, 18:0-18:1 PC, 13:0 PC, 16:0-14:0 PC, 18:0-14:0 PC, 14:0-16:0 PC, 15:0 PC, 14:0-18:0 PC, and 16:0-18:0 PC, or a combination thereof.
  • PC phosphatidylcholine
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica microrods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, one or more T-cell co-stimulatory molecules, loaded into and/or presented on the scaffold; and, optionally, one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the fluid supported lipid bilayer (SLB) comprises a lipid comprising 14 to 23 carbon atoms.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the lipid is phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylserine (PS), or phosphoinositide, or a derivative thereof.
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • PA phosphatidic acid
  • PS phosphatidylserine
  • phosphoinositide or a derivative thereof.
  • the APC- MS comprises fluid supported lipid bilayer (SLB) comprises a lipid which is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE) or a combination thereof.
  • DMPC dimyristoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • POPC palmitoyl-oleoylphosphatidylcholine
  • DOPC di
  • the lipid bilayer comprises a lipid composition that mimics the lipid composition of a mammalian cell membrane (e.g., a human cell plasma membrane).
  • a mammalian cell membrane e.g., a human cell plasma membrane.
  • the lipid composition of many mammalian cell membranes have been characterized and are readily ascertainable by one of skill in the art (see, e.g., Essaid et al. Biochim. Biophys. Acta 1858(11): 2725- 36 (2016), the entire contents of which are incorporated herein by reference).
  • the lipid bilayer comprises cholesterol.
  • the lipid bilayer comprises a sphingolipid.
  • the lipid bilayer comprises a phospholipid.
  • the lipid is a phosphatidylethanolamine, a phosphatidylcholine, a phosphatidylserine, a phosphoinositide a phosphosphingolipid with saturated or unsaturated tails comprising 6-20 carbons, or a combination thereof.
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica microrods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, one or more T-cell co-stimulatory molecules, loaded into and/or presented on the scaffold; and, optionally, one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a fluid architecture for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, one or more T-cell co-stimulatory molecules, loaded into and/or presented on the scaffold; and, optionally, one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a fluid architecture for at least 7 days.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, one or more T-cell co-stimulatory molecules, loaded into and/or presented on the scaffold; and, optionally, one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a fluid architecture for at least 14 days.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the MSR-SLB scaffold retains a fluid architecture for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 50 days, or more.
  • the MSR of the MSR-SLB scaffold degrade in about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 50 days, or more.
  • the lipid bilayer of the MSR-SLB scaffold degrades in about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 50 days, or more.
  • the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a fluid architecture for at least 7 days.
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica microrods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a plurality of T-cell activating ligand and/or the T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and a plurality of one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between about 1:1 to about 1000:1, about 1:1 to about 700:1, about 1:100 to about 700:1, or about 1:150 to about 600:1.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC- MS), comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a plurality of T-cell activating ligand and/or the T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and a plurality of one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between about 1: 1 to about 500:1.
  • APC- MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a plurality of T-cell activating ligand and/or the T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and a plurality of one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between about 1: 1 to about 250:1.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a plurality of T-cell activating ligand and/or the T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and a plurality of one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between about 1: 1 to about 100:1.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a plurality of T-cell activating ligand and/or the T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and a plurality of one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between about 1: 1 to about 50:1.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a plurality of T-cell activating ligand and/or the T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and a plurality of one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between about 1: 1 to about 10:1.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the ratio of MSR to T-cell activating/co-stimulatory molecules is reflective of the weight of the MSR to the weight of the antibodies which are used as T-cell activating/co-stimulatory molecules.
  • the MSR:antibody weight ratio is adjusted to achieve the desired scaffold composition.
  • the weight ratio of the SLB to the antibody composition is between about 2:1 and about 20:1, between about 3:1 and about 10:1, between about 4:1 and about 8:1, including all ratios in between, e.g., about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 40:1.
  • the present invention relates to antigen presenting cell-mimetic scaffolds (APC-MS), comprising a base layer comprising high surface area mesoporous silica microrods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a plurality of T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and a plurality of one or more T-cell homeostatic agents loaded into and/or presented on the scaffold, wherein the scaffolds are stacked to selectively permit infiltration of T-cells into the mesoporous silica micro-rods (MSR).
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the instant invention further provides APC-MS wherein the T-cell activating and/or co- stimulatory molecules are present on the scaffolds at a concentration sufficient to permit in situ manipulation of T-cells.
  • spaces between the mesoporous silica micro-rods (MSR) permit T cell infiltration.
  • the present invention relates to pharmaceutical compositions comprising antigen presenting cell-mimetic scaffolds (APC-MS) comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or the T-cell inhibiting ligand, optionally, loaded into and/or presented on the scaffold; and a pharmaceutically acceptable carrier.
  • the present invention relates to pharmaceutical compositions comprising antigen presenting cell-mimetic scaffolds (APC-MS) further comprising one or more T-cell co-stimulatory molecules and/or one or more T-cell homeostatic agents.
  • the present invention relates to pharmaceutical compositions comprising antigen presenting cell-mimetic scaffolds (APC-MS) further comprising one or more T-cell costimulatory molecules loaded into and/or presented on the scaffold and/or one or more T-cell homeostatic agents loaded into and/or presented on the scaffold.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the instant invention further provides pharmaceutical compositions that are formulated for intravenous administration, subcutaneous administration, intraperitoneal administration, or intramuscular administration.
  • the present invention relates to pharmaceutical compositions comprising antigen presenting cell-mimetic scaffolds (APC-MS) comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and, optionally, one or more T- cell homeostatic agents loaded into and/or presented on the scaffold; and a pharmaceutically acceptable carrier.
  • the instant invention further provides pharmaceutical compositions that are formulated for intravenous administration, subcutaneous administration, intraperitoneal administration, or intramuscular administration.
  • the present invention relates to compositions comprising antigen presenting cell-mimetic scaffolds (APC-MS) comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and/or a T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and one or more T-cell homeostatic agents loaded into and/or presented on the scaffold; and T-cells clustered therein.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • the instant invention further provides compositions that contain APC-MS and T-cells selected from the group consisting of natural killer T-cells (NKT), a CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combination thereof.
  • APC-MS natural killer T-cells
  • Tregs regulatory T-cells
  • the present invention relates to compositions comprising antigen presenting cell-mimetic scaffolds (APC-MS) comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell activating ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and one or more T-cell homeostatic agents loaded into and/or presented on the scaffold; and T-cells clustered therein.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • the instant invention further provides compositions that contain APC-MS and T-cells selected from the group consisting of natural killer T-cells (NKT), a CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combination thereof.
  • APC-MS natural killer T-cells
  • Tregs regulatory T-cells
  • the present invention relates to compositions comprising antigen presenting cell-mimetic scaffolds (APC-MS) comprising a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell inhibiting ligand and one or more T-cell co-stimulatory molecules loaded into and/or presented on the scaffold; and one or more T-cell homeostatic agents loaded into and/or presented on the scaffold; and T-cells clustered therein.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • the instant invention further provides compositions that contain APC-MS and T-cells selected from the group consisting of natural killer T-cells (NKT), a CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combination thereof.
  • APC-MS natural killer T-cells
  • Tregs regulatory T-cells
  • the instant invention provides methods of manipulating a population of T- cells, optionally a population of engineered T-cells, in a subject.
  • the instant invention provides methods of manipulating a population of T-cells, in a subject, comprising administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a T-cell ligand (e.g., a T-cell activating ligand or a T-cell inhibiting ligand), wherein the subject was administered a population of T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby manipulating the administered population of T- cells in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • manipulating a population of T-cells can comprise increasing and/or decreasing the level of T-cell activity and/or function as compared to a control level.
  • the method disclosed herein results in a decrease in the level of T-cell activity and/or function in the subject.
  • a decrease in the level of T-cell activity and/or function of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 100% or more as compared to a control level.
  • the method disclosed herein results in an increase in the level of T-cell activity and/or function in the subject.
  • the population of T-cells may be a population of engineered T-cells, such as a population of T-cells comprising a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the instant invention provides methods of treating a disease in a subject.
  • the instant invention provides methods of treating a cancer in a subject, comprising administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC- MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a T-cell ligand (e.g., a T-cell activating ligand or a T-cell inhibiting ligand), wherein the subject was administered a population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby enhancing the systemic anti-tumor activity of the administered population of engineered T-cells in the subject.
  • the subject was administered a population of engineered T-cells prior to administration of the APC-MS.
  • the instant invention provides methods of enhancing anti-tumor activity of an engineered T-cell therapy in a subject, comprising administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell ligand (e.g., a T-cell activating ligand or a T-cell inhibiting ligand), wherein the subject was administered an engineered T-cell therapy comprising a population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby enhancing the systemic anti-tumor activity of an engineered T-cell therapy in the subject.
  • the subject was administered a population of engineered T-cells prior to administration of the APC-MS.
  • the instant invention provides methods in which the APC-MS may further comprise one or more T-cell co-stimulatory molecules, optionally, wherein the T-cell costimulatory molecule may be loaded into and/or presented on the APC-MS.
  • the instant invention provides methods in which the APC-MS may further comprise one or more T-cell homeostatic agents.
  • the T-cell homeostatic agent may be (i) loaded into and/or presented on the SLB layer, or (ii) loaded into and/or presented on the base layer.
  • the instant invention provides methods in which (i) the T-cell homeostatic agent is selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL- 17, IL-21, and transforming growth factor beta (TGF-P), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof; (ii) the T-cell homeostatic agent is IL -2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof with at least one additional homeostatic agent selected from the group consisting of IL-7, IL-21, IL-15, and IL-15 superagonist; and/or (iii) the T-cell homeostatic agent is selected from the group consisting of an N-terminal IL-2 fragment comprising the first 30 amino acids of IL -2 (pl-30), an IL-2 superkine peptide,
  • the instant invention provides methods wherein the subject was administered a population of engineered T-cells at a therapeutic dose and/or a subtherapeutic dose prior to, simultaneous with, or after to administration of the APC-MS.
  • the subject is administered an engineered T-cell therapy comprising a population of engineered T-cells at a subtherapeutic dose prior to, simultaneous with or after administration of the APC-MS.
  • the subject is administered an engineered T-cell therapy comprising a population of engineered T-cells prior to administration of the APC-MS.
  • the subject is administered an engineered T-cell therapy comprising a population of engineered T-cells at a subtherapeutic dose prior to administration of the APC-MS.
  • the engineered T-cells are selected from the group consisting of natural killer T-cells (NKT), CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combination thereof, optionally, wherein the engineered T-cells comprise chimeric antigen receptor (CAR) T-cells and/or T-cell receptor (TCR) T-cells. In some embodiments, the engineered T-cells comprise CAR T-cells.
  • the instant invention provides methods wherein the one or more T- cell ligands, optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand, and/or the T- cell co-stimulatory molecule is (i) loaded into and/or presented on the fluid supported lipid bilayer (SLB); (ii) coated onto the fluid supported lipid bilayer (SLB); (iii) partly embedded onto the fluid supported lipid bilayer (SLB); and/or (iv) loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • SLB fluid supported lipid bilayer
  • MSR mesoporous silica micro-rods
  • the instant invention provides methods wherein the T-cell activating ligand is (i) loaded into and/or presented on the fluid supported lipid bilayer (SLB); (ii) coated onto the fluid supported lipid bilayer (SLB); (iii) partly embedded onto the fluid supported lipid bilayer (SLB); and/or (iv) loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • the T-cell activating ligand is (i) loaded into and/or presented on the fluid supported lipid bilayer (SLB); (ii) coated onto the fluid supported lipid bilayer (SLB); (iii) partly embedded onto the fluid supported lipid bilayer (SLB); and/or (iv) loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • the instant invention provides methods wherein the T-cell inhibiting ligand is (i) loaded into and/or presented on the fluid supported lipid bilayer (SLB); (ii) coated onto the fluid supported lipid bilayer (SLB); (iii) partly embedded onto the fluid supported lipid bilayer (SLB); and/or (iv) loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • the T-cell inhibiting ligand is (i) loaded into and/or presented on the fluid supported lipid bilayer (SLB); (ii) coated onto the fluid supported lipid bilayer (SLB); (iii) partly embedded onto the fluid supported lipid bilayer (SLB); and/or (iv) loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • the instant invention provides methods wherein the T-cell co-stimulatory molecule is (i) loaded into and/or presented on the fluid supported lipid bilayer (SLB); (ii) coated onto the fluid supported lipid bilayer (SLB); (iii) partly embedded onto the fluid supported lipid bilayer (SLB); and/or (iv) loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • the T-cell co-stimulatory molecule is (i) loaded into and/or presented on the fluid supported lipid bilayer (SLB); (ii) coated onto the fluid supported lipid bilayer (SLB); (iii) partly embedded onto the fluid supported lipid bilayer (SLB); and/or (iv) loaded into and/or presented on the mesoporous silica micro-rods (MSR).
  • the instant invention provides methods wherein the one or more T- cell ligands, optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand, and/or the T- cell co-stimulatory molecule is loaded into and/or presented on the fluid supported bilayer (SLB) or the mesoporous silica micro-rods (MSR) via affinity pairing or chemical coupling.
  • SLB fluid supported bilayer
  • MSR mesoporous silica micro-rods
  • affinity coupling comprises a biotin-streptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an affinity pair, a capture protein pair, an Fc receptor-IgG pair, a metalchelating lipid pair, or a combination thereof, optionally, wherein the chemical coupling comprises azide-alkyne chemical (AAC) reaction, dibenzo- cyclooctyne ligation (DCL), or tetrazine-alkene ligation (TAL).
  • AAC azide-alkyne chemical
  • DCL dibenzo- cyclooctyne ligation
  • TAL tetrazine-alkene ligation
  • the one or more T-cell ligands optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand is loaded into and/or presented on the APC-MS.
  • the one or more T-cell ligands, optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand, and/or the T-cell co-stimulatory molecule are antibody molecules or antigen-binding fragments thereof, optionally, wherein the T-cell activating ligands are anti-idiotype antibodies or derivatives thereof.
  • the anti-idiotype antibodies or derivatives thereof bind to the antigen-binding domain of a CAR molecule of the administered population of engineered T-cells in the subject.
  • the one or more T-cell ligands, optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand is selected from the group consisting of an anti-idiotype CD3 antibody (aCD3) or an antigen-binding fragment thereof; an anti-idiotype CD5 antibody (aCD5) or an antigen-binding fragment thereof; an anti-idiotype CD7 antibody (aCD7) or an antigen-binding fragment thereof; an anti-idiotype CD28 antibody (aCD28) or an antigen-binding fragment thereof; an anti-idiotype CD19 antibody (aCD19) or an antigen-binding fragment thereof; an anti-idiotype CD20 antibody (aCD20) or an antigen-binding fragment thereof; an anti-idiotype CD22 antibody (aCD22) or an antigen-binding fragment thereof; an anti-idiotype CD3 antibody
  • the one or more T-cell ligands optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand, comprises a T-cell ligand or derivative thereof that bind to a CAR molecule of the administered population of engineered T-cells in the subject.
  • the one or more T-cell ligands may be selected from the group consisting of a CD3 molecule or a fragment thereof; a CD5 molecule or a fragment thereof; a CD7 molecule or a fragment thereof; a CD28 molecule or a fragment thereof; a CD 19 molecule or a fragment thereof; a CD20 molecule or a fragment thereof; a CD22 molecule or a fragment thereof; a CD70 molecule or a fragment thereof; a CD123 molecule or a fragment thereof; a CS1 molecule or a fragment thereof; a BCMA molecule or a fragment thereof; a SLAMF7 molecule or a fragment thereof; a Claudin-6 molecule or a fragment thereof; a NKG2D molecule or a fragment thereof; a NKG2DL molecule or a fragment thereof; a GD2 molecule or a fragment thereof; a
  • the one or more T-cell ligands can be selected from the group consisting of an anti-CD3 antibody or an antigen-binding fragment thereof, anti-macrophage scavenger receptor (MSR1) antibody or an antigen-binding fragment thereof, an anti-T-cell receptor (TCR) antibody or an antigen-binding fragment thereof, an anti-CD2 antibody or an antigen-binding fragment thereof, an anti-CD47 antibody or an antigen-binding fragment thereof, a major histocompatibility complex (MHC) molecule loaded with an MHC peptide or a multimer thereof, and an MHC-immunoglobulin (Ig) conjugate or a multimer thereof, ICAM-1, or a combination thereof.
  • MHC major histocompatibility complex
  • the one or more T-cell co-stimulatory molecules can be antibodies, or an antigen-binding fragments thereof, which specifically bind to a co-stimulatory antigen selected from the group consisting of CD28, 4.1BB (CD137), 0X40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LT(3R (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRT AM (CD355),TIM1 (HAVCR1, KIMI), CD2 (LFA2, 0X34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), LylO8 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), ICAM-1, and CRACC (CD319, BLAME).
  • a co-stimulatory antigen selected from
  • the one or more T-cell ligands optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand, and/or the one or more T-cell co-stimulatory molecules comprise bispecific antibodies or antigen binding fragments thereof.
  • the one or more T- cell ligands comprises clones of antibodies against CD3/CD28, CD3/ICOS, CD3/CD27, and/or CD3/CD137.
  • the T-cell activating ligand comprises activating clones of antibodies against CD3/CD28, CD3/ICOS, CD3/CD27, and/or CD3/CD137; and/or (ii) the one or more T-cell ligands comprises an activating clone of antibodies against CD28.
  • the T-cell inhibiting ligand comprises inhibiting clones of antibodies against CD3/CD28, CD3/ICOS, CD3/CD27, and/or CD3/CD137; and/or (ii) the one or more T-cell ligands comprises an activating clone of antibodies against CD28.
  • the scaffold further comprises an immunoglobulin molecule that binds specifically to an Fc-fusion protein.
  • the scaffold further comprises a recruitment compound selected from the group consisting of granulocyte macrophage -colony stimulating factor (GM-CSF), chemokine (C- C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), Chemokine (C-X-C Motif) ligand 12 (CXCL12), interferon gamma (IFNy), or a FMS-like tyrosine kinase 3 (Flt-3) ligand.
  • GM-CSF granulocyte macrophage -colony stimulating factor
  • C-C motif chemokine
  • C-C motif chemokine
  • CXCL12 Chemokine (C-X-C Motif) ligand 12 (CXCL12), interferon gamma (IFNy), or a FMS-like tyrosine kinase 3 (Flt-3) ligand.
  • the T-cell activating ligand is an antigen, or the scaffold further comprises an antigen.
  • the antigen comprises a tumor antigen, optionally, wherein the tumor antigen comprises a protein, a glycoprotein, a glycolipid, or a carbohydrate expressed on the surface of a tumor cell.
  • the T-cell activating ligand is a tumor antigen selected from the group consisting of MAGE-1, MAGE-2, MAGE-3, CEA, Tyrosinase, midkin, BAGE, CASP-8, P-catenin, - catenin, y-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gplOO, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf 112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L,
  • the instant invention provides APC-MS wherein the weight ratio of the supported lipid bilayer (SLB) to the mesoporous silica micro-rods (MSR) is between about 10:1 and about 1:20.
  • the fluid supported lipid bilayer comprises a phospholipid.
  • the fluid supported lipid bilayer comprises a phosphatidylcholine (PC).
  • phosphatidylcholine or "PC” is used herein to refer to a class of phospholipids that is composed of a choline head group and glycerophosphoric acid, with a variety of fatty acids, including saturated fatty acids and unsaturated fatty acids.
  • a phosphatidylcholine may comprise acyl chains having lengths ranging from about 10 to about 24 carbons (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 carbons).
  • the fluid supported lipid bilayer comprises a lipid within melting temperature (Tm) about -2°C to about 100°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 75°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 55°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about - 2°C to about 25°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 15°C.
  • Tm melting temperature
  • the fluid supported lipid bilayer comprises a lipid within Tm range about -2°C to about 10°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 5°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about 27°C to about 35°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within melting temperature (Tm) of about 1°C to about 50°C, about 25°C to about 45°C, about 25°C to about 75°C, about 50°C to about 75°C.
  • Tm melting temperature
  • the fluid supported lipid bilayer comprises a lipid within Tm range of about -2°C, about -1°C, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C
  • the fluid supported lipid bilayer comprises a lipid selected from the group consisting of cholesterol, 18:0-18:1 PC, 13:0 PC, 16:0-14:0 PC, 18:0-14:0 PC, 14:0-16:0 PC, 15:0 PC, 14:0-18:0 PC, and 16:0-18:0 PC, or a combination thereof.
  • the fluid supported lipid bilayer comprises a lipid selected from the group consisting of (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE) or a combination thereof.
  • DMPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • POPC palmitoyloleoylphosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • DOPE dioleoylphosphatidylethanolamine
  • DMPE dimyristoylphosphatid
  • the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a fluid architecture for at least 7 days, optionally, at least 14 days.
  • the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between 1:1 to 1000:1, about 1:1 to about 700:1, about 1:100 to about 700:1, or about 1:150 to about 600:1.
  • the scaffolds are stacked to selectively permit infiltration of engineered T-cells into the mesoporous silica micro-rods (MSR).
  • spaces between the mesoporous silica micro-rods (MSR) permit T cell infiltration.
  • the one or more T-cell ligands optionally, the T-cell activating ligand and/or the T-cell inhibiting ligand, and/or the co-stimulatory molecule is present on the scaffolds at a concentration sufficient to permit in situ manipulation of engineered T-cells.
  • the engineered T-cell comprise CAR T-cells and the method results in (a) improved activation, growth, division, differentiation, expansion, proliferation, systemic antitumor activity, cytotoxic activity, metabolic activity, and/or viability of the population of CAR T-cells in the subject; (b) co-stimulating the population of CAR T-cells in the subject; (c) homeostatically maintaining the population of CAR T-cells in the subject; (d) re-stimulating the population of CAR T- cells in the subject; (e) an elevated IFN-y and IL-2 expression in the population of CAR T-cells in the subject, optionally, wherein direct stimulation of the CAR-construct by a T-cell activating ligand results in improved T cell-mediated cytotoxic responses and elevated IFN-y and IL-2 expression in the population of CAR T-cells in the subject as compared to CAR T-cells re-stimulated with anti- CD3/anti-CD28-presenting materials;
  • the persistent, memory-like differentiation profile comprises an effector memory (EM) profile, optionally, wherein the EM profile comprises CD45RA-/CCR7- T- cells, optionally, wherein the EM profile comprises elevated expression of CD25 in both CD4+ and CD8+ T cells, optionally, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or more of the CAR T-cells in the subject have an effector memory (EM) profile.
  • EM effector memory
  • the engineered T-cells physically rearrange and assemble the individual mesoporous silica micro-rods (MSR) into a 3D scaffold to support engineered T-cell activation.
  • MSR mesoporous silica micro-rods
  • the subject is human, optionally, wherein the subject is a pediatric or adult human, optionally, wherein the subject has or is a risk of having impaired in vivo expansion, persistence and/or anti-tumor activity of the administered population of engineered T-cells.
  • the instant invention provides methods wherein the administered population of engineered T-cells comprises a CD3 CAR T-cell; a CD5 CAR T-cell; a CD7 CAR T- cell; a CD28 CAR T-cell; a CD19 CAR T-cell; a CD20 CAR T-cell; a CD22 CAR T-cell; a CD70 CAR T-cell; a CD123 CAR T-cell; a CS1 CAR T-cell; a BCMA CAR T-cell; a SLAMF7 CAR T-cell; a Claudin-6 CAR T-cell; a NKG2D CAR T-cell; a NKG2DL CAR T-cell; a GD2 CAR T-cell; a Her2 CAR T-cell; and a mesothelin (MSLN) CAR T-cell; or a combination thereof.
  • MSLN mesothelin
  • the instant invention provides methods wherein (i) the therapeutic dosage of the engineered T-cells which are CAR T-cells comprises about 1 x 10 4 to about 1 x 10 9 of the CAR T cells; and/or (ii) the subtherapeutic dosage of the engineered T-cells which are CAR T- cells comprises about 75%, about 50%, about 25%, about 20%, about 15%, about 10%, or about 5% or less of the therapeutic dosage of the CAR T-cells, optionally, wherein the subtherapeutic dosage of the engineered T-cells which are CAR T-cells is insufficient to elicit a systemic anti-tumor effect in itself in the absence of the antigen presenting cell-mimetic scaffold (APC-MS), and/or comprises an amount of CAR T-cells that is quantitatively less than an established therapeutic dose for the CAR T- cell.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the instant invention provides methods wherein the administered population of engineered T-cells was administered as a fixed dose, in a dose escalation regime, or as a fractional dose of a total treatment dosage, optionally, wherein the fractional dose comprises between about 1% to about 99% of a therapeutic dosage of the engineered T-cells.
  • the instant invention provides methods which further comprise administering an additional population of engineered T-cells to the subject after or concurrently with the administration of the antigen presenting cell-mimetic scaffold (APC-MS).
  • API-MS antigen presenting cell-mimetic scaffold
  • the instant invention provides scaffolds which comprises at least one cytotoxic T-cell specific ligand and/or at least one cytotoxic T-cell specific co-stimulatory molecule.
  • the instant invention provides methods of treating a cancer in a subject, wherein the cancer is selected from the group consisting of hematological cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, esophageal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, glioblastoma, leukemia, lymphoma, mantle cell lymphoma, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer, optionally, wherein the cancer is a hematological cancer of lymphoid origin expressing a CD 19 surface antigen, optionally, wherein the cancer is an acute lymphoblastic leukemia (ALL), a mature B-cell lymphoma, a chronic lymphocytic leukemia (CLL), a diffuse large B-cell lymphoma (DLBCL), a mantle cell lymphoma (MCL), or a follicle cell lymphoma (FCL).
  • ALL acute lymph
  • the instant invention provides methods wherein the antigen presenting cell-mimetic scaffold (APC-MS) is administered into the subject by injection or implantation, optionally, wherein APC-MS is administered into the subject subcutaneously, optionally, wherein APC-MS is administered into the subject at, or within proximity, of a tumor lesion, optionally, wherein the scaffold is maintained in the subject for a period of at least about 7 days.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the instant invention provides methods wherein the administered population of CAR T-cells is re-stimulated by contact with the scaffold for a period of between 1 day to about 20 days, optionally, at least about 7 days.
  • the instant invention provides methods wherein prior to administration of the APC-MS, the circulating T-cell frequencies in the subject were on average about 0.1% to about 3% of live cells.
  • embodiments of the instant invention relate to methods to improve the efficacy of a T-cell immunotherapy and to boost the systemic antitumor responses of the T-cell immunotherapy administered prior to, simultaneous with, or after administration of the scaffold materials.
  • the instant invention provides methods of treating a disease in a subject in need thereof, comprising administering a population of T-cells (e.g., a population of engineered T-cells) to a subject prior to, simultaneous with, or after administration of the scaffold materials, contacting the population of T-cells (e.g., the population of engineered T-cells) in the subject with the antigen presenting cell-mimetic scaffold (APC-MS), thereby activating, costimulating, and homeostatically maintaining the population of T-cells in vivo-, optionally expanding the population of T-cells in vivo-, optionally re-stimulating the population of T-cells in vivo, thereby treating the disease in the subject.
  • a population of T-cells
  • the instant invention further provides methods of treating a disease in a subject in need thereof, wherein the method further comprises re-stimulating the population of T-cells in vivo after the administration step.
  • the method includes expanding the population of T-cells after contacting the population of T-cells (e.g., the population of engineered T-cells) administered to the subject with the scaffold for a period between 1 day to 30 days, such as about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days or more.
  • the method includes expanding the population of T-cells after contacting the population
  • the subject is a human subject.
  • the method provides for the treatment of a cancer.
  • the cancer is selected from the group consisting of head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, esophageal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, glioblastoma, leukemia, lymphoma, mantle cell lymphoma, pre- neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.
  • the instant invention relates to methods for the manipulation of T- cells in vivo, comprising contacting the antigen presenting cell-mimetic scaffold (APC-MS) with a population of T-cells (e.g., the population of engineered T-cells) in vivo, thereby activating, costimulating, homeostatically maintaining and optionally expanding and/or re-stimulating a population of T-cells present within a subject, thereby manipulating the T-cells.
  • the manipulation may include stimulation, activation, changes in viability, promotion of growth, division, differentiation, expansion, proliferation, exhaustion, anergy, quiescence, apoptosis, death of T-cells in vivo.
  • the manipulation preferably includes promoting expansion and/or restimulating and/or proliferation of T-cells in vivo.
  • the manipulated T- cells may be engineered T-cells.
  • the manipulated T-cells may be further transformed.
  • the T-cells may be transformed to express a chimeric antigen receptor (CAR).
  • the CAR T-cell product may be expanded in vivo by administering and/or associating with the antigen presenting cell-mimetic scaffolds (APC-MS) containing an antigen which is specific to the CAR molecule and/or a T-cell ligand or derivative thereof that binds to the CAR molecule.
  • APC-MS antigen presenting cell-mimetic scaffolds
  • the CAR T-cell-specific antigen and/or the one or more T-cell ligands is selected from the group consisting of CD3, CD5, CD7, CD28, CD19, CD20, CD22, CD70, CD123, CS1, BCMA, SLAMF7, Claudin-6, NKG2D, NKG2DL, GD2, Her2, and mesothelin (MSLN), or a fragment thereof or a variant thereof or a combination thereof.
  • the CAR T-cell-specific antigen is a cancer antigen, such as a tumor antigen.
  • Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE- la, p53, prostein, PSMA, Her2/neu, survivin and telomerase, prostate -carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF- II, IGF-I receptor and mesothelin.
  • the T cell ligand is a T-cell activating ligand or a the T-cell inhibiting ligand.
  • the T-cell activating ligand and/or the T-cell inhibiting ligand may be an antibody molecule or antigen-binding fragments thereof.
  • the T-cell activating ligand and/or the T-cell inhibiting ligand may be an anti-idiotype antibody or derivative thereof.
  • the T-cell activating ligand and/or the T-cell inhibiting ligand may be selected from the group consisting of an anti-idiotype CD3 antibody (aCD3) or an antigen-binding fragment thereof; an anti-idiotype CD5 antibody (aCD5) or an antigen-binding fragment thereof; an anti-idiotype CD7 antibody (aCD7) or an antigen-binding fragment thereof; an anti-idiotype CD28 antibody (aCD28) or an antigen-binding fragment thereof; an anti-idiotype CD19 antibody (aCD19) or an antigen-binding fragment thereof; an anti-idiotype CD20 antibody (aCD20) or an antigen-binding fragment thereof; an anti-idiotype CD22 antibody (aCD22) or an antigenbinding fragment thereof; an anti-idiotype CD70 antibody (aCD70) or an antigen-binding fragment thereof; an anti-idiotype CD123 antibody (aCD123) or an antigen-binding fragment thereof; an antiidiotype CS1 antibody (aCSl) or an antigen
  • the instant invention relates to methods for the manipulation of T- cells in vivo, comprising contacting the antigen presenting cell-mimetic scaffold (APC-MS) comprising a T-cell activating ligand and, optionally one or more T-cell co-stimulatory molecules, with a population of T-cells (e.g., a population of engineered T-cells) in vivo, wherein the method confers increased expansion of the population of T-cells after about 1 week of contact with the scaffold in vivo compared to a control scaffold comprising the base layer comprising high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell activating ligand and, optionally one or more T-cell co-stimulatory molecules.
  • APC-MS antigen presenting cell-mimetic scaffold
  • SLB fluid supported lipid bilayer
  • the method confers about a 5-fold to 1500-fold increase in the expansion of the population of T-cells after about 1 week of contact with the scaffold in vivo compared to a control scaffold comprising the base layer comprising high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell activating ligand and, optionally the T-cell co-stimulatory molecule.
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • the instant invention relates to methods for improving the metabolic activity of T-cells, comprising contacting the antigen presenting cell-mimetic scaffold (APC-MS) comprising a T-cell activating ligand and, optionally one or more T-cell co-stimulatory molecules, with a population of T-cells (e.g., a population of engineered T-cells) in vivo, thereby activating, costimulating, homeostatically maintaining and optionally expanding a population of T-cells present within a subject, thereby improving the metabolic activity of T-cells.
  • APC-MS antigen presenting cell-mimetic scaffold
  • a population of T-cells e.g., a population of engineered T-cells
  • the method confers improved metabolic activity of the population of T-cells after about 1 week of contact with the scaffold in vivo compared to a control scaffold comprising the base layer comprising high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell activating ligand and, optionally the T-cell co-stimulatory molecule.
  • a control scaffold comprising the base layer comprising high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell activating ligand and, optionally the T-cell co-stimulatory molecule.
  • the method confers about a 5-fold to 20-fold improved metabolic activity of the population of T-cells after about 1 week of contact with the scaffold compared to a control scaffold comprising the base layer comprising high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell activating ligand and, optionally the T-cell co-stimulatory molecule.
  • the antigen presenting cell-mimetic scaffold is contacted with a population of T-cells (e.g., a population of engineered T-cells) in vivo for a period from about 1 day to about 30 days.
  • the antigen presenting cellmimetic scaffold is contacted with a population of T-cells (e.g., a population of engineered T-cells) in vivo for a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days or more.
  • a population of T-cells e.g., a population of engineered T-cells
  • the method may result in the production of one or more cytokines or cytotoxins by the manipulated T- cells, such as one or more cytokines or cytotoxins selected from the group consisting of interferon gamma (IFNy), tissue necrosis factor alpha (TNFa), IL-2, IL-1, IL -4, IL-5, IL-10, and IL-13, and IL- 17 or a combination thereof.
  • IFNy interferon gamma
  • TNFa tissue necrosis factor alpha
  • the method involves further detecting the production of a cytokine selected from the group consisting of interferon gamma (IFNy), tissue necrosis factor alpha (TNFa), IL-2, IL-1, IL -4, IL-5, IL-10, and IL-13, IL-17 or a combination thereof by the manipulated T-cells.
  • a cytokine selected from the group consisting of interferon gamma (IFNy), tissue necrosis factor alpha (TNFa), IL-2, IL-1, IL -4, IL-5, IL-10, and IL-13, IL-17 or a combination thereof by the manipulated T-cells.
  • the instant invention relates to methods for the manipulation of T-cells in vivo in accordance with the foregoing methods, wherein the scaffold is administered to the subject to permit the population of T-cells (e.g., the population of engineered T-cells) to come into contact with the scaffold in vivo.
  • the scaffold may be maintained in the subject for a period between about 3 days to about 15 days, preferably for a period between about 7 days to about 11 days.
  • the scaffold may be maintained in the subject for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, or more.
  • the instant invention relates to methods for making the antigen presenting cell-mimetic scaffold (APC-MS), comprising (a) providing a base layer comprising high surface area mesoporous silica micro-rods (MSR); (b) optionally loading the one or more T-cell homeostatic agents on the MSR; (c) layering a fluid supported lipid bilayer (SLB) on the base layer comprising the MSRs, thereby generating an MSR-SLB scaffold; (d) loading the one or more T-cell homeostatic agents on the MSR-SLB scaffold if step (b) is not carried out; (e) optionally blocking one or more non-specific integration sites in the MSR-SLB scaffold with a blocker; and (f) loading the T-cell activating ligand and/or the T-cell inhibiting ligand and, optionally, the one or more T-cell co-stimulatory molecules onto the MSR-SLB scaffold, thereby making the APC-MS.
  • APC-MS antigen presenting
  • the methods may further involve assembling a plurality of scaffolds to generate stacks with sufficient porosity to permit infiltration of T cells.
  • the method may include loading at least one additional agent selected from the group consisting of a growth factor, a cytokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule, or a fragment thereof or a combination thereof.
  • FIG. 1 shows a schematic representation of the local macroscale materials-approach to modulate systemic T-cell activity.
  • the materials are based on our antigen-presenting cell-mimetic scaffold (APC-ms) system and are assembled and injected subcutaneously.
  • APC-ms antigen-presenting cell-mimetic scaffold
  • Mesoporous silica rods are loaded with IL-2 and then coated in a biotinylated (0.1 mol % biotin: POPC) supported lipid bilayer.
  • Various T-cell ligands e.g., aCD3, aCD28, or aCD19 idiotype
  • T-cell ligands are then conjugated to the APC-ms via biotin-streptavidin linkages.
  • circulating therapeutic T cells may continuously infiltrate into the scaffold, become activated, proliferate, and egress to carry out antitumor functions.
  • the inset is a zoom-in representation of the material scaffold, highlighting the physiological presentation of membrane-bound ligands for T cell stimulation and costimulation and paracrine cytokine release of interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • FIGS. 2A-2I shows properties of in vitro restimulated chimeric antigen receptor (CAR)-T cells.
  • FIGS. 2A-2B show representative scanning electron microscopy (SEM) images of individual high-aspect ratio mesoporous silica microrods (FIG. 2A) and following material assembly and co-culture with naive T cells (FIG. 2B).
  • FIG. 2C shows representative brightfield images of 2 nd generation CD19-directed CAR-T cells interacting with the material scaffolds presenting 0.1 mol% aCD3 (top) and anti-CD3/anti-CD28 (bottom) after 2 days of culture. Interacting T cells physically rearrange and assemble the individual rods into a 3D scaffold to support T-cell activation.
  • FIG. 2D shows fold expansion of CAR-T cells following co-culture with either IL-2 alone (hereafter denoted as “mock”) or material scaffolds presenting 0.1 mol% anti-CD3/anti-CD28 after 6 days.
  • FIG. 2E shows frequency of CAR-expressing T cells following material co-culture.
  • FIG. 2F shows CD4-to-CD8 ratio following material co-culture.
  • FIG. 2G shows proportion of various memory T-cell subpopulations following material co-culture, gated using CD45RA and CCR7.
  • Naive/stem memory CD45RA-CCR7+
  • Central memory CD45RA-CCR7+
  • Effector memory EM
  • Effector CD45RA+CCR7-.
  • CAR-T cells restimulated with the material scaffold were predominantly CD45RA-CCR7- (EM).
  • Restimulated CAR-T cells were co-cultured with CD19-expressing Raji target cells to evaluate their response to in vitro tumor antigen stimulation.
  • FIG. 21 shows luciferized Raji cell cytotoxicity following co-culture with restimulated CAR-T cells.
  • FIGS. 3A-3E shows subcutaneously-injected material scaffolds are biodegradable and retain lipid membranes.
  • FIG. 3A shows in vivo degradation of material scaffolds following subcutaneous injection. C57BL/6 albino mice were subcutaneously administered 5 mg of material (coated with lipid membranes containing 2.5 mol % Cy5 -labeled lipid or unlabeled lipid). Biodegradation of the scaffolds was tracked longitudinally via in vivo fluorescence imaging using IVIS. Representative IVIS images are shown.
  • FIG. 3B shows quantified fluorescence signal (as a mean fluorescence intensity measured by drawing a rectangular region-of-interest around the scaffold) over time. Each line represents one animal.
  • FIG. 3A shows in vivo degradation of material scaffolds following subcutaneous injection. C57BL/6 albino mice were subcutaneously administered 5 mg of material (coated with lipid membranes containing 2.5 mol % Cy5 -labeled lipid or unlabeled lipid). Biodegradation of the scaffold
  • FIG. 3C shows gross nodule size containing the material scaffold (calculated by measuring the length and width of the nodule).
  • FIG. 4A-4F shows functional properties of subcutaneously-injected material scaffolds.
  • FIG. 4A Showa material scaffolds presenting anti-CD3 and anti-CD28 were subcutaneously injected into NSG mice and then excised at the indicated timepoints. The scaffolds were gently disrupted and co-cultured with CFSE-labeled CAR-T cells in the absence of IL-2 for 3 days to evaluate the material’s capacity to restimulate T cells. Proliferation was measured via CFSE dilution.
  • FIG. 4C shows proportion of recovered scaffolds - scaffolds decreased dramatically in size over a period of 21 days.
  • FIG. 4D shows mass of explanted scaffolds (either blank or presenting 0.1 mol% anti-CD3/anti-CD28) and surrounding tissue. The fraction denotes the number of scaffolds recovered of total scaffolds injected at each timepoint.
  • FIG. 4E shows CFSE mean fluorescence intensity. The “+ Ctrl” and Ctrl” represents fresh, never before-injected materials presenting anti-CD3/anti-CD28 or not, respectively. The “+scaffold” and “-scaffold” represents explanted materials. Similar CFSE intensities were observed in scaffolds explanted at 7 days, but not 14 or 21 days.
  • FIG. 4F shows expression of CD25 in CD8+ T cells following 3 days of ex vivo coculture with explanted scaffolds.
  • FIGS. 5A-5H shows injected material scaffolds presenting ligands for stimulation and costimulation facilitates CAR T-cell infiltration, activation, and egress.
  • FIG. 5A shows naive, unstimulated T cells were labeled with CFSE and co-delivered subcutaneously with materials presenting T-cell ligands and IL-2 or without. After 5 days, the scaffolds were explanted, and T- cell proliferation was measured by CFSE dilution.
  • FIG. SB shows CFSE mean fluorescence intensity of T cells. ICAM-1 was added to improve T-cell adhesion. Representative histogram of diluted CFSE in corresponding CD8+ T cells (right).
  • FIG. 5A shows naive, unstimulated T cells were labeled with CFSE and co-delivered subcutaneously with materials presenting T-cell ligands and IL-2 or without. After 5 days, the scaffolds were explanted, and T- cell proliferation was measured by CFSE
  • SC shows scaffolds injected into NSG mice were resected after 7 and 14 days, enzymatically digested, and subjected to FACS analysis.
  • Human T cells were detected in anti-CD3/anti-CD28 presenting scaffolds, while very few human T cells were detected in non-ligand-presenting “blank” scaffolds.
  • FIG. 5D shows quantification of human T cells detected in resected scaffolds.
  • FIG. 5E shows the capacity of injected material scaffolds to boost T-cell proliferation was evaluated in a xenograft lymphoma model. Animals were inoculated with 5e5 luciferized Raji-cells and treated 4 days afterwards with a curative dose of le6 CD19 CAR-T cells.
  • FIG. 5F shows concentration of CAR-T cells circulating in the blood after subcutaneous injection of materials. CAR-T cell proliferation was boosted by scaffolds containing T-cell ligands, peaking ⁇ 11 days after injection.
  • FIG. 5G shows representative FACs plots showing increased frequencies of T cells at day 11 relative to blank scaffolds. Data in FIGS. 5F-5G represents mean ⁇ s.e.m and is representative of at least two experimental replicates.
  • FIGS. 6A-6C shows in vitro restimulation of CAR-T cell products.
  • FIG. 6A shows representative brightfield images of CD 19 CAR-T cells restimulated with various materials presenting activating anti-CD3/anti-CD28 antibodies at the indicated densities. 4x magnification after 2 days co-culture.
  • FIG. 6B shows fold expansion after 6 days of in vitro culture.
  • FIGS. 7A-7G shows a single local injection of scaffolds enhances systemic anti-tumor activity in an aggressive liquid tumor model.
  • FIG. 7A shows 5e5 luciferized Raji tumor cells were injected into NSG mice via the tail vein. Mice were injected with 5e5 CD19.BBz CAR+T cells at day 4. At day 9, mice were subcutaneously injected with scaffolds.
  • FIG. 7B shows IVIS images of NSG mice injected intraperitoneally with luciferin to measure tumor burden. Color indicates tumor burden. Time points are measured relative to the date of tumor injection.
  • FIG. 7C shows quantification of total photon flux (photons/second) for mice from FIG. 7B.
  • FIG. 7D shows Kaplan Meier curve depicting survival of the mice in FIG. 7B (note that the time scale shown in FIG. 7D is longer than that depicted in FIG. 7B).
  • FIG. 7E shows percentage (%) of CD3+ human T cells and
  • FIG. 7F shows CAR+ T cells detected via FACS within scaffolds that were explanted from the animals at the time of death.
  • FIG. 7G shows percentage (%) of CD8+ T cell within the explanted scaffolds. Data in e-g represents mean ⁇ s.e.m. Significance in FIG. 7D was calculated using a log-rank Mantel-cox test.
  • FIGS. 8A-8J shows scaffolds are capable of antigen-specifically stimulating CAR + T cells.
  • FIG. 8A shows representative brightfield images of 2 nd generation CD19-directed CAR-T cells interacting with the material scaffolds presenting 0.1 mol% aCD3, CD 19 protein, or anti- CD19 scFv idiotype with (top) or without anti-CD28 (bottom). Interacting T cells physically rearrange and assemble the individual rods into a 3D scaffold to support T-cell activation. This effect is particularly pronounced when scaffolds co-present aCD3, CD19 protein, or anti-CD19 scFv idiotype with aCD28 antibody at a 1:1 ratio (bottom row).
  • FIG. 8A shows representative brightfield images of 2 nd generation CD19-directed CAR-T cells interacting with the material scaffolds presenting 0.1 mol% aCD3, CD 19 protein, or anti- CD19 scFv idiotype with (top) or without anti-CD28 (bottom).
  • FIG. 8B shows fold expansion of CD19.BBz CARs cultured with material scaffolds presenting the indicated ligands for 7 days
  • FIG. 8C shows percentage (%) of CAR + cells of CD19.BBz CARs cultured with material scaffolds presenting the indicated ligands for 7 days.
  • FIG. 8D shows CD4-CD8 ratio of CD19.BBz CARs cultured with material scaffolds presenting the indicated ligands for 7 days.
  • FIG. 8E shows representative FACS plots following stimulation with material scaffolds for 6 days.
  • FIG. 8F shows quantification of T cell effector/memory phenotypes following stimulation with material scaffolds for 6 days.
  • FIG. 8G shows FACS quantification of the percentage of scaffold-stimulated T cells expressing the high-affinity IL-2 receptor CD25.
  • FIG. 8H shows in vitro scaffold stimulation provokes effector responses (e.g., IFNg and IL-2 release) as measured by intracellular cytokine staining following 4 hours of T-cell stimulation by the scaffold.
  • FIG. 81 shows quantification of cytokines measured via FACS following in vitro stimulation.
  • FIG. 8J shows in vitro killing of CD19+ Raji-luc target cells following co-culture with CD19.BBz T cells that were previously stimulated with scaffolds presenting various composition of the material scaffold. Data in FIGS. 8B-8J represents mean ⁇ s.d. of four technical replicates and is representative of at least two experimental replicates.
  • FIGS. 9A-9E shows additional characterization of T-cell activation and egress.
  • Naive, unstimulated T cells were labeled with CFSE with and co-delivered subcutaneously with materials presenting T-cell ligands and IL-2 or without.
  • FIG. 9A shows tSNE analysis of single cell immune infiltrate, highlighting CD4 and CD8 T cells. The plot of FIG. 9A is colored by CD3 expression.
  • FIGS. 9B-9C shows frequency of CD3+ T cells among live cells (FIG. 9B) and their CFSE mean fluorescence intensity (FIG. 9C).
  • FIG. 9D shows analysis of T-cell boosting capacity in different tumor contexts.
  • FIG. 9E shows number of CD3+ T cells following subcutaneous scaffold injection under sub-curative CAR-T dosing conditions. Data in FIG. 9D represents mean ⁇ s.e.m.
  • FIG. 10 shows no difference in animal survival following administration of scaffolds with or without collagen.
  • Material scaffolds were formulated to present anti-CD3/anti-CD28/IL-2 and co-administered with or without 1 mg/mL collagen. No significant differences in animal survival were observed.
  • FIG. 11 shows functional responsiveness of in vitro restimulated CAR-T cells.
  • CD 19 CAR-T cells were co-cultured with materials presenting either anti-CD19 idiotype or CD 19 protein with anti-CD28 costimulation at either 0.1, 0.3, or 0.5 mol% densities. Frequency of IL-2 expressing CD8+ CAR-T cells. Despite the presence of anti-CD28 costimulation, the activating anti-CD19 idiotype promoted robust IL-2 signaling.
  • the present invention provides antigen presenting cell-mimetic scaffolds (APC-MS), which are useful in the manipulation of engineered T-cells, for example, CAR T-cells, in vivo.
  • the scaffolds include mesoporous silica rods (MSR), which incorporate or are coated with a fluid supported lipid bilayer (SLB) thereby forming MSR-SLB scaffolds.
  • the MSR-SLB scaffold further contains a T-cell ligand, such as, a T-cell activating ligand and/or a T-cell inhibiting ligand, and may optionally further comprise one or more T-cell co-stimulatory molecules and/or one or more T-cell homeostatic agents, which together make up a structure that mimics antigen-presenting cells (APC) and allows the scaffolds to elicit various effector functions on target cells, e.g., T-cells, in vivo.
  • the scaffold mediates these effects via direct or indirect interaction between the cell surface molecules residing in target cells and the various binding partners presented by the scaffolds.
  • the scaffold regulates survival and growth of the targeted cells through the physical or chemical characteristics of the scaffold itself.
  • the scaffold composition may be modified to contain certain a T-cell ligand, such as, a T-cell activating ligand and/or a T-cell inhibiting ligand, as well as, optionally, T- cell co-stimulatory and/or homeostatic signaling molecules, which act together to mediate various effector functions, e.g., activation, division, promote differentiation, growth, expansion, reprogramming, anergy, quiescence, senescence, apoptosis or death, of target cells.
  • the scaffolds were found to surprisingly enhance the activity of prior-administered CAR- T cells.
  • the scaffolds were found to be able to improve subject survival, for example, in a therapeutic xenograft setting.
  • elevated numbers of CAR-T cells were found in scaffolds delivering both surface and soluble ligands which was accompanied by more circulating effector T cells.
  • the persistence of CAR-T cells in the scaffolds several weeks following subcutaneous injection described herein demonstrates that CAR-T cells may continuously infiltrate and egress from the scaffolds, which may have important implications in the solid tumor setting.
  • a local subcutaneous scaffold of the invention injected close to a tumor lesion may provide more optimal tumor-reactive T-cell stimulation while providing desirable survival and proliferation signals. Scaffolds presenting a CAR-specific ligand were also found to promote CAR-T cell restimulation, resulting in CAR antigen-specific expansion and improved functionality in vitro.
  • a scaffold of the invention Compared to CAR-T cells restimulated with anti- CD3/anti-CD28-presenting materials, it was found that direct stimulation of the CAR-construct by a scaffold of the invention led to improved T cell-mediated cytotoxic responses and elevated IFN-y and IL-2 expression, demonstrating a benefit in stimulating the CAR specifically under optimal immune microenvironments.
  • the injectable material scaffolds were found to surprisingly provide local stimulation to prior-administered CAR-T cells to improve their systemic response. Accordingly, the materials described herein may serve as a cost-effective “booster” for T-cell immunotherapies.
  • the present invention provides antigen-presenting cell-mimetic scaffolds (APC-MS).
  • the scaffolds contain a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a T-cell ligand (e.g., a T-cell activating ligand or a T-cell inhibiting ligand).
  • the scaffold may further include, one or more T-cell co-stimulatory molecules and/or one or more T-cell homeostatic agents.
  • the components of the scaffolds of the invention include mesoporous silica.
  • Mesoporous silica is a porous body with hexagonal close-packed, cylinder-shaped, uniform pores. This material is synthesized by using a rod-like micelle of a surfactant as a template, which is formed in water by dissolving and hydrolyzing a silica source such as alkoxysilane, sodium silicate solution, kanemite, silica fine particle in water or alcohol in the presence of acid or basic catalyst. See, US Pub. No. 2015-0072009 and Hoffmann et al., Angewandte Chemie International Edition, 45, 3216-3251, 2006.
  • a mesoporous silica having the greatest specific surface area and a pore volume. See, U.S. Publication No. 2013/0052117 and Katiyar et al. Journal of Chromatography 1122 (1-2): 13- 20).
  • the terms “mesoscale,” “mesopore,” “mesoporous” and the like, as used in this specification, may refer to structures having feature sizes in the range of 5 nm to 100 nm, in particular in the range of 2 nm to 50 nm.
  • a mesoporous material includes pores, which may be ordered or randomly distributed, having a diameter in the range of 5 nm to 100 nm.
  • the mesoporous silica used in the scaffolds of the invention may be provided in various forms, e.g., microspheres, irregular particles, rectangular rods, round nanorods, etc., although structured rod forms (MSR) are particularly preferred.
  • the particles can have various predetermined shapes, including, e.g., a spheroid shape, an ellipsoid shape, a rod-like shape, or a curved cylindrical shape.
  • Methods of assembling mesoporous silica to generate microrods are known in the art. See, Wang et al., Journal of Nanoparticle Research, 15:1501, 2013.
  • mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate with a template made of micellar rods. The result is a collection of nano-sized spheres or rods that are filled with a regular arrangement of pores. The template can then be removed by washing with a solvent adjusted to the proper pH.
  • hydrophilic silica nanoparticles characterized by a uniform, ordered, and connected mesoporosity are prepared with a specific surface area of, for example, about 600 m 2 /g to about 1200 m 2 /g, particularly about 800 m 2 /g to about 1000 m 2 /g and especially about 850 m 2 /g to about 950 m 2 /g.
  • the mesoporous particle could be synthesized using a simple sol-gel method or a spray drying method. Tetraethyl orthosilicate is also used with an additional polymer monomer (as a template).
  • one or more tetraalkoxy-silanes and one or more (3-cyanopropyl)trialkoxy-silanes may be co-condensed to provide the mesoporous silicate particles as rods. See, US Publication Nos. 2013-0145488, 2012-0264599 and 2012-0256336, which are incorporated by reference.
  • the mesoporous silica rods may comprise pores of between 2-50 nm in diameter, e.g., pores of between 2-5 nm, 10-20 nm, 10-30 nm, 10-40 nm, 20-30 nm, 30-50 nm, 30-40 nm, 40-50 nm.
  • the microrods comprise pores of approximately 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, or more in diameter.
  • the pore size may be altered depending on the type of application.
  • the length of the micro rods is in the micrometer range, ranging from about 5 pm to about 500 pm.
  • the microrods comprise a length of 5-50 pm, e.g., 10-20 pm, 10-30 pm, 10-40 pm, 20-30 pm, 30-50 pm, 30-40 pm, 40-50 pm.
  • the rods comprise length of 50 pm to 250 pm, e.g., about 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 120 pm, 150 pm, 180 pm, 200 pm, 225 pm, or more.
  • MSR compositions having a higher aspect ratio e.g., with rods comprising a length of 50 pm to 200 pm, particularly a length of 80 pm to 120 pm, especially a length of about 100 pm or more.
  • the MSR provide a high surface area for attachment and/or binding to target cells, e.g., T-cells.
  • target cells e.g., T-cells.
  • Methods of obtaining high surface area mesoporous silcates are known in the art. See, e.g., US patent No. 8,883,308 and US Publication No. 2011-0253643, the entire contents of which are incorporated by reference herein.
  • the high surface area is due to the fibrous morphology of the nanoparticles, which makes it possible to obtain a high concentration of highly dispersed and easily accessible moieties on the surface.
  • the high surface area MSRs have a surface area of at least about 100 m 2 /g, at least 150 m 2 /g, or at least 300 m 2 /g. In other embodiments, the high surface area MSRs have a surface area from about 100 m 2 /g to about 1000 m 2 /g, including all values or sub-ranges in between, e.g., 50 m 2 /g, 100 m 2 /g, 200 m 2 /g, 300 m 2 /g, 400 m 2 /g, 600 m 2 /g, 800 m 2 /g, 100-500 m 2 /g, 100-300 m 2 /g, 500-800 m 2 /g or 500-1000 m 2 /g.
  • the scaffolds of the invention comprise a fluid supported lipid bilayer (SLB) on the base layer.
  • SLB fluid supported lipid bilayer
  • lipid generally denotes a heterogeneous group of substances associated with living systems which have the common property of being insoluble in water, can be extracted from cells by organic solvents of low polarity such as chloroform and ether.
  • lipid refers to any substance that comprises long, fatty-acid chains, preferably containing 10-30 carbon units, particularly containing 14-23 carbon units, especially containing 16-18 carbon units.
  • the lipid is provided as a monolayer. In another embodiment, the lipid is provided as a bilayer.
  • a lipid bilayer is a thin polar membrane made of two layers of lipid molecules. Preferably, the lipid bilayer is fluid, wherein individual lipid molecules able to diffuse rapidly within the monolayer.
  • the membrane lipid molecules are preferably amphipathic.
  • the lipid layers are continuous bilayers, e.g., resembling those found in natural biological membranes such as cellular plasma membranes.
  • the lipid is provided in the form of a supported bilayer (SLB).
  • SLB is a planar structure sitting on a solid support, e.g., mesoporous silica rods (MSR).
  • MSR-SLB scaffolds are stable and remain largely intact even when subject to high flow rates or vibration and can withstand holes, e.g., holes that are aligned with the pores of the mesoporous silica base layer. Because of this stability, experiments lasting weeks and even months are possible with supported bilayers.
  • SLBs are also amenable to modification, derivatization and chemical conjugation with many chemical and/or biological moieties.
  • the SLB may be immobilized on the base layer using any known methods, including covalent and non-covalent interactions. Types of non-covalent interactions include, for example, electrostatic interactions, van der Waals’ interactions, n-effects, hydrophobic interactions, etc.
  • the lipids are adsorbed on the base layer.
  • the SLBs are attached or tethered to the MSR base layer via covalent interactions. Methods for attaching lipids to silicates are known in the art, e.g., surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material.
  • the lipid bilayers are layered onto the MSR base layer.
  • a lipid film (containing for example, a solution of DPPC/cholesterol/DSPE-PEG at a molar ratio of 77.5:20:2.5 in chloroform) may be spotted onto the mesoporous silica and the solvent is evaporated using a rotary evaporator.
  • the lipid bilayer can be prepared, for example, by extrusion of hydrated lipid films through a filter with pore size of, for example, about 100 nm, using standard protocols. The filtered lipid bilayer films can then be fused with the porous particle cores, for example, by a pipette mixing.
  • covalent coupling via alkylating or acylating agents may be used to provide a stable, structured and long-term retention of the SLB on the MSR layer.
  • the lipid bilayers may be reversibly or irreversibly immobilized onto the MSR layers using known techniques.
  • the MSR base layer can be hydrophilic and can be further treated to provide a more hydrophilic surface, e.g., with ammonium hydroxide and hydrogen peroxide.
  • the lipid bilayer can be fused, e.g., using known coupling techniques, onto the porous MSR base layer to form the MSR-SLB scaffolds.
  • the scaffolds may be further processed and derivatized with additional moieties to allow attachment and/or immobilization of other secondary agents onto the structure.
  • the instant invention provides MSR-SLB scaffolds, wherein the SLB component is a phospholipid.
  • SLB component is a phospholipid.
  • Representative examples of such lipids include, but are not limited to, amphoteric liposomes described in U.S. Patent Nos. 9,066,867 and 8,3676,28.
  • the lipid bilayer may comprise a lipid selected from dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoyl-phosphatidylethanolamine (DPPE) or a combination thereof.
  • DMPC dimyristoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • POPC palmitoyl-oleoylphosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • the lipid bilayer comprises a lipid composition that mimics the lipid composition of a mammalian cell membrane (e.g., a human cell plasma membrane).
  • a mammalian cell membrane e.g., a human cell plasma membrane.
  • the lipid composition of many mammalian cell membranes have been characterized and are readily ascertainable by one of skill in the art (see, e.g., Essaid et al. Biochim. Biophys. Acta 1858(11): 2725- 36 (2016), the entire contents of which are incorporated herein by reference).
  • the composition of the lipid bilayer may be altered to modify the charge or fluidity of the lipid bilayer.
  • the lipid bilayer comprises cholesterol.
  • the lipid bilayer comprises a sphingolipid.
  • the lipid bilayer comprises a phospholipid.
  • the lipid is a phosphatidylethanolamine, a phosphatidylcholine, a phosphatidylserine, a phosphoinositide a phosphosphingolipid with saturated or unsaturated tails comprising 6-20 carbons, or a combination thereof.
  • the lipid is DIYNE PC lipid.
  • lipids include, but are not limited to, l-Palmitoyl-2-10,12 Tricosadiynoyl-sn-Glycero-3-Phosphocholine (16:0-23:2 DIYNE PC) and l,2-bis(10,12-tricosadiynoyl)-SN-Glycero-3-Phosphocholine (23:2 Diyne PC).
  • the fluid supported lipid bilayer comprises a lipid within melting temperature (Tm) about -2°C to about 100°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 75°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 55°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 25°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 15°C.
  • Tm melting temperature
  • the fluid supported lipid bilayer comprises a lipid within Tm range about -2°C to about 10°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about -2°C to about 5°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within Tm range about 27°C to about 35°C. In some embodiments, the fluid supported lipid bilayer (SLB) comprises a lipid within melting temperature (Tm) of about 1°C to about 50°C, about 25°C to about 45°C, about 25°C to about 75°C, about 50°C to about 75°C.
  • Tm melting temperature
  • the fluid supported lipid bilayer comprises a lipid within Tm range of about -2°C, about -1°C, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C
  • Such a lipid may be selected, without limitation, from the group consisting of cholesterol, 18:0-18:1 PC, 13:0 PC, 16:0-14:0 PC, 18:0-14:0 PC, 14:0- 16:0 PC, 15:0 PC, 14:0-18:0 PC, and 16:0-18:0 PC, or a combination thereof.
  • the MSR-SLB scaffold of the invention retains a fluid architecture for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, or more.
  • the architecture of the MSR-SLB scaffold may be studied with any known techniques, including, the microscopic visualization techniques illustrated in the Examples below.
  • the MSR-SLB scaffold may contain one or more functional molecules.
  • the antigen presenting cell-mimetic scaffold may comprise a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a functional molecule selected from the group consisting of a T-cell ligand, a T-cell co-stimulatory molecule, a T-cell homeostatic agent and a combination thereof.
  • Each of the aforementioned one or more functional molecules may be independently loaded into and/or presented on the APC-MS.
  • the term “functional molecule” includes any molecule which possesses biologically desirable properties.
  • examples of such functional molecules include proteins, peptides, antigens, antibodies, nucleic acids, DNA, RNA, carbohydrates, lipids, haptens, and other small molecules, e.g., drugs.
  • the term “functional molecule” may refer to an agent, e.g. , a functional molecule or moiety, that exhibits a function, e.g., a catalytic activity, a binding activity, a therapeutic activity, or an activity as a detectable label.
  • a functional molecule is a protein having a catalytic activity, e.g., an enzyme or a fragment thereof.
  • a functional molecule is a protein having a binding activity, e.g., a binding agent such as an antibody or an antigen-binding antibody fragment.
  • a functional molecule is a small molecule or a nucleic acid having a binding activity.
  • a functional molecule is a small molecule, a peptide or protein, or a nucleic acid having a therapeutic activity, e.g., a cytotoxic, cytostatic, anti-angiogenic, or pro-apoptotic activity; or a pro-survival, anti-apoptotic, or pro-angiogenic activity.
  • the one or more functional molecules is one or more T-cell ligands, such as one or more T-cell activating ligands and/or one or more T-cell inhibiting ligands.
  • the one or more functional molecules is one or more T-cell co-stimulatory molecules.
  • the one or more functional molecules is one or more T-cell homeostatic agents.
  • the MSR-SLB scaffolds comprise a plurality of functional molecules, e.g., at least one T-cell ligand, at least one T-cell co-stimulatory molecule, and/or at least one T-cell homeostatic agent.
  • the MSR-SLB scaffolds comprise at least one T-cell ligand and at least one T-cell co-stimulatory molecule. In certain embodiments, the MSR-SLB scaffolds comprise at least one T-cell ligand, at least one T- cell co-stimulatory molecule, and at least one T-cell homeostatic agent.
  • the term “loaded” or “load”, is used to refer to a functional molecule’s mechanisms of attachment, or association with, the scaffolds of the instant disclosure.
  • loaded include, but are not limited to, covalent and non-covalent interactions.
  • mechanisms encompassing loaded may involve techniques known in the art, such as immobilizing, affixing, attaching, adhering, adsorbing, bonding, tethering, coupling, interacting, mixing, conjoining, connecting, integrating, contacting, conjugating, sticking, hybridizing, coating, containing, imbedding, linking, associating, fusing, embedding, partially embedding, and/or binding, and are envisaged within the scope of the instant disclosure.
  • the functional molecules e.g., a functional molecule selected from the group consisting of a T-cell ligand, a T-cell co-stimulatory molecule, a T-cell homeostatic agent, and a combination thereof
  • a functional molecule selected from the group consisting of a T-cell ligand, a T-cell co-stimulatory molecule, a T-cell homeostatic agent, and a combination thereof may each independently or in combination be loaded into and/or presented on the APC-MS and/or the SLB.
  • one or more T-cell ligands, a T-cell co-stimulatory molecule, and a T-cell homeostatic agent may each independently or in combination be loaded into and/or presented on the APC-MS and/or the SLB.
  • one or more T-cell ligands may be loaded into and/or presented on the APC-MS and/or the SLB.
  • one or more T-cell co-stimulatory molecules may be loaded into and/or presented on the APC-MS and/or the SLB.
  • one or more T-cell homeostatic agents may be loaded into and/or presented on the APC-MS and/or the SLB.
  • the instant invention provides for MSR-SLB scaffolds containing one or more T-cell ligands (e.g., one or more T-cell activating ligands or one or more T-cell inhibiting ligands).
  • the instant invention provides for MSR-SLB scaffolds containing one or more T-cell activating ligands.
  • T-cell ligands may mediate direct, indirect, or semi-direct activation of a target population of T-cells. See, Benichou et al. , Immunotherapy, 3(6): 757-770, 2011.
  • the T-cell ligands mediate direct activation of T-cells.
  • the instant invention provides for MSR-SLB scaffolds containing molecules which directly activate T-cells, e.g., via binding to cell surface receptors on target T- cells.
  • the direct activation may be mediated via cluster of differentiation-3 (CD3), which is a T-cell co-receptor that helps to activate cytotoxic T-cells.
  • CD3 cluster of differentiation-3
  • T- cells may be directly activated without concomitant participation of CD3, e.g., in a CD3- independent manner.
  • a T-cell ligand e.g., a T-cell activating ligand or a T-cell inhibiting ligand
  • a T-cell activating ligand or a T-cell inhibiting ligand can be an antibody molecule or antigen-binding fragment thereof.
  • the T-cell activating ligands and/or the T-cell inhibiting ligands can be anti-idiotype antibodies or derivatives thereof.
  • the anti-idiotype antibodies or derivatives thereof may bind to the antigen-binding domain of a CAR molecule of the administered population of engineered T-cells in the subject.
  • the T-cell activating ligands and/or the T-cell inhibiting ligands are selected from the group consisting of an anti-idiotype CD3 antibody (aCD3) or an antigen-binding fragment thereof; an anti-idiotype CD5 antibody (aCD5) or an antigen-binding fragment thereof; an anti-idiotype CD7 antibody (aCD7) or an antigen-binding fragment thereof; an anti-idiotype CD28 antibody (aCD28) or an antigen-binding fragment thereof; an anti-idiotype CD19 antibody (aCD19) or an antigen-binding fragment thereof; an anti-idiotype CD20 antibody (aCD20) or an antigen-binding fragment thereof; an anti-idiotype CD22 antibody (aCD22) or an antigen-binding fragment thereof; an anti-idiotype CD70 antibody (aCD70) or an antigen-binding fragment thereof; an anti-idiotype CD123 antibody (aCD123) or an antigen-binding fragment thereof; an anti-idiotype CS1 antibody (aCSl) or
  • the T-cell activating ligands and/or the T-cell inhibiting ligands are T-cell ligands or derivatives thereof that bind to a CAR molecule of the administered population of engineered T-cells in the subject.
  • the T-cell activating ligands and/or the T-cell inhibiting ligands are selected from the group consisting of a CD3 molecule or a fragment thereof; a CD5 molecule or a fragment thereof; a CD7 molecule or a fragment thereof; a CD28 molecule or a fragment thereof; a CD 19 molecule or a fragment thereof; a CD20 molecule or a fragment thereof; a CD22 molecule or a fragment thereof; a CD70 molecule or a fragment thereof; a CD123 molecule or a fragment thereof; a CS1 molecule or a fragment thereof; a BCM A molecule or a fragment thereof; a SLAMF7 molecule or a fragment thereof; a Claudin-6 molecule or a fragment thereof; a NKG2D molecule or a fragment thereof; a NKG2DL molecule or a fragment thereof; a GD2 molecule or a fragment thereof; a Her2 molecule or a fragment thereof; and a me
  • the target T-cells are activated in a CD3-dependent manner. It is generally believed that T cell activation requires a T cell receptor (TCR) to recognize its cognate peptide in the context of an MHC molecule.
  • TCR T cell receptor
  • the association of CD3 with the TCR- peptide-MHC complex transmits the activation signal to intracellular signaling molecules to initiate a signaling cascade in the T cell. See, Ryan et al., Nature Reviews Immunology 10, 7, 2010.
  • the CD3 receptor complex found on T-cells contains a CD3y chain, a CD35 chain, and two CD3a chains, which associate with TCR and the ⁇ -chain (zeta-chain; CD247) to generate an activation signal in T cells.
  • TCR T cell receptor
  • the T-cell ligand is an antibody or an antigen binding fragment thereof. Where the T-cell ligand acts in a CD3-dependent manner, the T-cell ligand is preferably an anti-CD3 antibody or an antigen-binding fragment thereof. In another embodiments, the T-cell ligand may include, for example, an anti-CD2 antibody or an antigen-binding fragment thereof, an anti-CD47 antibody or an antigen-binding fragment thereof, anti-macrophage scavenger receptor (MSR1) antibody or an antigen-binding fragment thereof, an anti-T-cell receptor (TCR) antibody or an antigen-binding fragment thereof, etc.
  • MSR1 anti-macrophage scavenger receptor
  • TCR anti-T-cell receptor
  • the T-cell ligand is a major histocompatibility complex (MHC) molecule or a multimer thereof that is optionally loaded with an MHC peptide. Still further, the T-cell ligand is a conjugate containing MHC and immunoglobulin (Ig) or a multimer thereof.
  • the T-cell activating antibody used in the compositions and methods of the disclosure is the anti-CD3 antibody selected from the group consisting of muromonab (0KT3), otelixizumab (TRX4), teplizumab (hOKT3yl(Ala- Ala)), visilizumab, an antibody recognizing 17-19 kD s-chain of CD3 within the CD3 antigen/T cell antigen receptor (TCR) complex (HIT3a), and an antibody recognizing a 20 kDa subunit of the TCR complex within CD3e (UCHT1), or an antigen-binding fragment thereof.
  • TCR CD3 antigen/T cell antigen receptor
  • HIT3a CD3 antigen/T cell antigen receptor
  • UCHT1 an antibody recognizing a 20 kDa subunit of the TCR complex within CD3e
  • Other anti-CD3 antibodies, including, antigen-binding fragments thereof are described in US patent pub. No. 2014-0088295, which is incorporated by
  • Embodiments of the invention include “full-length” antibodies.
  • each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Immunoglobulin molecules can be of any type ⁇ e.g., IgG, IgE, IgM, IgD, IgA and IgY), class ⁇ e.g., IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen e.g., IL-13). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 Al herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigenbinding portion” of an antibody.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994)).
  • Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001 ) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
  • Antibody fragments comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, and typically most or all, of the functions normally associated with that portion when present in an intact antibody.
  • an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
  • an antibody fragment for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement binding.
  • an antibody fragment is a monovalent antibody that has an in vivo half-life substantially similar to an intact antibody.
  • such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
  • antibody construct refers to a polypeptide comprising one or more the antigen binding portions of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain.
  • Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions.
  • Such linker polypeptides are well known in the art (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al. , Structure 2: 1121-1123 (1994)).
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art and disclosed in Table 2 of U.S. Patent No. 7,915,388, the entire contents of which are incorporated herein by reference.
  • an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol.
  • Antibody portions such as Fab and F(ab’)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • an “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD3 is substantially free of antibodies that specifically bind antigens other than CD3).
  • An isolated antibody that specifically binds CD3 may, however, have cross-reactivity to other antigens, such as CD3 molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in U.S. Patent No. 7,915,388, the contents of which are incorporated herein by reference), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom et al. , TIB Tech. 15:62-70 (1994); Azzazy et al., Clin. Biochem.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • One embodiment provides fully human antibodies capable of binding human CD3 which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries such as those disclosed in Jermutus et al., PCT publication No. WO 2005/007699 A2.
  • chimeric antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
  • Methods for producing chimeric antibodies are known in the art and discussed in to detail in Example 2.1. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties.
  • chimeric antibodies may be produced by art-known techniques. See, Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851- 855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454 which are incorporated herein by reference in their entireties.
  • telomere binding in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • the antibodies used in the scaffolds of the present invention may be “monospecific,” “bispecific,” or “multispecific.”
  • the expression “antibody” herein is intended to include both monospecific antibodies e.g., anti-CD3 antibody) as well as bispecific antibodies comprising an arm that binds to an antigen of interest (e.g., a CD3-binding arm) and a second arm that binds a second target antigen.
  • the target antigen that the other arm of the CD3 bispecific antibody binds can be any antigen expressed on or in the vicinity of a cell, tissue, organ, microorganism or virus, against which a targeted immune response is desired.
  • the CD3-binding arm binds human CD3 and induces human T cell proliferation.
  • antibodies which bind to different regions of the CD3 molecule e.g., an arm that binds to a 17-19 kD 8-chain of CD3 within the CD3 antigen/T cell antigen receptor (TCR) complex (e.g., derived from HIT3a), and arm that binds to a 20 kDa subunit of the TCR complex within CD3e (e.g., derived from UCHT1).
  • TCR CD3 antigen/T cell antigen receptor
  • the anti-CD3 antibody is OKT3 or a CD3-binding fragment thereof.
  • the antibody molecule used in the scaffolds of the invention is a bispecific antibody.
  • Bispecific antibodies may be employed in the context of the invention to bring a cell of interest, e.g., a cancer cell or a pathogen, in close proximity with the target effector cell of the invention, e.g., a cytotoxic T-cell, such that the effector function of the target effector cell is mediated specifically upon the cell of interest.
  • the invention provides scaffolds containing bispecific antibodies, wherein one arm of the antibody binds CD3 and the other arm binds a target antigen which is a tumor- associated antigen.
  • Non-limiting examples of specific tumor- associated antigens include, e.g., AFP, ALK, BAGE proteins, P-catenin, brc-abl, BRCA1 , BORIS, CA9, carbonic anhydrase IX, caspase-8, CCR5, CD19, CD20, CD30, CD40, CDK4, CEA, CTLA4, cyclin-Bl , CYP1 Bl , EGFR, EGFRvlll, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1 , F0LR1 , GAGE proteins (e.g., GAGE-1 , -2), GD2, GD3, GloboH, glypican-3, GM3, gplOO, Her2, HLA/B-raf, HLA/k-ras, H LA/MAG E-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1 , - 2,
  • the cancer antigen is a member of the epidermal growth factor receptor (EGFR) family, e.g., a receptor selected from the group consisting of EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4), or a mutant thereof.
  • EGFR epidermal growth factor receptor
  • the invention in another embodiment, relates to scaffolds containing a bispecific T-cell engager (BiTE) molecule.
  • the BiTE molecule is specifically an antibody that recognizes at least one of the aforementioned tumor antigens and at least one T-cell cell surface molecule, e.g., CD3.
  • T-cell cell surface molecule e.g., CD3.
  • bispecific T-cell engager molecules include, but are not limited to, solitomab (CD3xEpCAM), blinatumomab (CD3xCD19), MAB MT-111 (CD3xCEA), and BAY- 2010112 (CD3xPSMA).
  • Bispecific antibodies may also be used in the context of the invention to target effector cells such as T-cells or B-cells to mediate effect on pathogens, e.g., bacteria, viruses, fungus, protists, and other microbes, either directly or indirectly.
  • pathogens e.g., bacteria, viruses, fungus, protists, and other microbes, either directly or indirectly.
  • the pathogen is a virus.
  • the pathogen is a bacteria.
  • Bispecific antibodies have been used to treat bacterial infections, e.g., drug resistant Pseudomonas aeruginosa. See, DiGiandomenico et al., Sci Transl Med., 6(262), 2014; Kingwell et al., Nat Rev Drug Discov., 14(1): 15, 2015.
  • the invention provides scaffolds containing bispecific antibodies, wherein one arm of the antibody binds CD3 and the other arm binds a target antigen which is an infectious disease-associated antigen (e.g., a bacterial, protozoal, viral, or fungal antigen).
  • a target antigen which is an infectious disease-associated antigen (e.g., a bacterial, protozoal, viral, or fungal antigen).
  • infectious disease-associated antigens include, e.g., an antigen that is expressed on the surface of a virus particle, or preferentially expressed on a cell that is infected with a virus, wherein the virus is selected from the group consisting of HIV, hepatitis (A, B or C), herpes virus (e.g., HSV-1 , HSV-2, CMV, HAV- 6, VZV, Epstein Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.
  • viruses e.g., an antigen that is expressed on the surface of a virus particle, or preferentially
  • the target antigen can be an antigen that is expressed on the surface of a bacterium, or preferentially expressed on a cell that is infected with a bacterium, wherein the bacterium is from a genus selected from the group consisting of Chlamydia, Rickettsia, Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococci, Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, Clostridium, and Leptospira.
  • a genus selected from the group consisting of Chlamydia, Rickettsia, Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococci, Gonococci, Klebsiella, Proteus, Serratia, Pseudomon
  • the bacteria causes cholera, tetanus, botulism, anthrax, plague, or Lyme disease.
  • the target antigen is an antigen that is expressed on the surface of a fungus, or preferentially expressed on a cell that is infected with a fungus, wherein the fungus is selected from the group consisting of Candida (e.g., C. albicans, C. krusei, C. glabrata, C. tropicalis, etc.). Crytococcus neoformans, Aspergillus (e.g. , A. fumigatus, A. niger, etc.), Mucorales (e.g., M. mucor, M.
  • the target antigen is an antigen that is expressed on the surface of a parasite, or preferentially expressed on a cell that is infected with a parasite, wherein the parasite is selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis, Taenia crassiceps, and Brugia malayi.
  • Non-limiting examples of specific pathogen-associated antigens include, e.g., HIV gpl20, HIV CD4, hepatitis B glucoprotein L, hepatitis B glucoprotein M, hepatitis B glucoprotein S, hepatitis C El , hepatitis C E2, hepatocyte-specific protein, herpes simplex virus gB, cytomegalovirus gB, and HTLV envelope protein.
  • the scaffold of the invention may be used for the treatment and/or prevention of an allergic reaction or allergic response.
  • the scaffold may be used to generate T-cells (e.g., Tregs) that suppress an allergic response or reaction.
  • the scaffolds comprise an anti-CD3 antibody and TGF-p.
  • the scaffolds comprise an anti-CD3 antibody and IL-10, in some embodiments, the scaffolds comprise an anti-CD3 antibody and rapamycin.
  • the scaffolds comprise an anti-CD3 antibody, TGF-P, IL-10 and rapamycin.
  • the scaffolds comprise an anti-CD3 antibody TGF-P, and IL-10.
  • the scaffolds comprise an anti-CD3 antibody and TGF-P and rapamycin.
  • the scaffolds comprise an anti- CD3 antibody and IL- 10 and rapamycin.
  • the scaffold of the invention may be used to selectively expand allergen reactive T-cells (e.g., Tregs).
  • the scaffold comprises a peptide derived from an allergen.
  • the peptide derived from an allergen is presented on (e.g., complexed with) an MHC molecule (e.g., an MHC class I or MHC class II molecule).
  • the MHC molecule is a monomer.
  • the allergen is a food allergen (e.g., a banana, milk, legumes, shellfish, tree nut, stone fruit, egg, fish, soy, or wheat allergen).
  • the allergen is selected from the group consisting of a food allergen, a plant allergen, an insect allergen, an animal allergen, a fungal allergen, a viral allergen, a latex allergen, and a mold spore allergen.
  • the allergen polypeptide is an insect allergen.
  • the insect allergen is a dust mite allergen (e.g., an allergen from Dermatophagoides farina or Dermatophagoides pteronyssinus).
  • the allergen polypeptide is an ovalbumin polypeptide.
  • the allergen polypeptide is a food allergen polypeptide.
  • the scaffold comprises a peptide derived from an allergen and a Th 1 -skewing cytokine (e.g., IL-12 or IFNy).
  • the allergen polypeptide is a food allergen polypeptide.
  • the scaffold comprises a peptide derived from an allergen presented on an MHC molecule and a Thl-skewing cytokine e.g., IL-12 or IFNy).
  • the present invention includes bispecific antigen-binding molecules that specifically bind CD3 and CD28. Such molecules may be referred to herein as, e.g., “anti-CD3/anti-CD28,” or “anti-CD3xCD28” or “CD3xCD28” bispecific molecules, or other similar terminology.
  • CD28 refers to the human CD28 protein unless specified as being from a non-human species (e.g., “mouse CD28,” “monkey CD28,” etc.).
  • the human CD28 protein has the amino acid sequence shown in GENBANK accession Nos. NP_001230006.1, NP_001230007.1, or NP_006130.1.
  • the mouse CD28 protein has the amino acid sequence shown in GENBANK accession No. NP_031668.3.
  • the various polypeptide sequences encompassed by the aforementioned accession numbers include, the corresponding mRNA and gene sequences, are incorporated by reference herein in their entirety.
  • an antigen-binding molecule means a protein, polypeptide or molecular complex comprising or consisting of at least one complementarity determining region (CDR) that alone, or in combination with one or more additional CDRs and/or framework regions (FRs), specifically binds to a particular antigen.
  • CDR complementarity determining region
  • FRs framework regions
  • an antigen-binding molecule is an antibody or a fragment of an antibody, as those terms are defined elsewhere herein.
  • the expression “bispecific antigen-binding molecule” means a protein, polypeptide or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain.
  • Each antigen-binding domain within the bispecific antigen- binding molecule comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen.
  • the first antigen-binding domain specifically binds a first antigen (e.g., CD3)
  • the second antigen-binding domain specifically binds a second, distinct antigen (e.g., CD28).
  • the first antigen-binding domain and the second antigen-binding domain of the bispecific antibodies may be directly or indirectly connected to one another.
  • the first antigenbinding domain and the second antigen- binding domain may each be connected to a separate multimerizing domain.
  • the association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule.
  • a “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution.
  • a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain.
  • a nonlimiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgGl, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.
  • Bispecific antigen-binding molecules of the present invention will typically comprise two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain.
  • the first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgGl/lgGl , lgG2/lgG2, lgG4/lgG4.
  • the first and second multimerizing domains may be of different IgG isotypes such as, e.g., lgGl/lgG2, lgGl/lgG4, lgG2/lgG4, etc.
  • the multimerizing domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residues. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine- containing peptide.
  • Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.
  • bispecific antibody format or technology may be used to make the bispecific antigenbinding molecules of the present invention.
  • an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule.
  • bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgGl/lgG2, dual acting Fab (DAF)-lgG, and Mab2 bispecific formats (see, e.g., Klein et al., mAbs 4:6, 1 -11, 2012 and references cited therein, for a review of the foregoing formats).
  • Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.
  • the anti-CD3 antibodies of the present invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein.
  • an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity.
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.
  • the multispecific antigenbinding molecules of the invention are derived from chimeric, humanized or fully human antibodies. Methods for making multispecific antibodies are well known in the art.
  • one or more of the heavy and/or light chains of the bispecific antigen-binding molecules of the present invention can be prepared using VELOCIMMUNETM technology.
  • VELOCIMMUNETM technology or any other human antibody generating technology
  • high affinity chimeric antibodies to a particular antigen e.g., CD3 or CD28 are initially isolated having a human variable region and a mouse constant region.
  • the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc.
  • the mouse constant regions are replaced with a desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the bispecific antigen-binding molecules of the present invention.
  • the multimerizing domains may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain.
  • the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn.
  • the bispecific antigen-binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0).
  • an acidic environment e.g., in an endosome where pH ranges from about 5.5 to about 6.0.
  • the present invention also includes bispecific antigenbinding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bispecific antibody lacking the amino acid difference.
  • the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype.
  • the T-cell ligand is a major histocompatibility complex (MHC) molecule which binds to CD3.
  • MHC major histocompatibility complex
  • Representative examples include, but are not limited to, MHC type I which binds to TCR and CD8 or MHC type II which binds to TCR and CD4.
  • the MHC molecules may be optionally loaded with antigens, e.g., biotinylated peptides.
  • the MHC molecules may be conjugated to immunoglobulins, e.g., Fc portion of an immunoglobulin G (IgG) chain.
  • IgG immunoglobulin G
  • a plurality of MHC -peptide complexes may be employed.
  • MHC multimers include, but are not limited to, MHC-dimers (contains two copies of MHC-peptide; IgG is used as multimerization domain, and one of the domains of the MHC protein is covalently linked to IgG); MHC -tetramers (contains four copies of MHC-peptide, each of which is biotinylated and the MHC complexes are held together in a complex by the streptavidin tetramer protein, providing a non-covalent linkage between a streptavidin monomer and the MHC protein); MHC pentamers (contains five copies of MHC-peptide complexes are multimerised by a self-assembling coiled-coil domain)., MHC dextramers (typically contains more than ten MHC complexes which are attached to a dex
  • MHC tetramers are described in U.S. Pat. No. 5,635,363; MHC pentamers are described in the US patent 2004209295; MHC -dextramers are described in the patent application WO 02/072631. MHC streptamers are described in Knabel M et al., Nature Medicine 6. 631-637, 2002).
  • the target T-cells may also be activated in a CD3-independent manner, for example, via binding and/or ligation of one or more cell-surface receptors other than CD3.
  • cell-surface molecules include, e.g., CD2, CD47, CD81, MSR1, etc.
  • CD2 is found on virtually all T cells (and also natural killer T-cells (NKT)) and is important in T-lymphocyte function.
  • CD2 is associated with several proteins including CD3, CD5 and CD45.
  • CD2-CD58 interaction facilitates cell-cell contact between T cells and APC, thereby enhancing antigen recognition through the TCR /CD3 complex.
  • CD2 also serves a signal transduction role.
  • Co-stimulation blockade using antibodies directed against CD2 may be a potent immunosuppressive strategy in organ transplantation.
  • the T-cells are activated via the use of an antibody or an antigen binding fragment thereof that specifically binds to CD2.
  • Representative examples of anti-CD2 antibodies include, for example, siplizumab (MEDI-507) and LO-CD2b (ATCC accession No. PTA-802; deposited June 22, 1999).
  • CD47 (IAP) belongs to the immunoglobulin superfamily and partners with membrane integrins and also binds the ligands thrombospondin- 1 (TSP-1) and signal-regulatory protein alpha (SIRPa). See Barclay et al., Curr. Opin. Immunol. 21 (1): 47-52, 2009; Br. J. Pharmacol., 167 (7): 1415-30, 2012. CD47 interacts with signal-regulatory protein alpha (SIRPa), an inhibitory transmembrane receptor present on myeloid cells.
  • SIRPa signal-regulatory protein alpha
  • the T- cells are activated via the use of an antibody or an antigen binding fragment thereof that specifically binds to CD47.
  • anti-CD47 antibodies include, for example, monoclonal antibody Hu5F9-G4, which is being investigated in various clinical trials against myeloid leukemia and monoclonal antibodies MABL-1 and MABL-2 (FERM Deposit Nos. BP-6100 and BP-6101). See, e.g., WO1999/12973, the disclosure in which is incorporated by reference herein.
  • CD 81 is a member of the tetraspanin superfamily of proteins. It is expressed on a broad array of tissues, including T cells and hematopoietic cells. CD 81 is known to play an immunomodulatory role. In particular, cross-linking of CD81 enhances CD3 mediated activation of aP and y5 T-lymphocytes and induces TCR-independent production of cytokines by y5 T cells in vitro.
  • the T-cells are activated via the use of an antibody or an antigen binding fragment thereof that specifically binds to CD81. See, Menno et al., J. Clin. Invest., 4:1265, 2010. Representative examples of anti-CD81 antibodies include, for example, monoclonal antibody 5A6. See, e.g., Maecker et al., BMC Immunol., 4:1, 2003., the disclosure in which is incorporated by reference herein.
  • MSR1 (CD204) belongs to the family of class A macrophage scavenger receptors, which include three different types (1, 2, 3) generated by alternative splicing of the MSR1 gene. These receptors or isoforms are trimeric integral membrane glycoproteins and have been implicated in many macrophage-associated physiological and pathological processes including atherosclerosis, Alzheimer's disease, and host defense. See, Matsumoto et al., Proc. Natl. Acad. Sci. U.S.A. SI (23): 9133-7, 1990. Recent studies demonstrate that dendritic (DC) MSR1 impacts the activation and proliferation of CD8 T cells and antibody-mediated blocking of MSR1 increased proliferation and expansion of T-cells in vitro.
  • DC dendritic
  • the T-cells are activated via the use of an antibody or an antigen binding fragment thereof that specifically binds to MSR1.
  • anti-MSRl antibodies include, for example, rat anti-human CD204 antibody (Thermo Catalog No. MA5-16494) and goat anti-human CD204/MSR1 antibody (Biorad Catalog No. AHP563).
  • the T-cells are activated by ligating/binding to a T-cell receptor (TCR) molecule, which is expressed ubiquitously in T-cells.
  • TCR T-cell receptor
  • the TCR is a heterodimer composed of two different protein chains. In humans, in 95% of T cells the TCR consists of an alpha (a) and beta (P) chain, whereas in 5% of T cells the TCR consists of gamma and delta (y/5) chains.
  • TCR engages with antigenic peptide and MHC (peptide/MHC)
  • the T lymphocyte is activated through signal transduction.
  • the T-cells are activated via the use of an antibody or an antigen binding fragment thereof that specifically binds to TCR.
  • anti-TCR antibodies include, for example, mouse anti-human TCR monoclonal antibody IMMU510 (Immunotech, Beckman Coulter, Fullerton, CA)(described in Zhou et al. , Cell Mol Immunol., 9(1): 34-44, 2012) and monoclonal antibody defining alpha/beta TCR WT31 (described in Gupta et al. , Cell Immunol. , 132(l):26-44, 1991).
  • IMMU510 Immunotech, Beckman Coulter, Fullerton, CA
  • monoclonal antibody defining alpha/beta TCR WT31 described in Gupta et al. , Cell Immunol. , 132(l):26-44, 1991.
  • the T-cell ligand is a major histocompatibility complex (MHC) molecule that is optionally loaded with an MHC peptide.
  • MHC major histocompatibility complex
  • pMHC cytotoxic T lymphocytes
  • Class II MHC molecules are found mainly on antigen-presenting immune cells (APCs), which ingest polypeptide antigens (in, for example, microbes) and digest them into peptide fragments.
  • APCs antigen-presenting immune cells
  • the MHC-II molecules then present the peptide fragments to helper T cells, which, after activation, provide generally required helper activity for responses of other cells of the immune system (e.g., CTL or antibody-producing B cells).
  • TCR T cell receptor
  • the instant invention relates to MSR-SLB scaffolds containing a human MHC molecule optionally loaded with a peptide.
  • MHC molecules include HLA-A, HLA-B, HLA-C, DP, DQ and DR, or a combination thereof.
  • the MHC molecules may be monovalent or bivalent. In some embodiments, bivalency or multivalency of the MHC molecules is desirable for signal delivery (either activation or inhibition signals) to the T cell. Therefore, in some embodiments, the MSR-SLB scaffolds of the present invention include at least two identical MHC molecules attached to a linker.
  • the linker of the bivalent MHC molecule serves three functions. First, the linker contributes the required bivalency or multivalency. Second, the linker increases the half-life of the entire fusion protein in vivo. Third, the linker determines whether the fusion protein will activate or suppress T cells. T cell priming requires stimulation via the TCR and an additional second signal generally delivered by the APC. In the absence of a second signal, T cell hyporesponsiveness may result. By constructing a fusion protein in which the linker allows delivery of a second signal, T cell stimulation results in enhanced T cell immunity. By constructing a fusion protein in which the linker does not provide for delivery of a second signal, T cell suppression results in immunosuppression.
  • a fusion protein with T cell stimulatory properties can be constructed by using a linker which allows for delivery of a second signal to the T cell in addition to the signal delivered via the TCR. This can be accomplished by using a linker that has binding affinity for a cell surface structure on another cell, that cell being capable of delivering a second signal to the T cell. Thus, the linker serves to bridge the T cell and the other cell. By bringing the other cell into close proximity to the T cell, the other cell can deliver a second signal to the T cell. Examples include linkers that can bind to Fc receptors on other cells such as certain immunoglobulin chains or portions of immunoglobulin chains. Specific examples include IgG, IgA, IgD, IgE, and IgM.
  • the entire protein is not required.
  • the immunoglobulin gene can be cleaved at the hinge region and only the gene encoding the hinge, CH2, and CH3 domains of the heavy chain is used to form the fusion protein.
  • the linker may bind other cell surface structures.
  • the linker can include a cognate moiety for many cell surface antigens which can serve as a bridge to bring the second cell into close proximity with the T cell.
  • the linker might also deliver a second signal independently.
  • a linker with binding affinity for the T cell antigen CD28 can deliver a second signal.
  • the linker can increase the half-life of the entire fusion protein in vivo.
  • a fusion protein with T cell inhibitory properties can be constructed by using a linker that does not result in delivery of a second signal.
  • linker that does not result in delivery of a second signal.
  • examples include Ig chains that do not bind Fc receptor, Ig F(ab')2 fragments, a zinc finger motif, a leucine zipper, and non-biological materials.
  • non-biological materials include plastic microbeads, or even a larger plastic member such as a plastic rod or tube, as well as other physiologically acceptable carriers which are implantable in vivo.
  • the MHC molecules are not attached to a linker.
  • a linker it is believed that the fluid nature of the lipid bilayer allows T cells to reorganize the membrane and form multivalent clusters. These clusters can subsequently be disassembled, which would not be possible if the signaling molecules were attached together with a linker. Inability to un-form these multivalent clusters can potentially lead to overstimulation and T cell exhaustion or anergy (see, e.g., Fee K-H et al. Science 302(5648): 1218-22 (2003)).
  • the lipid bilayer of the APC-MS comprises a lipid compositions that favor the spontaneous partitioning of lipid species into liquid-ordered domains (see, e.g., Wang T-Y et al. Biochemistry 40(43): 13031-40 (2001)).
  • the MHC molecules may be loaded with a specific peptide (e.g., a peptide derived from a viral antigen, a bacterial antigen, or an allergen).
  • the specific peptide of the fusion protein can be loaded into the MHC molecules after the fusion protein has been made.
  • the peptide may also be subsequently covalently attached to the MHC, for example by UV cross-linking.
  • a peptide sequence can be incorporated into the DNA sequence encoding the fusion protein such that the peptide is loaded into the MHC molecules during generation of the fusion protein.
  • the peptide can be attached with a tether, such as polylysine, which allows it to complex with the MHC portion of the fusion protein.
  • the specific peptides to be loaded into the MHC molecules are virtually limitless and are determined based on the desired application.
  • peptides from various sources e.g., viral, fungal and bacterial infections, or to tumors, can be used.
  • autoreactive peptides can be used.
  • self-peptides which are presented by alloantigens can be used.
  • Toxins, such as ricin and diphtheria toxin, and radioisotopes may be complexed to the fusion protein (for example, using 5-methyl-2-iminothiolane) to kill the specific T cell clones.
  • These toxins can be chemically coupled to the linker or to the MHC portion of the fusion protein, or they can be incorporated into the DNA sequence encoding the fusion protein such that the toxin is complexed to the fusion protein during generation of the fusion protein.
  • the MHC-peptide/immunoglobulin fusion protein can be prepared by constructing a gene which encodes for the production of the fusion protein.
  • the components of the fusion protein can be assembled using chemical methods of conjugation. Sources of the genes encoding the MHC molecules and the linkers can be obtained from various databases.
  • the MHC fragment can be attached to the linker and P2 microglobulin can be allowed to self-associate.
  • the fusion protein gene can be constructed such that P2 microglobulin is attached to the MHC fragment by a ether.
  • either the alpha or the beta chain can be attached to the linker and the other chain can be allowed to selfassociate.
  • the fusion protein gene can be constructed such that the alpha and beta chains are connected by a tether.
  • Peptides can be prepared by encoding them into the fusion protein gene construct or, alternatively, with peptide synthesizers using standard methodologies available to one of ordinary skill in the art. The resultant complete fusion proteins can be administered using routine techniques.
  • the instant invention provides MSR-SLB scaffolds containing a plurality of one or more T-cell co-stimulatory molecules.
  • These co-stimulatory molecules may mediate direct, indirect, or semi-direct stimulation of a target population of T-cells.
  • the co-stimulatory molecules mediate activation of T-cells in the presence of one or more T-cell ligands.
  • co-stimulatory molecule refers to a group of immune cell surface receptor/ligands which engage between T cells and antigen presenting cells and generate a stimulatory signal in T cells which combines with the stimulatory signal (z.e. , “co-stimulation”) in T cells that results from T cell receptor (“TCR”) recognition of antigen on antigen presenting cells.
  • a soluble form of a co-stimulatory molecule “derived from an APC” refers to a co-stimulatory molecule normally expressed by B cells, macrophages, monocytes, dendritic cells and other APCs.
  • a “co-stimulator of T cells activation” refers to the ability of a co-stimulatory ligand to bind and to activate T cells which have been activated via any of the aforementioned mechanisms or pathways, e.g., via CD3-dependent or CD3-independent T-cell activation.
  • Co- stimulatory activation can be measured for T cells by the production of cytokines as is well known and by proliferation assays that are well known (e.g., CFSE staining) and/or as described in the examples below.
  • the instant invention provides for MSR-SLB scaffolds containing molecules that specifically bind to a co-stimulatory antigen.
  • the MSR-SLB scaffolds contain a plurality of T-cell costimulatory molecules which specifically bind to CD28, 4.
  • IBB CD137
  • 0X40 CD134
  • CD27 TNFRSF7
  • GITR CD357
  • CD30 CD30
  • HVEM CD270
  • LT 3R
  • DR3 TNFRSF25
  • CD278 CD226
  • CD355 CRTAM
  • TIM1 HVCR1, KIMI
  • CD2 LFA2, 0X34
  • SLAM CD150, SLAMF1), 2B4 (CD244, SLAMF4)
  • LylO8 NTBA, CD352, SLAMF6)
  • CD84 SLAMF5
  • Ly9 CD229, SLAMF3
  • CD279 PD-1) and/or CRACC (CD319, BLAME).
  • the co-stimulatory molecule is an antibody or an antigen binding fragment thereof which binds specifically to one or more of the aforementioned co-stimulatory antigens.
  • CD28 is the prototypic T cell co-stimulatory antigen and binds to molecules of the B7 family expressed on APCs such as dendritic cells and activated B cells. Human CD28 is found on all CD4+ T cells and on about half of CD8+ T cells. T cell activities attributed to CD28 include prevention of energy, induction of cytokine gene transcription, stabilization of cytokine mRNA and activation of CD8+ cytotoxic T lymphocytes.
  • the ligands for CD28 identified as CD80(B7-l) and CD86(B7-2) are immunoglobulin superfamily monomeric transmembrane glycoproteins of 60 kd and 80 kd respectively.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD28.
  • anti-CD28 antibodies include, for example, lulizumab pegol and TGN1412. See also US patent No. 8,785,604.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to ICOS (CD278).
  • ICOS is a CD28-superfamily costimulatory molecule that is expressed on activated T cells. It is thought to be important for Th2 cells in particular.
  • Representative examples of anti-ICOS antibodies include, for example, monoclonal antibody 2C7, which recognizes the ICOS molecule expressed on activated T cells and induces the activation as well as proliferation of T cells prestimulated by anti-human CD3 monoclonal antibodies. See Deng et al., Hybrid Hybridomics., 23(3): 176-82, 2004.
  • the instant invention provides for MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD152 (CTLA4).
  • the antibody is preferably a neutralizing antibody or a blocking antibody.
  • CD 152 is expressed on activated CD4+ and CD8+ T cells, and on regulatory T-cells (Tregs). Its functions in T-cell biology, during immune responses to infection, and as a target for cancer immunotherapy have been well described (Egen et al., Nat. Immunol., 3(7):611-618, 2002).
  • CTLA-4 is a homologous counterpart to CD28, both of which bind to CD80 and CD86 on APCs.
  • CTLA- 4 for immune tolerance is clear (Waterhouse et al., Science, 270(5238):985-988, 1995). These include out-competing lower affinity CD28 molecules for ligand binding to minimize T-cell costimulation, recruitment of inhibitory phosphatases to the TCR complex to disrupt positive signaling cascades, and removing CD80 and CD86 from the surface of APC by trans-endocytosis, thereby diminishing the ability of APC to properly activate otherwise responsive T-cells. Accordingly, exploitation of the CTLA-4 receptor/pathway is an attractive strategy to modulate T-cell immunity.
  • anti-CTLA-4 was the first monoclonal antibody (ipilimumab) to be FDA-approved for checkpoint blockade treatment in cancer patients.
  • CTLA-4 antibodies that may be employed in accordance with the instant invention include tremelimumab and antigen-binding fragments thereof.
  • the instant invention provides for MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to programmed death-1 (PD-1; CD279).
  • PD1 is a member of the same family of receptors as CD28 and CTLA-4, and is broadly expressed on lymphoid and myeloid cells.
  • PD-1 binds uniquely to the B7 ligands PD-L1 and PD-L2 on APC and other surrounding tissues, greatly influencing the fate of responding CD8+ T cells in settings of chronic infections.
  • the antibody is preferably a neutralizing antibody or a blocking antibody.
  • anti-PD-1 antibodies include, for example, nivolumab, lambrolizumab (MK-3475), pidilizumab(CT-011) and AMP-224.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD 81.
  • Engagement of CD81 lowers the signaling threshold required to trigger T-Cell/CD3 mediated proviral DNA in CD4+ T cells (Tardif et al., J. Virol. 79 (7): 4316-28, 2005).
  • Representative examples of anti- CD81 antibodies include, for example, monoclonal antibody 5A6. See, e.g., Maecker et al., BMC Immunol., 4:1, 2003, the disclosure in which is incorporated by reference herein.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD 137.
  • Crosslinking of CD 137 enhances T cell proliferation, IL-2 secretion, survival and cytolytic activity. Further, it can enhance immune activity to eliminate tumors in vivo.
  • the antibodies that bind to CD137 are preferably agonistic antibodies.
  • Representative examples of anti-CD137 antibodies include, for example, monoclonal antibody utomilumab, which is a human IgG that is currently being investigated in clinical trials. See National Clinical Trials ID: NCT01307267.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to 0X40 (CD 134).
  • OX40L binds to 0X40 receptors on T-cells, preventing them from dying and subsequently increasing cytokine production.
  • 0X40 has a critical role in the maintenance of an immune response beyond the first few days and onwards to a memory response due to its ability to enhance survival.
  • 0X40 also plays a crucial role in both Thl and Th2 mediated reactions in vivo.
  • the antibodies that bind to 0X40 are preferably agonistic antibodies.
  • anti-OX40 antibodies include, for example, anti-OX40 monoclonal antibody utomilumab, which is being investigated in various clinical trials see National Clinical Trials ID: NCT01644968, NCT01303705 and NCT01862900).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD27 (TNFRSF7).
  • CD27 a member of the TNF-receptor superfamily and is required for generation and long-term maintenance of T cell immunity. It binds to ligand CD70, and plays a key role in regulating immunoglobulin synthesis.
  • CD27 supports antigen-specific expansion (but not effector cell maturation) of naive T cells, independent of the cell cycle-promoting activities of CD28 and IL2 (Hendriks et al., Nature Immunology 1, 433-440, 2000)).
  • the MSR-SLB scaffolds of the invention preferably include agonistic antibodies that bind to CD27.
  • Representative examples of anti-CD27 antibodies include, for example, the monoclonal antibody varlilumab. See Ramakrishna et al., Journal for ImmunoTherapy of Cancer, 3:37, 2015.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to glucocorticoid- induced TNF receptor family-regulated gene (GITR or CD357).
  • GITR is a 25 kD TNF receptor superfamily member which is expressed on activated lymphocytes. GITR is upregulated by T cell receptor engagement. The cytoplasmic domain of GITR is homologous to CD40, 4-1BB and CD27. GITR signaling has been shown to regulate T cell proliferation and TCR-mediated apoptosis, and to break immunological self-tolerance.
  • GITR further binds GITRL and is involved in the development of regulatory T cells and to regulate the activity of Thl subsets. Modulation of GITR with agonistic antibodies has been shown to amplify the antitumor immune responses in animal models via multiple mechanisms. Anti-GITR antibodies are designed to activate the GITR receptor thereby increasing the proliferation and function of effector T cells. At the same time, ligation of GITR on surface of Tregs could abrogate suppressive function of these cells on tumor specific effector T-cells thus further augmenting T-cell immune response.
  • anti-GITR antibodies include, for example, humanized, Fc disabled anti-human GITR monoclonal antibody TRX518, which induces both the activation of tumor-antigen-specific T effector cells, as well as abrogating the suppression induced by inappropriately activated T regulatory cells.
  • TRX518 is being investigated in various clinical trials (see National Clinical Trials ID: NCT01239134).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD30 (TNFRSF8).
  • CD30 antigen is a trans-membrane glycoprotein belonging to the tumor necrosis factor receptor superfamily, which, when stimulated, exerts pleiotropic effects on cell growth and survival.
  • CD30 expression is restricted to medium/large activated B and/or T- lymphocytes. It is expressed by activated, but not by resting, T and B cells (Guo et al., Infect. Immun., 81 (10), 3923-3934, 2013).
  • Stimulation of CD30L/CD30 signaling by in vivo administration of agonistic anti-CD30 monoclonal antibody (MAb) restored IL-17A production by Vyl- Vy4- y5 T cells in CD30L knockout mice.
  • anti-CD30 antibodies include, for example, brentuximab vedotin (Adcetris).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to HVEM (CD270).
  • CD270 is a member of the TNF-receptor superfamily. This receptor was identified as a cellular mediator of herpes simplex virus (HSV) entry. Mutations in this gene have been recurrently been associated to cases of diffuse large B-cell lymphoma.
  • Representative examples of anti-CD270 antibodies include, for example, the monoclonal antibody HVEM-122. See, Cheung et al., J. Immunol., 185:1949, 2010; Hobo et al., J Immunol., 189:39, 2012.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to lymphotoxin beta receptor (LTpR; TNFRSF3).
  • LTpR lymphotoxin beta receptor
  • TNFRSF3 lymphotoxin beta receptor
  • anti-LTpR antibodies include, for example, the monoclonal antibody BBF6 antibody. See also W02010/078526, which is incorporated by reference.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to DR3 (TNFRSF25).
  • DR3 is thought to be involved in controlling lymphocyte proliferation induced by T-cell activation. Specifically, activation of DR3 is dependent upon previous engagement of the T cell receptor. Following binding to TL1A, DR3 signaling increases the sensitivity of T cells to endogenous IL -2 via the IL -2 receptor and enhances T cell proliferation. Because the activation of the receptor is T cell receptor dependent, the activity of DR3 in vivo is specific to those T cells that are encountering cognate antigen.
  • DR3+ regulatory T cells At rest, and for individuals without underlying autoimmunity, the majority of T cells that regularly encounter cognate antigen are FoxP3+ regulatory T cells. Stimulation of TNFRSF25, in the absence of any other exogenous signals, stimulates profound and highly specific proliferation of FoxP3+ regulatory T cells from their 8-10% of all CD4+ T cells to 35-40% of all CD4+ T cells within 5 days.
  • Representative examples of DR3 agonists include, for example, antibodies binding specifically to DR3 (Reddy et al., J. Virol., 86 (19) 10606-10620, 2012) and the agonist 4C12 (Wolf et al., Transplantation, 27;94(6):569-74, 2012).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD226 (DNAM1).
  • CD226 is a ⁇ 65 kDa glycoprotein expressed on the surface of natural killer cells, platelets, monocytes and a subset of T cells. It is a member of the immunoglobulin superfamily and mediates cellular adhesion to other cells bearing its ligands, CD112 and CD155.
  • Cross-linking CD226 with antibodies causes cellular activation and ligation of CD226 and LFA-1 with their respective ligands cooperates in triggering cytotoxicity and cytokine secretion by T and NK cells (Tahara et al., Int. Immunol. 16 (4): 533-8, 2004).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CRTAM (CD355).
  • CTRAM is an MHC-class-I-restricted T-cell-associated molecule, which regulates late phase of cell polarity in some CD4+ T cells. CTRAM also regulates interferon-y (IFNy) and interleukin-22 (IL-22) production.
  • the MSR-SLB scaffolds comprise a monoclonal anti-CTRAM antibody.
  • Representative examples of CTRAM antibodies include, for example, the mouse anti human CTRAM antibody 21A9 (GENTEX Inc. USA, Irvine, CA).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to TIM1 (HAVCR1, KIMI).
  • TIM genes belong to type I cell-surface glycoproteins, which include an N-terminal immunoglobulin (Ig)-like domain, a mucin domain with distinct length, a single transmembrane domain, and a C-terminal short cytoplasmic tail. The localization and functions of TIM genes are divergent between each member.
  • TIM-1 is preferentially expressed on Th2 cells and has been identified as a stimulatory molecule for T-cell activation (Umetsu et al., Nat. Immunol. 6 (5): 447-54, 2005).
  • the MSR-SLB scaffolds comprise a monoclonal anti-TIMl antibody.
  • Representative examples of TIM1 antibodies include, for example, the rabbit anti human TIM1 antibody ab47635 (ABCAM, Cambridge, MA).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to SLAM (CD 150, SLAMF1).
  • SLAM CD150
  • CD150 is a self-ligand and cell surface receptor that functions as a costimulatory molecule and also a microbial sensor that controlled the killing of Gram-negative bacteria by macrophages.
  • SLAM regulated activity of the NADPH oxidase N0X2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins (Berger et al., Nature Immunology 11, 920-927, 2010).
  • the MSR- SLB scaffolds comprise a monoclonal anti-SLAMl antibody or an antigen-binding fragment thereof.
  • Representative examples of SLAM1 antibodies include, e.g., the rabbit anti human SLAM1 antibody 600-401-EN3 (Rockland Antibodies, Limerick, PA).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to 2B4 (CD244, SLAMF4).
  • CD244 is a cell surface receptor expressed on natural killer cells (NK cells) (and some T cells) mediating non-major histocompatibility complex (MHC) restricted killing. The interaction between NK-cell and target cells via this receptor is thought to modulate NK-cell cytolytic activity.
  • CD244 is a co-inhibitory SLAM family member which attenuates primary antigen-specific CD8(+) T cell responses in the presence of immune modulation with selective CD28 blockade. Recent studies reveal a specific up-regulation of 2B4 on antigen-specific CD8(+) T cells in animals in which CD28 signaling was blocked (Liu et al., J Exp Med. 2014 Feb 10;211(2):297-311).
  • the MSR-SLB scaffolds comprise a monoclonal anti-CD244 antibody or an antigen-binding fragment thereof.
  • CD244 antibodies include, e.g., anti-2B4 antibody C1.7 or PE-conjugated anti-2B4 (C1.7), which have been characterized in Sandusky et al. (Eur J Immunol. 2006 Dec;36(12):3268-76).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to Lyl08 (NTBA, CD352, SLAMF6).
  • SLAMF6 is a type I transmembrane protein, belonging to the CD2 subfamily of the immunoglobulin superfamily, which is expressed on natural killer (NK), T, and B lymphocytes.
  • NK natural killer
  • T T
  • B lymphocytes Co-stimulation of T lymphocytes through the SLAMF3/SLAMF6 pathways mediates more potent effects on IL-17A expression when compared with the canonical CD28 pathway.
  • the MSR-SLB scaffolds comprise a monoclonal anti-CD244 antibody or an antigen-binding fragment thereof.
  • CD244 antibodies include, e.g., anti NTB-A antibodies characterized in Flaig et al. J. Immunol. 2004. 172: 6524-6527) and Stark et al. (J. Immunol. Methods 2005. 296: 149-158).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD84 (SLAMF5).
  • CD84 is a member of the CD2 subgroup of the immunoglobulin receptor superfamily. Members of this family have been implicated in the activation of T cells and NK cells. CD84 increases proliferative responses of activated T-cells and homophilic interactions enhance interferon gamma secretion in lymphocytes. CD84 may also serve as a marker for hematopoietic progenitor cells . See the disclosure in the references with the PUB MED ID Nos.
  • the MSR-SLB scaffolds comprise a monoclonal anti-CD84 antibody or an antigen-binding fragment thereof.
  • CD84 antibodies include, e.g., PE anti-human CD84 antibody CD84.1.21, which is able to enhance CD3 induced IFN-y production and partially block CD84-Ig binding to lymphocytes (BioLegend, San Diego, CA; Catalog No. 326008).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to Ly9 (CD229, SLAMF3).
  • CD229 participates in adhesion reactions between T lymphocytes and accessory cells by homophilic interaction. It also promotes T-cell differentiation into a helper T-cell Th 17 phenotype leading to increased IL- 17 secretion; the costimulatory activity requires SH2D1A (Chatterjee et al., J Biol Chem., 287(45): 38168-38177, 2012).
  • the MSR-SLB scaffolds comprise a monoclonal anti-CD229 antibody or an antigen-binding fragment thereof.
  • CD229 antibodies include, e.g., PE anti-human CD229 antibody HLy- 9.1.25 (BIOLEGEND, San Diego, CA; Catalog No. 326108) or mouse anti-human CD229 antibody (R&D Systems Catalog No. AF1898).
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CD279 (PD-1).
  • PD-1 functions as an immune checkpoint and plays an important role in down regulating the immune system by preventing the activation of T-cells, which in turn reduces autoimmunity and promotes self-tolerance.
  • the inhibitory effect of PD-1 is accomplished through a dual mechanism of promoting apoptosis (programmed cell death) in antigen specific T-cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells).
  • Representative examples of CD229 antibodies include, e.g., nivolumab, pembrolizumab, pidilizumab (CT-011, Cure Tech), BMS936559, and atezolizumab.
  • the instant invention relates to MSR-SLB scaffolds containing an antibody or an antigen-binding fragment thereof which binds specifically to CRACC (CD319, BLAME).
  • CD319 mediates NK cell activation through a SH2DlA-independent extracellular signal-regulated ERK-mediated pathway (Bouchon et al., J Immunol. 2001 Nov 15; 167(10):5517- 21).
  • CD319 also positively regulates NK cell functions and may contribute to the activation of NK cells.
  • the MSR-SLB scaffolds comprise a monoclonal anti-CD319 antibody or an antigen-binding fragment thereof.
  • Representative examples of CD319 antibodies include, e.g., elotuzumab or an antigen-binding fragment thereof.
  • the instant invention provides for MSR-SLB scaffolds containing a binding pair containing at least one T-cell ligand and at least one T-cell co-stimulatory molecule.
  • Representative examples of such pairs include, but are not limited to, for example, antibodies binding to CD3/CD28, CD3/ICOS, CD3/CD27, and CD3/CD137, or a combination thereof.
  • the instant invention provides for MSR-SLB scaffolds containing a binding pair containing at least one T-cell ligand which is an antibody binding to CD3 and at least one T-cell co-stimulatory molecule which is an antibody binding to CD28, optionally together with at least one additional co-stimulatory molecule (e.g., a second co-stimulatory molecule) which is an antibody binding to an antigen selected from the group consisting of ICOS, CD27, and CD137.
  • a binding pair containing at least one T-cell ligand which is an antibody binding to CD3 and at least one T-cell co-stimulatory molecule which is an antibody binding to CD28, optionally together with at least one additional co-stimulatory molecule (e.g., a second co-stimulatory molecule) which is an antibody binding to an antigen selected from the group consisting of ICOS, CD27, and CD137.
  • the MSR-SLB scaffold contains a combination of functional molecules selected from the following combinations: (a) antibodies which bind to CD3, CD28 and ICOS, (b) antibodies which bind to CD3, CD28 and CD27, (c) antibodies which bind to CD3, CD28 and CD137, (d) antibodies which bind to CD3, CD28, ICOS and CD27.
  • stimulation of these secondary T-cell co-stimulation factors may stimulate differentiation of certain types of T-cells when applied with appropriate activation stimuli such as CD3+CD28.
  • ICOS stimulation favors differentiation of Th effector cells when cooperates with CD3+CD28+ stimulation, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.
  • anti-CD27 antibodies may be used to fine-tune the system.
  • anti-CD27 antibody 1F5 when used together with anti-CD3 antibodies did not trigger potentially dangerous polyclonal T-cell activation - a phenomena observed with co-stimulatory CD28-specific super-agonistic antibodies. See, Thomas et al., Oncoimmunology, 3: e27255, 2014.
  • the binding pair includes monospecific antibodies, wherein a first antibody binds to a first member of the pair, e.g., CD3, and a second antibody binds to a second member of the pair, e.g., CD28.
  • the pair includes bispecific antibodies, wherein a single antibody binds to the individual pair members, e.g., a bispecific antibody binding to CD3 and CD28.
  • bispecific antibodies are preferred due to their ability to confer enhanced T-cell activation. See, Willems et al., Cancer Immunol Immunother. 2005 Nov;54(ll):1059-71.
  • the binding pair includes monospecific antibodies, wherein a first antibody binds to CD3 and a second antibody binds to ICOS.
  • a first antibody binds to CD3
  • a second antibody binds to ICOS.
  • an antagonistic antibody that neutralizes ICOS.
  • a bispecific antibody containing an agonist CD3-binding antibody fragment and an antagonist ICOS-binding antibody fragment, may also be employed.
  • the binding pair includes monospecific antibodies, wherein a first antibody binds to CD3 and a second antibody binds to CD27.
  • both antibodies are preferably stimulatory or agonist antibodies. It has been reported that CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo (Song et al. , Blood, 119(3):696-706, 2012).
  • a bispecific antibody containing an agonist CD3-binding antibody fragment and an agonist CD27- binding antibody fragment may also be employed.
  • the binding pair includes monospecific antibodies, wherein a first antibody binds to CD3 and a second antibody binds to CD 137.
  • both antibodies are preferably stimulatory or agonist antibodies. It has been reported that CD 137 costimulation improves the expansion and function of CD8(+) melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy (Chacon et al., PLoS One. 2013;8(4):e60031, 2013).
  • a bispecific antibody containing an agonist CD3-binding antibody fragment and an agonist CD27-binding antibody fragment may also be employed.
  • the MSR-SLB scaffolds and/or the antigen-presenting cell mimetic scaffolds contains one or more T-cell homeostatic agents.
  • the one or more T-cell homeostatic agents may be selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL- 15, IL-17, IL-21, and transforming growth factor beta (TGF-P), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof.
  • the MSR-SLB scaffolds and/or the antigen-presenting cell mimetic scaffolds contains a plurality of one or more T-cell homeostatic agents selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, and transforming growth factor beta (TGF-P), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof.
  • TGF-P transforming growth factor beta
  • Functional fragments of these homeostatic agents which are characterized by their ability to modulate the activity of target cells, may also be employed.
  • Representative types of homeostatic agents including, NCBI accession numbers of human and/or mouse homologs thereof, are provided in Table 1.
  • T-cell homeostatic agents that may be employed in the scaffolds.
  • Fragments and variants of the aforementioned T-cell homeostatic agents are known in the art.
  • the UNIPROT database entry of each of the aforementioned homeostatic agents lists “natural variants,” including structural relationship between the variant and the wild-type biomarker.
  • the human IL-ip protein (UNIPROT: P01584) includes a natural variant (VAR_073951) having E— >-N amino acid substitution at amino acid residue 141 of the putative human IL-ip protein sequence. Fragments, if known, are similarly listed under this section.
  • the T-cell homeostatic agent is interleukin-2 (IL-2) or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof with one or more T-cell homeostatic agents listed in Table 1.
  • IL-2 agonists include, for example, BAY 50-4798 (Margolin et al. , Clin Cancer Res. 2007 Jun 1 ; 13( 11) : 3312-9).
  • IL-2 mimetics include, for example, peptide 1-30 (Pl-30), which acts in synergy with IL-2 (Eckenberg et al., J Immunol 2000; 165:4312-4318).
  • IL-2 fragments include, for example, a ballast portion containing the first 100 amino acids of IL-2 (see, US patent No. 5,496,924).
  • IL-2 variants include, for example, natural variant VAR_003967 and natural variant VAR_003968.
  • fusion proteins containing IL-2 e.g., F16-IL2, which is an scFv against the extra-domain Al of tenascin-C that is fused, via a short 5-amino acid linker, to a recombinant form of the human IL-2.
  • the monoclonal antibody portion of the Fl 6- IL2 fusion protein binds to tumor cells expressing the tumor associated antigen (TAA) tenascin- C.
  • TAA tumor associated antigen
  • the IL-2 moiety of the fusion protein stimulates natural killer (NK) cells, macrophages and neutrophils and induces T-cell antitumor cellular immune responses.
  • Other IL-2 mimetics that may be employed in accordance with the invention include, for example, an IL-2 superkine peptide (Levin et al., Nature 484, 529-533, 2012), and an IL -2 partial agonist peptide (Zurawski et al., EMBO Journal, 9(12): 3899-3905, 1990 and US patent No. 6,955,807), or a combination thereof.
  • Embodiments of the instant invention further include MSR-SLB scaffolds, including, APC-MS scaffolds made from such scaffolds, which further comprise a plurality of the aforementioned T-cell homeostatic agents.
  • the invention provides for MSR-SLB scaffolds containing a first T-cell homeostatic agent which is IL-2 and a second T-cell homeostatic agent which is IL-7, IL-21, IL-15, or IL-15 superagonist.
  • IL-15 superagonist IL- 15 SA
  • IL- 15 SA is a combination of IL- 15 with soluble IL- 15 receptor-a, which possesses greater biological activity than IL- 15 alone.
  • IL- 15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8+ T (mCD8+ T) lymphocytes. See, Guo et al., J Immunol. 2015 Sep l;195(5):2353-64.
  • Embodiments of the instant invention further relate to an scaffolds which comprise a plurality of T-cell stimulatory molecules, T-cell co-stimulatory molecules and T-cell homeostatic agents.
  • a typical scaffold may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or more of each of the aforementioned T-cell activating ligands, T-cell inhibiting ligands, T-cell stimulatory molecules, T-cell co-stimulatory molecules, and T-cell homeostatic agents.
  • any functional molecule for example, antigens, antibodies, proteins, enzymes, including fragments thereof, may be directly or indirectly immobilized onto the MSR base layer and/or the SLB using routine techniques.
  • the functional molecules may be provided in an organelle e.g., Golgi membrane or plasma membrane), a cell, a cell cluster, a tissue, a microorganism, an animal, a plant, or an extract thereof, which in turn is immobilized onto the MSR layer or the SLB layer.
  • a functional molecule may also be synthesized by genetic engineering or chemical reactions at the desired situs, e.g., outer face of the SLB layer.
  • the scaffolds described herein comprise and release signaling molecules, e.g., one or more T-cell homeostatic agents, to elicit functional T-cell responses.
  • the released one or more T-cell homeostatic agents are polypeptides that are isolated from endogenous sources or synthesized in vivo or in vitro.
  • endogenous IL-2 polypeptides may isolated from healthy human tissue.
  • synthetic functional molecules may be synthesized via transfection or transformation of template DNA into a host organism or cell, e.g., a cultured human cell line or a mammal e.g., humanized mouse or rabbit).
  • synthetic functional molecules in protein form may be synthesized in vitro by polymerase chain reaction (PCR) or other art -recognized methods Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), incorporated by reference herein).
  • PCR polymerase chain reaction
  • the functional molecules may be modified to increase protein stability in vivo.
  • the functional molecules are engineered to be more or less immunogenic.
  • the sequences may be modified at one or more of amino acid residues, e.g., glycosylation sites, to generate immunogenic variants.
  • the functional molecules are recombinant.
  • the functional molecules are humanized derivatives of mammalian counterparts.
  • Exemplary mammalian species from which the functional molecules are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate.
  • the functional molecules are human or humanized version of the aforementioned functional molecules.
  • Each of the aforementioned functional molecules may, independently from one another, be adsorbed or integrated into the MSR base layer or the SLB base layer. Therefore, in one embodiment, there is provided an APC-MS, wherein the T-cell stimulatory molecules are adsorbed or integrated into the MSR base layer. Preferably, there is provided an APC-MS, wherein the T-cell stimulatory molecules are adsorbed or integrated into the SLB layer.
  • an APC-MS wherein the T-cell stimulatory molecules are adsorbed or integrated into both the MSR base layer as well as the SLB layer.
  • an APC-MS wherein the one or more T-cell co-stimulatory molecules are adsorbed or integrated into the MSR base layer.
  • the one or more T-cell co-stimulatory molecules are adsorbed or integrated into the SLB layer.
  • an APC-MS wherein the one or more T-cell co-stimulatory molecules are adsorbed or integrated into both the MSR base layer as well as the SLB layer.
  • an APC-MS wherein the one or more T-cell homeostatic agents are adsorbed or integrated into the MSR base layer.
  • an APC-MS wherein the one or more T-cell homeostatic agents are adsorbed or integrated into the SLB layer.
  • an APC-MS wherein the one or more T-cell homeostatic agents are adsorbed or integrated into both the MSR base layer as well as the SLB layer.
  • the functional molecules and the MSR base layer and/or the SLB layer may be linked together through the use of reactive groups, which are typically transformed by the linking process into a new organic functional group or unreactive species.
  • the reactive functional group(s) may be located in any of the aforementioned components.
  • Reactive groups and classes of reactions useful in practicing the present invention are generally those that are well known in the art of bioconjugate chemistry. Currently favored classes of reactions available with reactive chelates are those that proceed under relatively mild conditions.
  • nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
  • electrophilic substitutions e.g., enamine reactions
  • additions to carbon-carbon and carbon-heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
  • Useful reactive pendant functional groups include, for example:
  • N- hydroxysuccinimide esters N-hydroxybenztriazole esters
  • acid halides e.g., I, Br, Cl
  • acyl imidazoles e.g., thioesters
  • p-nitrophenyl esters alkyl, alkenyl, alkynyl and aromatic esters
  • haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the functional group of the halogen atom;
  • a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion
  • dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups;
  • aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
  • amine or sulfhydryl groups which can be, for example, acylated, alkylated or oxidized;
  • alkenes which can undergo, for example, cycloadditions, acylation, Michael addition, etc;
  • the reactive functional groups can be chosen such that they do not participate in, or interfere with, the reactions necessary to assemble the reactive chelates.
  • a reactive functional group can be protected from participating in the reaction by the presence of a protecting group.
  • Those of skill in the art understand how to protect a particular functional group such that it does not interfere with a chosen set of reaction conditions. See, for example, Greene et al. , Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.
  • the functional molecules are loaded into and/or presented on the MSR base layer or the SLB or both the MSR layer and the SLB via affinity pairing or chemical coupling.
  • affinity pair includes antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin, biotin/biotin binding agent, Ni2+ or Cu2+/HisTag (6x histidine) and virus-receptor interactions.
  • RNA or DNA nucleic acid
  • biotin binding agent encompasses avidin, streptavidin and other avidin analogs such as streptavidin or avidin conjugates, highly purified and fractionated species of avidin or streptavidin, and non or partial amino acid variants, recombinant or chemically synthesized avidin analogs with amino acid or chemical substitutions which still accommodate biotin binding.
  • each biotin binding agent molecule binds at least two biotin moieties and more preferably at least four biotin moieties.
  • biotin encompasses biotin in addition to biocytin and other biotin analogs such as biotin amido caproate N-hydroxysuccinimide ester, biotin 4- amidobenzoic acid, biotinamide caproyl hydrazide and other biotin derivatives and conjugates.
  • biotin-dextran biotin-disulfide-N-hydroxysuccinimide ester, biotin-6 amido quinoline, biotin hydrazide, d-biotin-N hydroxysuccinimide ester, biotin maleimide, d-biotin p- nitrophenyl ester, biotinylated nucleotides and biotinylated amino acids such as Ns-biotinyl- 1 -lysine.
  • the ligands that may be functionalized via affinity pairing include, but are not limited to, receptors, monoclonal or polyclonal antibodies, viruses, chemotherapeutic agents, receptor agonists and antagonists, antibody fragments, lectin, albumin, peptides, proteins, hormones, amino sugars, lipids, fatty acids, nucleic acids and cells prepared or isolated from natural or synthetic sources.
  • any site-specific ligand for any molecular epitope or receptor to be detected through the practice of the invention may be utilized.
  • the ligand is a membrane-anchored protein.
  • the ligand may also be a derivative of a membrane-anchored protein, such as a soluble extracellular domain.
  • a ligand can be a receptor involved in receptor-receptor cellular interactions such as TCR binding to the MHC receptor.
  • the ligands of the instant invention can be expressed and purified by any method known in the art.
  • the proteins are expressed by a baculovirus-based insect expression system or a mammalian expression system. Fifteen residues of AVITAGTM peptide may be added to the C-terminals of all of the molecules.
  • the lysine residue in the AVITAGTM (Avidity, CO) can be specifically biotinylated by BirA enzyme (Avidity, CO).
  • the proteins may also be designed to be secreted into the supernatant of the cell culture.
  • the functional molecules can be any protein or peptide.
  • the proteins are involved in ligand-receptor interactions.
  • an important event of T cell activation is a result of membrane-membrane contact between T cells and APCs, wherein a variety of ligand-receptor interactions take place between the two opposing membranes, including, MHC- peptide and TCR, LFA-1 and ICAM-1, CD2 and CD48, as well as B7 or CTLA-4 and CD28. Understanding the valency requirements of these interactions will facilitate the design of therapeutics that enhance or inhibit the immune response to certain antigens.
  • the instant invention can also be used as a tool to study the subtle differences in T cell intracellular signaling pathways induced by agonist and antagonist antigens.
  • the scaffolds provide a clean physiological setting to test the subtle differences without using native antigen presenting cells that often complicate biochemical analyses.
  • the instant invention also provides a screening assay to quantify primary T cells responsive to a specific antigen.
  • biotinylated pep-MHC and costimulatory molecules may be coupled onto a streptavidin coated chips, and the chips are paired with the scaffolds of the invention.
  • the functional molecules are chemically coupled to the MSR base layer and/or the SLB layer.
  • the chemical coupling includes, click-chemistry reagents, for example, azide-alkyne chemical (AAC) reaction, dibenzo-cyclooctyne ligation (DCL), or tetrazine-alkene ligation (TAL).
  • AAC azide-alkyne chemical
  • DCL dibenzo-cyclooctyne ligation
  • TAL tetrazine-alkene ligation
  • a clickable reagent such as 3 -azidopropylamine or 10-undecynoic acid may be amide- bonded to the carboxy- or amino-terminus, respectively, of a peptide or protein via a click reaction with a corresponding alkyne or azido compound and appropriate catalyst to form the 1,2, 3 -triazole ring linking groups. See, e.g., U.S. Publication No. 2007/0060658.
  • aza-dibenzocyclooctyne (ADIBO) -containing compounds for azide -coupling reactions may be used for the site-specific covalent anchoring of protein functional molecules, e.g., antibodies, interluekins and cytokines.
  • the same metal-free click reaction is employed for the PEGylation of unfunctionalized areas of the surface. Such treatment allows for a dramatic reduction or complete elimination of non-specific binding.
  • the copper-free click immobilization methods can be applied to the preparation of various types of arrays, as well as to the derivatization of microbeads and nanoparticles. See, e.g., U.S. Patent No. 8,912,322.
  • the functional molecules are coupled to the MSR base layer and/or the SLB layer using a click reagent selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine and norbornene and variants thereof.
  • DBCO dibenzocyclooctyne
  • the functional molecule comprises azide and a lipid of the lipid bilayer of the MSR-SLB comprises DBCO.
  • click chemistry refers to a chemical philosophy introduced by K. Barry Sharpless of The Scripps Research Institute, describing chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together. Click chemistry does not refer to a specific reaction, but to a concept including reactions that mimic reactions found in nature. In some embodiments, click chemistry reactions are modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, exhibit a large thermodynamic driving force >84 kJ/mol to favor a reaction with a single reaction product, and/or can be carried out under physiological conditions. A distinct exothermic reaction makes a reactant “spring loaded”.
  • a click chemistry reaction exhibits high atom economy, can be carried out under simple reaction conditions, use readily available starting materials and reagents, uses no toxic solvents or use a solvent that is benign or easily removed (preferably water), and/or provides simple product isolation by non-chromatographic methods (crystallization or distillation).
  • click chemistry handle refers to a reactant, or a reactive group, that can partake in a click chemistry reaction.
  • a strained alkyne e.g., a cyclooctyne
  • click chemistry reactions require at least two molecules comprising click chemistry handles that can react with each other.
  • Such click chemistry handle pairs that are reactive with each other are sometimes referred to herein as partner click chemistry handles.
  • an azide is a partner click chemistry handle to a cyclooctyne or any other alkyne.
  • Exemplary click chemistry handles suitable for use according to some aspects of this invention are described herein, for example, US 2014/0249296. Other suitable click chemistry handles are known to those of skill in the art.
  • the instant invention provides APC-MS comprising a plurality of T- cell ligands and one or more T-cell co-stimulatory molecules optionally together with one or more T-cell homeostatic agents, which are adsorbed into the scaffold via metal-chelating lipid headgroups.
  • APC-MS comprising a plurality of T- cell ligands and one or more T-cell co-stimulatory molecules optionally together with one or more T-cell homeostatic agents, which are adsorbed into the scaffold via metal-chelating lipid headgroups.
  • Peterson et al. (US 5,674,677) describes a method for joining two amino acid sequences by coupling an organic chelator to an protein, e.g., an enzyme, and charging the chelator with a metal ion. This complex is then mixed with any protein containing a histidine tag to couple the complex with the histidine tagged protein. See also, US 6,087,452, which is incorporated by reference herein in its entirety.
  • the functional molecules of the invention are preferably proteins.
  • protein protein
  • peptide and “polypeptide” are used interchangeably, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds.
  • the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
  • a protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins.
  • One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide.
  • a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
  • conjugation refers to an association of two molecules, for example, two proteins, with one another in a way that they are linked by a direct or indirect covalent or non-covalent interaction.
  • conjugation is via a covalent bond formed by the reaction of the click chemistry handles.
  • the association is covalent, and the entities are said to be “conjugated” to one another.
  • a protein is post-translationally conjugated to another molecule, for example, a second protein, by forming a covalent bond between the protein and the other molecule after the protein has been translated, and, in some embodiments, after the protein has been isolated.
  • the post-translational conjugation of the protein and the second molecule is effected via installing a click chemistry handle on the protein, and a second click chemistry handle, which can react to the first click chemistry handle, on the second molecule, and carrying out a click chemistry reaction in which the click chemistry handles react and form a covalent bond between the protein and the second molecule, thus generating a chimeric protein.
  • two proteins are conjugated at their respective C-termini, generating a C-C conjugated chimeric protein.
  • two proteins are conjugated at their respective N-termini, generating an N — N conjugated chimeric protein.
  • a plurality of detectable labels may be used to analyze and/or study the conjugation process.
  • a “detectable label” refers to a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the molecule, e.g., a protein or polypeptide, or other entity, to which the label is attached. Labels can be directly attached (z.e.
  • a tether such as, for example, an optionally substituted alkylene; an optionally substituted alkenylene; an optionally substituted alkynylene; an optionally substituted heteroalkylene; an optionally substituted heteroalkenylene; an optionally substituted heteroalkynylene; an optionally substituted arylene; an optionally substituted heteroarylene; or an optionally substituted acylene, or any combination thereof, which can make up a tether).
  • the label may be attached to or incorporated into a molecule, for example, a protein, polypeptide, or other entity, at any position.
  • a label can fall into any one (or more) of five classes: a) a label which contains isotopic moieties, which may be radioactive or heavy isotopes, including, but not limited to, 2 H, 3 H, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 67 Ga, "mTc (Tc-99 m), U1 ln, 125 I, 131 I, 153 Gd, 169 Yb, and 186 Re; b) a label which contains an immune moiety, which may be antibodies or antigens, which may be bound to enzymes (e.g., such as horseradish peroxidase); c) a label which is a colored, luminescent, phosphorescent, or fluorescent moieties e.g., such as the fluorescent label fluorescein isothiocyanate (FITC) or carboxyfluorescein); d) a label which has one or more photo affinity moieties; and e) a label which
  • a label comprises a radioactive isotope, preferably an isotope which emits detectable particles.
  • the label comprises a fluorescent moiety.
  • the label is the fluorescent label fluorescein isothiocyanate (FITC).
  • the label comprises a ligand moiety with one or more known binding partners.
  • the label comprises biotin.
  • a label is a fluorescent polypeptide (e.g., GFP or a derivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla, or Gaussia luciferase).
  • a label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal.
  • a suitable substrate e.g., a luciferin
  • fluorescent proteins include GFP and derivatives thereof, proteins comprising chromophores that emit light of different colors such as red, yellow, and cyan fluorescent proteins, etc.
  • Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFPl, mUkGl, mAGl, AcGFPl, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mK02, mOrange, m0range2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima.
  • a label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.
  • the functional molecules may be loaded into and/or presented on mesoporous silica and/or the lipid bilayer using art known, covalent or non-covalent loading techniques.
  • the functional molecules are loaded non-covalently.
  • Lei et al. U.S. Publication No. 2011-0256184
  • the scaffolds of the invention may be formulated with such silicates.
  • the functional molecules are chemically coupled onto the MSR.
  • the coupling may be conducted by utilizing one or more of the following molecules and the reactive groups contained therein: cysteine (thiol group), serine or threonine (hydroxyl group), lysine (amino group), aspartate or glutamate (carboxyl group).
  • the functional molecules may be loaded into and/or presented on the MSR via utilization of polyhistidine-tag (His-tag), a peptide containing polyhistidine-tag or an antibody containing polyhistidine-tag.
  • the polyhistidine-tag consists of at least four, five, six or seven histidine (His) residues.
  • an anchor is used to connect the functional molecule to a pore wall.
  • the anchor is not an essential component.
  • each pore of the mesoporous silica accommodates at least one functional molecule.
  • the pores must have a size appropriate to immobilize a biological substance. The pore size depends on the size of the functional molecule to be immobilized.
  • the functional molecule can be adsorbed on an inner surface of the pore by electrostatic bonding.
  • a functional molecule may also be held in a pore by a noncovalent bonding, such as van der Waals forces, hydrogen bonding, or ionic bonding.
  • the anchor may have an effect of reducing a large structural change of the functional molecule to hold it stably.
  • the anchor is composed of substantially the same component as the mesoporous material.
  • the anchor may comprise one or more functional groups to permit binding to a desired functional molecule: a hydroxyl group, an amide group, an amino group, a pyridine group, a urea group, a urethane group, a carboxyl group, a phenol group, an azo group, a hydroxyl group, a maleimide group, a silane derivative, or an aminoalkylene group.
  • Embodiments of the invention further relate to MSR-SLB scaffolds of the invention, including, scaffolds containing such scaffolds, comprising, a plurality of the aforementioned functional molecules which are adsorbed in the lipid matrix.
  • the functional molecules are adsorbed into the supported lipid bilayer via physical insertion.
  • Techniques for inserting proteins into the bilayer of amphipathic molecules are known in the art.
  • proteins in the environment of the bilayer for example in the hydrophobic medium and/or in the hydrophilic body and/or in the hydrated support, may insert spontaneously into the bilayer.
  • proteins may be driven into the bilayer by the application of a voltage and/or by fusion of protein loaded vesicles with the bilayer.
  • the vesicles may be contained within or introduced to the hydrophilic body.
  • proteins may be introduced into the membrane by using the probe method disclosed in PCT Publication No. WO 2009/024775.
  • the inserted protein may be a known membrane-associated protein, e.g., one or more of the aforementioned T-cell ligands and/or T-cell co-stimulatory molecules.
  • the functional molecule may be an antigen that is used in expansion of T-cells.
  • antigens usable in T-cell expansion include, full-length CD 19 or a fragment thereof or a variant thereof.
  • CD 19 is a prototypical antigen used in the expansion of chimeric antigen receptor (CAR) T-cells. See, Turtle et al., Blood, 126:184, 2015; Turtle et al., J Clin Invest., 126, 2123-38, 2016.
  • the antigen is full-length CD22 or a fragment thereof or a variant thereof, which are also useful in the expansion of CAR T-cells. See, Haso et al., Blood, 121(7): 1165-1174, 2013; Qin et al., Blood, 122:1431, 2013.
  • the functional molecule may be a membrane-associated protein which is anchored directly or indirectly to the bilayer.
  • Other functional molecules e.g., selective or non-selective membrane transport proteins, ion channels, pore forming proteins or membrane-resident receptors, etc. may also be inserted into the SLB via this method.
  • the functional molecules may be conjugated to membrane- associated proteins which associate with and/or insert into the SLB, e.g. gramicidin; a-helix bundles, e.g. bacteriorhodopsin or K+ channels; and P-barrels, e.g., a-hemolysin, leukocidin or E. coli porins; or combinations thereof.
  • membrane-associated proteins e.g. gramicidin
  • a-helix bundles e.g. bacteriorhodopsin or K+ channels
  • P-barrels e.g., a-hemolysin, leukocidin or E. coli porins; or combinations thereof.
  • the fabricated SLB (containing one or more functional molecules) may be stabilized by compounds such as ionic or non-ionic surfactants.
  • Suitable surfactants include, but are not limited to, the following examples: synthetic phospholipids, their hydrogenated derivatives and mixtures thereof, sphingolipids and glycosphingolipids, saturated or unsaturated fatty acids, fatty alcohols, polyoxyethylene-polyoxypropylene copolymers, ethoxylated fatty acids as well as esters or ethers thereof, dimyristoyl phosphatidyl choline, dimyristoyl phosphatidyl glycerol or a combination of two or more of the above mentioned.
  • a preferred surfactant according to the invention is the dimyristoyl phosphatidyl glycerol.
  • the fabricated SLBs may be optionally stabilized by at least one co-surfactant selected in the group comprising or consisting of butanol, butyric acid, hexanoic acid, sodium cholate, sodium taurocholate and sodium glycocholate, more particularly sodium cholate.
  • the fabricated SLBs may also include other excipients, such as polymers having bioadhesive or absorption enhancing properties and selected from the group comprising or consisting of acrylic polymers (CARBOPOL®, Polycarbophil, NOVEON®), medium chain fatty acids and polyethylene glycols.
  • excipients are the above-mentioned acrylic polymers.
  • the SLB may be modified with reagents for detecting membrane-associated proteins.
  • the membrane-associated proteins are ion channel proteins and/or pore forming proteins.
  • the membrane-associated proteins diffuse into and/or associate with the bilayer causing a detectable change in the properties at the bilayer.
  • the properties changed may be physical, optical, electrical or biochemical.
  • the MSR-SLB scaffolds and/or the antigen-presenting cell mimetic scaffolds comprises a small molecule drug. In some embodiments, the MSR-SLB scaffolds and/or the antigen-presenting cell mimetic scaffolds comprises a thalomid analog. In some embodiments, the MSR-SLB scaffolds and/or the antigen-presenting cell mimetic scaffolds comprises a IDO/MEK inhibitor. In some embodiments, the MSR-SLB scaffolds and/or the antigen-presenting cell mimetic scaffolds comprises a small molecule drug that has immunomodulatory effects. Small molecule drugs with immunomodulatory effects are known the art (see, e.g. , Murphy et al. Hum. Vaccin. Immunother. 11(10): 2463-8 (2015), the entire contents of which are expressly incorporated herein by reference).
  • the MSR-SLB scaffolds containing the functional molecules may be used to detect cells which are capable of interaction with amphipathic molecules in the bilayer and/or with the functional molecule in the bilayer.
  • the interaction may be specific or non-specific in nature.
  • the cells may interact with the functional molecule or with the lipid bilayer to cause physical, optical, electrical, or biochemical changes.
  • Such interaction may be detected in many different ways, including, but limited to, by visual changes, via activation of fluorescently labelled lipids or proteins in the SLB, or changes in capacitance of the SLB.
  • Embodiments of the invention further relate to biodegradable scaffolds.
  • the scaffold structure may substantially degrade when exposed to a biological milieu.
  • the biological milieu is a tissue culture condition, e.g., tissue culture media that has been optionally adapted to culture lymphocytes such as T-cells.
  • the biological milieu is a biological fluid, e.g., blood, lymph, CSF, peritoneal fluid, or the like.
  • the biological milieu is the tissue environment at the site of implant, e.g., blood vessels, lymphatic system, adipose tissue, or the like.
  • the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vivo over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7, days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45 days, 60 days, 90 days, or more.
  • the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vivo in less than 1 week.
  • the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vitro over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7, days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45 days, 60 days, 90 days, or more.
  • the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vitro in less than 1 week.
  • substantial degradation it is meant that at least 30%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or more of the scaffold composition is degraded when the scaffold composition is contacted with the biological milieu.
  • biodegradable scaffolds it may be advantageous to use biodegradable scaffolds. For instance, by fabricating the scaffold composition such that it substantially degrades during the administration and/or association period (e.g. , when the T-cells are allowed to expand), it may be possible to use the expanded T-cells without subjecting them to additional purification and/or formulation steps. Avoiding downstream purification and/or formulation steps would ensure that the T-cells are fit and possess the desired functionality for the desired application.
  • the degradation kinetics of the scaffold compositions may be tailored by modifying the properties of mesoporous silica rods, such as size, geometry, porosity.
  • the degradation kinetics of the scaffold compositions may be modified by changing the culture conditions (e.g., by adjusting the pH of the media).
  • embodiments of the invention relate to MSR-SLB scaffolds comprising a plurality of functional molecules which are optionally biodegradable.
  • the scaffolds of the instant invention may be encapsulated into other biodegradable scaffolds.
  • Reagents and techniques that are useful in making such composite biodegradable scaffold compositions are known in the art. See, Liao et al., J. Biomed. Mater. Res. B. Appl. Biomater., 102(2):293-302, 2014.
  • the scaffolds are made up of physiologically-compatible and optionally biodegradable polymers. Examples of polymers that are employable in the scaffolds are known in the art. See, for example, U.S. Publication No.
  • polymers include, but are not limited to, poly(lactide)s, poly(glycolide)s, poly(lactic acid)s, poly(glycolic acid)s, polyanhydrides, polyorthoesters, polyetheresters, polycaprolactones, polyesteramides, polycarbonates, polycyanoacrylates, polyurethanes, polyacrylates, and blends or copolymers thereof.
  • Biodegradable scaffolds may comprise biodegradable materials, e.g., collagen, alginates, polysaccharides, polyethylene glycol (PEG), poly (glycolide) (PGA), poly(L-lactide) (PLA), or poly(lactide-co-glycolide) (PLGA) or silk.
  • biodegradable materials e.g., collagen, alginates, polysaccharides, polyethylene glycol (PEG), poly (glycolide) (PGA), poly(L-lactide) (PLA), or poly(lactide-co-glycolide) (PLGA) or silk.
  • Methods for fabricating the scaffold compositions are known in the art. See, for example, Martinsen et al. Biotech. & Bioeng., 33 (1989) 79-89), (Matthew et al. (Biomaterials, 16 (1995) 265-274), Atala et al. (J Urology, 152 (1994) 641-643), and S
  • Exemplary scaffolds utilize glycolides or alginates of a relatively low molecular weight, preferably of size which, after dissolution, is at the renal threshold for clearance by humans, e.g., the alginate or polysaccharide is reduced to a molecular weight of 1000 to 80,000 Daltons.
  • the molecular mass is 1000 to 60,000 Daltons, particularly preferably 1000 to 50,000 Daltons.
  • an alginate material of high guluronate content since the guluronate units, as opposed to the mannuronate units, provide sites for ionic cross-linking through divalent cations to gel the polymer.
  • U.S. Patent No. 6,642,363 which incorporated herein by reference, discloses methods for making and using polymers containing polysaccharides such as alginates.
  • the scaffolds of the invention may be porous such that the scaffolds can sustain antigen presentation and attract and manipulate immune cells.
  • the scaffolds contain porous matrices, wherein the pores have a diameter between 10 nm to 500 pm, particularly between 100 nm and 100 pm.
  • the invention utilizes scaffolds comprising mesoporous scaffolds. Methods of making polymer matrices having the desired pore sizes and pore alignments are described in the art, e.g., US pub. No. 2011/0020216 and US patent No. 6,511,650, which are incorporated herein by reference.
  • the mesoporous silica rods can be modified into multifunctional delivery platforms for delivering drugs such as chemotherapeutic agents and DNA/siRNA, antibody and protein biologies, cells, etc.
  • drugs such as chemotherapeutic agents and DNA/siRNA, antibody and protein biologies, cells, etc.
  • drugs such as chemotherapeutic agents and DNA/siRNA, antibody and protein biologies, cells, etc.
  • drugs such as chemotherapeutic agents and DNA/siRNA, antibody and protein biologies, cells, etc.
  • Embodiments described herein further relate to MSR-SLB scaffolds, including, scaffolds containing such scaffolds, wherein the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between about 1:1 to about 100:1, preferably between about 10:1 to about 50:1, particularly between about 20:1 to about 50:1. In some embodiments, the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules of the MSR-SLB scaffolds is between about 10,000:1 to about 1:1.
  • MSR mesoporous silica micro-rods
  • the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T- cell activating/co-stimulatory molecules of the MSR-SLB scaffolds is between about 5,000:1 to about 1:1, between about 1,000:1 to about 1:1, between about 500:1 to about 1:1, between about 100:1 to about 1:1.
  • the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules of the MSR-SLB scaffolds is about 10,000:1, about 5,000:1, about 2,500:1, about 1,000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 10:1, or about 1:1.
  • Embodiments described herein further relate to compositions and devices containing aforementioned scaffolds containing the MSR-SLB scaffolds together with the functional molecules, e.g., T-cell activating ligand, T-cell co-stimulatory molecule, and/or T-cell homeostatic agent, optionally together with one or more additional agents (listed below).
  • the invention provides for compositions comprising the scaffold and a population of T- cells, optionally, clustered therein.
  • the T-cells are selected from the group consisting of natural killer T-cells (NKT), a CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combination thereof.
  • the composition may be a pharmaceutical composition, which may be produced using methods that are well-known in the art.
  • pharmaceutical compositions may be produced by those of skill, employing accepted principles of medicinal chemistry.
  • the compositions, scaffolds, and devices may be provided with one or more reagents for selecting, culturing, expanding, sustaining, and/or transplanting the cells of interest.
  • Representative examples of cell selection kits, culture kits, expansion kits, transplantation kits for T-cells, B -cells and antigen presenting cells are known in the art.
  • inventions relate to T-cell manipulating devices.
  • the devices contain the scaffolds of the invention together with a plurality of molecules which attract/bind to target T cells.
  • the invention relates to devices containing scaffolds that are stacked to selectively permit infiltration of T-cells into the mesoporous silica micro-rods (MSR).
  • MSR mesoporous silica micro-rods
  • spaces between the mesoporous silica micro-rods (MSR) permit T cell infiltration.
  • the scaffold contains at least 10% more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 80% more, 90% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500% more, 600% more, 800% more, 1000% more, or greater number of target T -cells after a period of administration and/or association compared to that which is present in whole blood.
  • the period of administration and/or association is between 1-30 days, preferably between 4-15 days, particularly between 7-12 days.
  • selective infiltration relates to retention and/or expansion of T -cells compared to other blood cells, e.g., B-cells, dendritic cells, macrophages, red blood cells or platelets that are present in whole blood.
  • other blood cells e.g., B-cells, dendritic cells, macrophages, red blood cells or platelets that are present in whole blood.
  • the scaffolds of the invention permit selective infiltration of a specific sub-population of T-cells, e.g., natural killer T-cells (NKT), CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, or regulatory T-cells (Tregs).
  • T-cells e.g., natural killer T-cells (NKT), CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, or regulatory T-cells (Tregs).
  • the scaffold contains at least 10% more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 80% more, 90% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500% more, 600% more, 800% more, 1000% more, or greater number of target T-cells after 4-14 days administration and/or association compared to that which is present in whole blood.
  • NK cells 7% range: 2-13%
  • helper T cells 46% range: 28-59%
  • cytotoxic T cells 19% range: 13-32%
  • y5 T cells 5% range: 2%- 8%
  • B cells 23% range: 18-47%)
  • the genetically-modified T-cell immunotherapy may comprise a chimeric antigen receptor T cell (CAR T cell).
  • the engineered T-cell or population of engineered T-cells may comprise a chimeric antigen receptor T cell (CAR T cell).
  • a subject is genetically modified with an engineered T-cell receptor, such as a chimeric antigen receptor (Sadelain et al., Cancer Discov.3:388-398, 2013).
  • a T cell is provided and recombinant nucleic acid encoding an engineered T-cell receptor, such as a chimeric antigen receptor, is introduced into the patient-derived T cell to generate a CAR T cell.
  • T cells not derived from the subject are genetically modified with an engineered T-cell receptor, such as a chimeric antigen receptor.
  • the T cells are allogeneic cells.
  • Non-limiting examples of methods for introducing nucleic acid into a T cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.
  • lipofection e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)
  • CRISPR/Cas9 genome editing technology can be used to introduce CAR nucleic acids into T cells and/or to introduce other genetic modifications into T cells to enhance CAR T cell activity (for use of CRISPR/Cas9 technology in connection with CAR T cells, see e.g., US 9,890,393; US 9,855,297; US 2017/0175128; US 2016/0184362; US 2016/0272999; WO 2015/161276; WO 2014/191128; CN 106755088; CN 106591363; CN 106480097; CN 106399375; CN 104894068).
  • an engineered T-cell may refer, in some embodiments, to a T-cell comprising an engineered T-cell receptor.
  • An “engineered T-cell receptor” may comprise (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain.
  • an engineered T-cell receptor is a genetically modified TCR, a modified TCR, a recombinant TCR, a transgenic TCR, a partial TCR, a chimeric fusion protein, a CAR, a first generation CAR, a second generation CAR, a third generation CAR, or a fourth generation TRUCK.
  • the engineered T-cell receptor comprises an antibody or a fragment of an antibody.
  • the engineered T-cell receptor is a genetically modified TCR or a CAR.
  • T-cell receptor refers to a cell surface molecule found on T-cells that functions to recognize and bind antigens presented by antigen presenting molecules.
  • a TCR is a heterodimer of an alpha chain (TRA) and a beta chain (TRB).
  • TRG alternative gamma
  • TRD delta
  • T-cells expressing this version of a TCR are known as y5 T-cells.
  • TCRs are part of the immunoglobulin superfamily. Accordingly, like an antibody, the TCR comprises three hypervariable CDR regions per chain. There is also an additional area of hypervariability on the beta-chain (HV4).
  • the TCR heterodimer is generally present in an octomeric complex that further comprises three dimeric signaling modules Ou3y/a, CD35/8, and CD247 i, or /r
  • a “chimeric antigen receptor” is an artificially constructed hybrid protein or polypeptide comprising a specificity or recognition (i.e. binding) domain linked to an immune receptor responsible for signal transduction in lymphocytes.
  • CAR chimeric antigen receptor
  • the term “CAR” may be understood to refer to an artificial transmembrane protein receptor comprising an extracellular domain capable of binding to a predetermined CAR ligand or antigen, an intracellular segment comprising one or more cytoplasmic domains derived from signal transducing proteins different from the polypeptide from which the extracellular domain is derived, and a transmembrane domain.
  • the binding domain is typically derived from a Fab antibody fragment that has been fashioned into a single chain scFv via the introduction of a flexible linker between the antibody chains within the specificity domain.
  • Other possible specificity domains can include the signaling portions of hormone or cytokine molecules, the extracellular domains of receptors, and peptide ligands or peptides isolated by library (e.g. phage) screening (see Ramos and Doth, (2011) Expert Opin Bio Ther 11(7): 855).
  • antigen-binding domains may include antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments.
  • antibodies and antigen-binding fragments thereof include, but are not limited to: single-chain Fvs (scFvs), Fab fragments, Fab’ fragments, F(ab’)2, disulfide -linked Fvs (sdFvs), Fvs, and fragments containing either a VL or a VH domain. Flexibility between the signaling and the binding portions of the CAR may be a desirable characteristic to allow for more optimum interaction between the target and the binding domain, so often a hinge region is included.
  • One example of a structure that can be used is the CH2-CH3 region from an immunoglobulin such as an IgG molecule.
  • the signaling domain of the typical CAR comprises intracellular domains of the TCR-CD3 complex such as the zeta chain. Alternatively, the y chain of an Fe receptor may be used.
  • the transmembrane portion of the typical CAR can comprise transmembrane portions of proteins such as CD4, CD8 or CD28 (Ramos and Doth, ibid). Characteristics of some CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner. The non-MHC -restricted target recognition gives T- cells expressing CARs the ability to recognize a target independent of antigen processing, thus bypassing a major mechanism of tumor escape.
  • chimeric antigen receptor is sometimes called a “chimeric receptor,” a “T-body,” or a “chimeric immune receptor (OR).” Typically these receptors are used to impart the antigen specificity of a monoclonal antibody onto a T cell.
  • CARs contain three domains: 1) an ectodomain typically comprising a signal peptide, a ligand or antigen recognition region (e.g. scFv), and a flexible spacer; 2) a transmembrane (TM) domain; 3) an endodomain (alternatively known as an “activation domain”) typically comprising one or more intracellular signaling domains.
  • the “intracellular signaling domain” means any oligopeptide or polypeptide domain known to function to transmit a signal causing activation or inhibition of a biological process in a cell, for example, activation of an immune cell such as a T cell or a NK cell. Examples include ILR chain, CD28 and/or CD3 ⁇ .
  • the ectodomain of the CAR resides outside of the cell and exposed to the extracellular space, whereby it is accessible for interaction with its cognate ligand.
  • the TM domain allows the CAR to be anchored into the cell membrane of the effector cell.
  • the third endodomain also known as the “activation domain” aids in effector cell activation upon binding of the CAR to its specific ligand.
  • effector cell activation comprises induction of cytokine and chemokine production, as well as activation of the cytolytic activity of the cells.
  • the CARs redirect cytotoxicity toward tumor cells.
  • chimeric antigen receptors comprise a ligand- or antigenspecific recognition domain that binds to a specific target ligand or antigen (also referred to as a binding domain).
  • the binding domain is a single -chain antibody variable fragment (scFv), a tethered ligand or the extracellular domain of a co-receptor, fused to a transmembrane domain, which is linked, in turn, to a signaling domain.
  • the signaling domain is derived from CD3 ⁇ or FcRy.
  • the CAR comprises one or more co-stimulatory domains derived from a protein such as CD28, 4.
  • IBB CD137
  • 0X40 CD134
  • CD27 TNFRSF7
  • GITR CD357
  • CD30 CD30
  • HVEM CD270
  • LT 3R
  • DR3 TNFRSF25
  • CD278 CD226
  • CD355 CRT AM
  • TIM1 HVCR1, KIMI
  • CD2 LFA2, 0X34
  • SLAM CD150, SLAMF1
  • 2B4 CD244, SLAMF4
  • Lyl08 NBA, CD352, SLAMF6)
  • CD84 SLAMF5
  • Ly9 CD229, SLAMF3
  • ICAM-1 ICAM-1
  • CRACC CD319, BLAME
  • engagement of the antigen binding domain of the CAR with its target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell.
  • the main characteristic of CARs are their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific co-receptors.
  • MHC major histocompatibility
  • scFv-based CARs engineered to contain a signaling domain from CD3 ⁇ or FcRy have been shown to deliver a potent signal for T cell activation and effector function, they are not sufficient to elicit signals that promote T cell survival and expansion in the absence of a concomitant co-stimulatory signal.
  • a new generation of CARs containing a binding domain, a hinge, a transmembrane and the signaling domain derived from CD3 ⁇ or FcRy together with one or more co-stimulatory signaling domains has been shown to more effectively direct antitumor activity as well as increased cytokine secretion, lytic activity, survival and proliferation of CAR expressing T cells in vitro, in animal models and cancer patients (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).
  • co-stimulatory signaling domains e.g., intracellular co-stimulatory domains derived from CD28, CD137, CD134 and CD278
  • chimeric antigen receptor-expressing T cells are cells that are derived from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with specificity for a ligand.
  • the cells perform at least one effector function (e.g. induction of cytokines) that is stimulated or induced by the specific binding of the ligand to the CAR and that is useful for treatment of the same patient’s disease or condition.
  • the effector cell is a T cell (e.g. a cytotoxic T cell) that exerts its effector function (e.g. a cytotoxic T cell response) on a target cell when brought in contact or in proximity to the target or target cell (e.g. a cancer cell) (see e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450).
  • T cells Prolonged exposure of T cells to their cognate antigen can result in exhaustion of effector functions, enabling the persistence of infected or transformed cells.
  • Recently developed strategies to stimulate or rejuvenate host effector function using agents that induce an immune checkpoint blockade have resulted in success towards the treatment of several cancers. Emerging evidence suggests that T cell exhaustion may also represent a significant impediment in sustaining long-lived antitumor activity by chimeric antigen receptor-expressing T cells (CAR T cells).
  • CAR T cells chimeric antigen receptor-expressing T cells
  • the differentiation status of the patient-harvested T cells prior to CAR transduction and the conditioning regimen a patient undergoes before reintroducing the CAR T cells can profoundly affect the persistence and cytotoxic potential of CAR T cells.
  • CAR T cells e.g., addition or exclusion of alkylating agents, fludarabine, total-body irradiation
  • cytokines such as IL -2
  • T cell populations can also alter the differentiation status and effector function of CAR T cells (Ghoneim et al., (2016) Trends in Molecular Medicine 22(12): 1000-1011).
  • CAR ligand used interchangeably with “CAR antigen” and “T-cell ligand” means any natural or synthetic molecule (e.g., small molecule, protein, peptide, lipid, carbohydrate, nucleic acid) or part or fragment thereof that can specifically bind to the CAR.
  • Such ligands may be T-cell activating ligands or T-cell inhibiting ligands.
  • the one or more T-cell ligands is loaded into and/or presented on the APC-MS.
  • the one or more T-cell ligands is an antibody molecule or antigenbinding fragment thereof. In some embodiments, the one or more T-cell ligands is an anti-idiotype antibody or derivative thereof that binds to the antigen-binding domain of a CAR molecule of the administered population of engineered T-cells in the subject.
  • the one or more T-cell ligands is selected from the group consisting of an anti-idiotype CD3 antibody (aCD3) or an antigen-binding fragment thereof; an anti-idiotype CD5 antibody (aCD5) or an antigen-binding fragment thereof; an anti-idiotype CD7 antibody (aCD7) or an antigen-binding fragment thereof; an anti-idiotype CD28 antibody (aCD28) or an antigen-binding fragment thereof; an anti-idiotype CD19 antibody (aCD19) or an antigen-binding fragment thereof; an anti-idiotype CD20 antibody (aCD20) or an antigen-binding fragment thereof; an anti-idiotype CD22 antibody (aCD22) or an antigenbinding fragment thereof; an anti-idiotype CD70 antibody (aCD70) or an antigen-binding fragment thereof; an anti-idiotype CD123 antibody (aCD123) or an antigen-binding fragment thereof; an antiidiotype CS1 antibody (aCSl) or an antigen-binding fragment thereof; an anti-idiotype CD3 antibody (
  • the one or more T-cell ligands comprises a T-cell antigen or derivative thereof that binds to a CAR molecule of the administered population of engineered T-cells in the subject.
  • the one or more T-cell ligands is selected from the group consisting of a CD3 molecule or a fragment thereof; a CD5 molecule or a fragment thereof; a CD7 molecule or a fragment thereof; a CD28 molecule or a fragment thereof; a CD 19 molecule or a fragment thereof; a CD20 molecule or a fragment thereof; a CD22 molecule or a fragment thereof; a CD70 molecule or a fragment thereof; a CD123 molecule or a fragment thereof; a CS1 molecule or a fragment thereof; a BCMA molecule or a fragment thereof; a SLAMF7 molecule or a fragment thereof; a Claudin-6 molecule or a fragment thereof; a NKG2D molecule or a fragment thereof; a N
  • the one or more T-cell ligands is selected from the group consisting of an anti-CD3 antibody or an antigen-binding fragment thereof, anti-macrophage scavenger receptor (MSR1) antibody or an antigen-binding fragment thereof, an anti-T-cell receptor (TCR) antibody or an antigen-binding fragment thereof, an anti-CD2 antibody or an antigen-binding fragment thereof, an anti-CD47 antibody or an antigen-binding fragment thereof, a major histocompatibility complex (MHC) molecule loaded with an MHC peptide or a multimer thereof, and an MHC-immunoglobulin (Ig) conjugate or a multimer thereof, ICAM-1, or a combination thereof.
  • MHC major histocompatibility complex
  • the one or more T-cell ligands comprises a cancer antigen or derivative thereof that binds to a CAR molecule of the administered population of engineered T-cells in the subject.
  • cancer antigen refers to (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) cells that express tumor-specific antigens, (iv) cells that express tumor-associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor-specific membrane antigens, (viii) tumor-associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.
  • an exogenous T cell may be modified to express a receptor (e.g., TCR or CAR), such as a receptor that recognizes a cancer antigen (also referred to as a tumor antigen).
  • a cancer antigen typically is an antigen that is expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells) and in some instances is expressed solely by cancer cells.
  • the cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell.
  • Non-limiting examples of cancer antigens include, without limitation, an epithelial cancer antigen, (e.g., breast, gastrointestinal, lung), a prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a bladder cancer antigen, a lung (e.g., small cell lung) cancer antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, or a colorectal cancer antigen.
  • an epithelial cancer antigen e.g., breast, gastrointestinal, lung
  • PSA prostate specific cancer antigen
  • PSMA prostate specific membrane antigen
  • bladder cancer antigen e.g., a lung cancer antigen
  • a lung e.g., small cell lung cancer antigen
  • colon cancer antigen e.g., an
  • the tumor antigen is a lymphoma antigen (e.g., non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemia antigen, a myeloma (e.g.., multiple myeloma or plasma cell myeloma) antigen, an acute lymphoblastic leukemia antigen, a chronic myeloid leukemia antigen, or an acute myelogenous leukemia antigen.
  • a lymphoma antigen e.g., non-Hodgkin's lymphoma or Hodgkin's lymphoma
  • a B-cell lymphoma cancer antigen e.g., a B-cell lymphoma cancer antigen
  • a leukemia antigen e.g., a myeloma (e.g., multiple myeloma or plasma cell myeloma)
  • Exemplary CAR include, without limitation, a CD3 CAR, a CD5 CAR, a CD7 CAR, a CD28 CAR, a CD 19 CAR, a CD20 CAR, a CD22 CAR, a CD70 CAR, a CD 123 CAR, a CS1 CAR, a BCMA CAR, a SLAMF7 CAR, a Claudin-6 CAR, a NKG2D CAR, a NKG2DL CAR, a GD2 CAR, a Her2 CAR, and a mesothelin (MSLN) CAR, or a combination thereof.
  • the therapeutic efficacy of CAR T cells is enhanced by contact with the material scaffolds, described herein, in vivo.
  • the scaffolds of the invention include one or more agents, which may be naturally- occurring, synthetically produced, or recombinant compounds, e.g., peptides, polypeptides, proteins, nucleic acids, small molecules, haptens, carbohydrates, or other agents, including fragments thereof or combinations thereof.
  • the agents are antigens.
  • the antigens are peptides or proteins or immunologically active fragments thereof.
  • the antigens described herein are purified. Purified compounds contain at least 60% by weight (dry weight) of the compound of interest. Particularly, the antigens are at least 75% pure, preferably at least 90% pure, and more preferably at least 99% pure. Purity is measured by any appropriate standard method, for example, by column chromatography, gel electrophoresis, or HPLC analysis.
  • the antigens may be self-antigens or non-self antigens.
  • non-self antigens include, for example, antigens derived from a pathogen selected from the group consisting of a virus, a bacteria, a protozoan, a parasite, and a fungus.
  • the antigens may be optionally loaded into and/or presented on MHC molecules, e. g., HLA- A, HLA-B, HLA-C, DP, DQ and DR, which are then incorporated into the scaffolds.
  • the scaffolds contain a plurality of self-antigens, which are optionally linked to or associated with a disease or disorder.
  • the self-antigens are specifically associated with a human disease or a disorder.
  • the self-antigen is associated with an autoimmune disorder selected from the group consisting of rheumatoid arthritis , lupus, celiac disease, inflammatory bowel disease or Crohn’s disease, sjdgren’s syndrome polymyalgia rheumatic, multiple sclerosis, ankylosing spondylitis, Type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, etc.
  • rheumatoid arthritis-related antigen is a 47kDa protein (RA-A47). See Hattori et al, J Bone Miner Metab., 18(6):328-34 (2000). In Crohn’s disease, the antigen may be bacterial flagellin. See, Lodes et al. , J Clin Invest. 113(9): 1296-306 (2004).
  • myelin basic protein MBP
  • proteolipid protein PLP
  • MBP myelin basic protein
  • PGP proteolipid protein
  • IGRP islet-specific glucose-6-phosphatase
  • GAD65 glutamate decarboxylase
  • IA-2 insulinoma antigen-2
  • chromogranin A heat shock protein 60. See Roep et al., Cold Spring Harb Perspect Med.2(A), 2012 (PMID: 22474615).
  • the self-antigens are associated with a cancer.
  • cancer antigens include, for example, MAGE-1, MAGE-2, MAGE-3, CEA, Tyrosinase, midkin, BAGE, CASP-8, P-catenin, - catenin, y-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gplOO, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf 112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1
  • the antigen is a target of modified T-cells, e.g., CAR T-cells described above.
  • the antigen is CD 19 or a fragment thereof or a variant thereof.
  • the antigen is CD22 or a fragment thereof or a variant thereof.
  • the aforementioned antigens may be combined with the scaffold compositions using any known methods, including covalent and non-covalent interactions. Some of these methods have been outlined above in sections relating to fabricating the MSR-SLB scaffolds with the functional molecules of the invention. Examples of non-covalent interactions include, for example, electrostatic interactions, van der Waals’ interactions, n-effects, hydrophobic interactions, physical insertion etc. For example, full length transmembrane protein antigens can be incorporated into the lipid bilayer via physical insertion using routine methods. See, Cymer et al., Journal of Molecular Biology , 427.5: 999-1022, 2015 and US Patent No. 7,569,850, which are incorporated by reference herein.
  • the antigens may also be attached or tethered to scaffold compositions via covalent interactions.
  • Methods for attaching antigens to scaffolds/surfaces are known in the art, e.g., surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material.
  • covalent coupling via alkylating or acylating agents may be used to provide a stable, long-term presentation of an antigen on the scaffold in a defined conformation.
  • Exemplary reagents and methods for covalently coupling peptides/proteins to polymers are known in the art. See, for example, U.S. Patent No. 6,001,395, which is incorporated herein by reference.
  • the antigens are encapsulated into the scaffolds.
  • Methods for encapsulating antigens into suitable scaffolds e.g., PLGA microspheres, are known in the art. See, for example, US Patent No. 6,913,767 and International Publication No. WO 1995/011010, the disclosures of each of which are incorporated herein by reference.
  • the antigens may be formulated to interact with the immune cell via direct binding or indirect binding.
  • Types of direct binding include, for example, engagement or coupling of the antigen with the cognate receptor, e.g., T-cell receptor.
  • Indirect binding may occur through the intermediacy of one or more secondary agents or cell-types.
  • the antigen may first bind to a B-cell or an antigen-presenting cell (APC), get processed e.g., degraded) and presented on cell-surface major-histocompatibility complexes (MHC), to which the target cell population, e.g., T-cell, binds.
  • APC antigen-presenting cell
  • MHC major-histocompatibility complexes
  • the antigen may recruit other intermediary cells that secrete various cytokines, growth factors, chemokines, etc., which in turn attract the target immune cell population.
  • the recited components act in concert to manipulate or modify the immune cells.
  • the antigen may be derived from a cell lysate, a fractionated cell lysate, freshly harvested cells, biological fluids (including blood, serum, ascites), tissue extracts, etc.
  • the antigens are derived from lysates of target cells to which the desired immune cells, e.g., T cells, bind.
  • the antigens are first fractionated in the cell lysate prior to loading the scaffolds.
  • the lysates may be derived from a desired target tissue, e.g., an autoimmune disease-specific cells obtained from primary tissues.
  • the lysates may be derived from cancer cells, e.g., individual cells obtained from tumor samples or tissue cultures or tumor cells obtained from biopsies histologies.
  • the scaffolds of the invention may also contain one or more recruiting agents.
  • the recruiting agent may be an agent selected from the group consisting of a T-cell recruiting agent, a B-cell recruiting agent, a dendritic cell recruiting agent, and a macrophage recruiting agent.
  • the scaffolds contain T-cell recruiting agents.
  • T-cell recruiting agents include, e.g., granulocyte macrophage -colony stimulating factor (GM- CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), or a FMS-like tyrosine kinase 3 (Flt-3) ligand, granulocyte-colony stimulating factor (G-CSF), IFNy, a C- X-C Motif chemokine ligand (CXCL) selected from the group consisting of CXCL12 and CXCR4, or a fragment thereof, a variant thereof, or a combination thereof.
  • GM- CSF granulocyte macrophage -colony stimulating factor
  • C-C motif chemokine
  • C-C motif chemokine ligand 21
  • C-C motif ligand 19
  • T-cell recruiting agents include, ligands for CCR5 and CXCR3 receptors for recruiting T helper type 1 (Thl) subset.
  • the CCR5 ligands, CCL5 and macrophage inflammatory proteins (MIP-la), are known.
  • ligands for CCR3, CCR4, CCR8 and CXCR4 may be employed for specific recruitment of the Th2 subset.
  • a combination of the ligands may also be employed.
  • homologs of the aforementioned T-cell recruiting agents including functional fragments thereof, or variants thereof, are known in the art.
  • Representative examples of homologs include related proteins from fly, mouse, rat, pig, cow, monkey, humans or the like.
  • the homologs preferably include human or mouse homologs of the aforementioned recruiting agents.
  • the scaffolds of the instant invention are adapted for the preferential recruitment of a single type or single sub-type of cell, for example, preferential recruitment of T-cells and particularly a subset of Treg cells or NK cells.
  • Preferential recruitment is characterized by an accumulation of at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold, or greater increase in one or more of a particular type of immune cells (e.g., T cells, B-cells, DC/macrophages) in the device compared to other types of immune cells in the device (or in control scaffolds that are devoid of recruitment agents).
  • a particular type of immune cells e.g., T cells, B-cells, DC/macrophages
  • preferential recruitment is characterized where the total percentage of recruited cells is at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 2-fold (z.e., 200%), at least 5-fold, at least 8-fold, at least 10-fold, or greater than other types of immune cells in the device (or in control scaffolds).
  • preferential recruitment is characterized by 1-10 fold increase in the number of the cells of interest compared to other immune cells.
  • the instant invention relates to MSR-SLB scaffolds further comprising a recruitment agent which is GM-CSF, an agonist thereof, a mimetic thereof, a fragment thereof, a variant thereof, or a combination thereof.
  • the recruitment agent is GM-CSF in combination with at least one of CCL-21, CCL-19, Flt-3 or GCSF.
  • Representative examples of such recruitment agents include, e.g., human GM-CSF (NCBI Accession # NP_000749.2) and mouse GM- CSF (NCBI Accession # NP_034099.2).
  • the instant invention relates to MSR-SLB scaffolds containing fragments of GM-CSF, e.g., a polypeptide containing amino acids 18 -144 of the hGM-CSF sequence.
  • the invention relates to scaffolds containing GM-CSF variants including, for example, VAR_013089 and VAR_001975, the sequences of which have been accessioned in UNIPROT (Accession No. P04141).
  • the invention relates to MSR-SLB scaffolds containing GM-CSF mimetics including, for example, antibodies binding to GM-CSF receptor, e.g., those described by Monfardini et al., Carr Pharm Des., 8(24): 2185-99, 2002.
  • Embodiments of the invention further provide for scaffolds for manipulating immune cells which comprise a plurality of additional agents.
  • the additional agent may comprise a growth factor, a cytokine, a chemokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule or a fragment thereof or a combination thereof.
  • growth factors/cytokines include, but are not limited to, adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic proteins (BMPs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), foetal Bovine Somatotrophin (FBS) glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF), migration-stimulating factor (MSF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PD), bone
  • interleukins include, but are not limited to, IL-1 (activates T cells, B- cells, NK cells, and macrophages), IL-2 (activates B-cells and NK cells), IL-3 (stimulates nonlymphoid cells), IL-4 (growth factor for activated B cells, resting T cells, and mast cells), IL-5 (for differentiation of activated B cells), IL-6 (growth factor for plasma cells and T-cells), IL-7 (growth factor for pre B-cells/pre T-cells and NK cells), IL-10 (activates macrophages, B-cells, mast cells, Thl/Th2 cells), IL-12 (activates T cells and NK cells), IL-17 (activates Th cells). Functional fragments of interleukins, which are characterized by their ability to modulate the activity of target cells, may also be employed.
  • the scaffolds may contain adhesion molecules, which may also serve as signaling agents.
  • adhesion signaling molecules include, but are not limited to, fibronectin, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibronogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, von Willebrand Factor, polysaccharide heparin sulfate, connexins, collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly- Ser-Arg) peptides and cyclic peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), condroitin-6-sulfate,
  • adhesion molecules include neural cell adhesion molecules (NCAMs), intercellular adhesion molecules (ICAMs), vascular cell adhesion molecule (VCAM-1), platelet -endothelial cell adhesion molecule (PECAM-1), LI, and CHL1.
  • NCAMs neural cell adhesion molecules
  • ICAMs intercellular adhesion molecules
  • VCAM-1 vascular cell adhesion molecule
  • PECAM-1 platelet -endothelial cell adhesion molecule
  • LI CHL1.
  • CHL1 functional fragments of the adhesion molecules, which are characterized by their ability to modulate the binding of target cells to the scaffolds of the invention, may also be employed.
  • adhesion molecules comprise peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD), which is known as a cell attachment ligand and found in various natural extracellular matrix molecules.
  • RGD arginine-glycine-aspartic acid
  • GFOGER peptides have been previously shown to be particularly good for T cell adhesion. See, Stephan et al, Nature Biotechnology 33, 2015.
  • a polymer matrix with such a modification provides cell adhesion properties to the scaffold of the invention, and sustains long-term survival of mammalian cell systems, as well as supporting cell growth and differentiation.
  • the adhesion molecules may be coupled to the polymer matrix is accomplished using synthetic methods which are in general known to one of ordinary skill in the art and are described in the examples. See, e.g., Hirano et al., Advanced Materials, 17-25, 2004; Hermanson et al. , Bioconjugate Techniques, p. 152-185, 1996; Massia and Hubbell, J. Cell Biol. 114:1089-1100, 1991; Mooney et al. , J. Cell Phys. 151:497-505, 1992; and Hansen et al. , Mol. Biol. Cell 5:967-975, 1994, the disclosures in which are incorporated by reference.
  • the scaffolds contain adhesion receptors that are useful in the binding/sequestration of T-cells.
  • the scaffolds may contain T-cell specific adhesion molecules, for example, a receptor selected from the group consisting of MHC class II (for CD4+ cells), MHC class I (for CD8+ cells), LFA-3 (CD2 ligand), ICAM1 (ligand for LFA-1) or a variant thereof, a fragment thereof or a combination thereof.
  • the scaffolds may be specifically formulated to contain a subset of recruitment agents and adhesion molecules so as to manipulate a particular subset of immune cells, e.g., a particular sub-population of T-cells.
  • the scaffolds may be formulated/fabricated using agents that specifically bind to cell-surface markers that are expressed in the target cells.
  • the scaffolds may be adapted for the preferential recruitment of helper T-cells (TH cells; which differentially express CD4+), cytotoxic T-cells (T c cells; which differentially express CD8+), memory T-cells (T m cells; which differentially express CD45RO), suppressor T-cells (T s which cells), regulatory T-cells (Tregs; further characterized as FOXP3+ Treg cells and FOXP3- Treg), natural killer T-cells (NK cells; differentially express CDld+), mucosal associated invariant (MAITs; differentially express MR1), gamma delta T cells, (y5 T cells; comprise TCRs containing one y-chain and one 5-chain).
  • helper T-cells TH cells
  • T c cells which differentially express CD8+
  • T m cells which differentially express CD45RO
  • suppressor T-cells T s which cells
  • Tregs regulatory T-cells
  • NK cells differentially express CDl
  • Such agents which bind to cell-surface markers may include, for example, haptens, peptides, ligands, antibodies, or the like.
  • Other routine techniques for enriching the isolates with one or more cell subtype may be optionally used in situ or ex situ.
  • the scaffolds may also be adapted for recruiting immune cells that are specific for a disease. For example, a plurality of T cells that are specific for a particular type of autoimmune disease may be recruited.
  • scaffolds that are useful in the diagnosis of autoimmune disorders may be formulated to contain recruitment agents that are specific to the immune cells implicated in the disorder. Such recruitment agents may, for example, be specific to regulatory T cells (Tregs), suppressor T cells (Ts) or a combination thereof.
  • scaffolds that are useful in the diagnosis of cancers may be formulated to contain recruitment agents for preferentially recruiting cancer-specific T-cell types, e.g., cytotoxic T cells (Tc), natural killer cells (NK) or a combination thereof.
  • Tc cytotoxic T cells
  • NK natural killer cells
  • the scaffold is useful to pan for disease-specific cells.
  • disease-specific cells may include, for example, cells that directly promote disease progression.
  • the disease may mediated and promoted via targeted killing of specific population of cells, e.g., beta cells of pancreas in T1D and neuronal cells in multiple sclerosis.
  • the disease may be precipitated by targeted attack of specific epitopes such as, for example, rheumatoid factors (RF) and citrullinated peptides (ACPA) in the context of rheumatoid arthritis and antigens present in the gut flora in the context of Crohn’s disease.
  • RF rheumatoid factors
  • ACPA citrullinated peptides
  • the targeted destruction of the cells generally involves specific type or subset of immune cells.
  • immune cells that are specific thereto may be preferentially manipulated using the scaffolds of the instant invention.
  • the scaffolds are provided with antigens to which disease-specific immune cells, e.g., T cells, bind.
  • T cells disease-specific immune cells
  • autoimmune cells can be manipulated and optionally re -programmed to a non-autoimmune phenotype.
  • Methods of reprogramming T- cells to pluripotency are known in the art. See, Nishimura et al., Stem Cell 12, 114-126 (2013); Themeli et al. , Nature Biotechnology 31, 928-933 (2013).
  • the reprogrammed cells may be rejuvenated to target the cancer.
  • the cells may be eliminated.
  • the scaffold of the invention are fabricated as porous structures that have been engineered to sustain antigen presentation.
  • Methods for fabricating porous scaffolds have been described in the art. See, for example, U.S. Publication Nos. 2011/0020216, 2013/0202707, 2011/0020216 and U.S. Patent No. 8,067,237, the disclosures in which are incorporated by reference herein.
  • Embodiments of the invention further provide for scaffolds containing MSR-SLB scaffolds that possess the desired stability for various in vivo applications.
  • the scaffolds are stable in as transplant material to be administered, for example, via injection, into subjects.
  • the invention relates to mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffolds which retain a fluid architecture for at least 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, or more in vivo.
  • the stability and/or fluid architecture of the scaffolds may be monitored using routine techniques, e.g., the microscopic visualization techniques illustrated in the Examples below.
  • Embodiments of the invention further relate to methods for making the antigen-presenting cell mimetic scaffolds (APC-MS) of the invention.
  • the method comprises providing a base layer comprising high surface area mesoporous silica micro-rods (MSR); optionally loading the one or more T-cell homeostatic agents on the MSR; layering a fluid supported lipid bilayer (SLB) on the base layer comprising the MSRs, thereby generating an MSR-SLB scaffold; loading the one or more T-cell homeostatic agents on the MSR-SLB scaffold if step (b) is not carried out; optionally blocking one or more non-specific integration sites in the MSR-SLB scaffold with a blocker; and loading the one or more T-cell ligands, optionally, the T-cell activating ligands and/or the T-cell inhibiting ligands, and, optionally the one or more T-cell co-stimulatory molecules, onto the MSR- SLB scaffold, thereby making the APC-MS
  • the method(s) may include further loading at least one additional agent which is a growth factor, a cytokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule, or a fragment thereof or a combination thereof in the scaffold.
  • additional agents which is a growth factor, a cytokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule, or a fragment thereof or a combination thereof in the scaffold.
  • a mixture of functional molecules containing a 1:1 mixture of the one or more T-cell ligands and the one or more T-cell co-stimulatory molecules is combined with the MSR-SLB scaffold such that the weight ratio of the functional molecules: MSR-SLB scaffold is between about 1:2 and about 1:20, preferably between about 1:4 and about 1:15, a particularly between about 1:5 to about 1:10.
  • the weight ratio of the T-cell ligand and the T-cell co-stimulatory molecule may be adjusted, e.g., between about 5:1 to about 1:5, while retaining the same dry weight ratio between the functional molecules and the MSR-SLB scaffold.
  • mesoporous-structured silica with a hexagonally ordered pore structure can be used as the base material for APC-MS assembly.
  • MSRs can be synthesized via a conventional sol-gel route, which uses a surfactant (i.e., Pluronic P123) to form hexagonally ordered micelles in acidic solution, and subsequently templates silica via a silica precursor (e.g., tetraethyl orthosilicate).
  • the microrods can be aged to condense the silica walls and further oxidized to remove excess surfactant.
  • the synthesized MSRs can have dimensions of - 70 pm x -5 pm, a pore size of -5 nm, and a surface area of -600 m 2 /g. These properties can support the adsorption of high quantities of soluble biomolecules and impart tunable biodegradability.
  • dried MSRs can be sieved, for example, through 63 pm test sieves (VWR), then calcinated at about 550 °C for about 4 h to remove excess surfactant and contaminants.
  • VWR test sieves
  • the materials can be resuspended in cell culture media, for example, to about 1.5 mg/mL, and added to CAR-T cell cultures.
  • materials can be prepared under aseptic conditions and used immediately after assembly.
  • embodiments of the invention further relate to methods of making the APC- MS by assembling a plurality of scaffolds to generate stacks with sufficient porosity to permit infiltration of T cells, more specifically, distinct sub-populations of helper T-cells or cytotoxic T- cells.
  • the scaffolds of the invention may be used for various applications, including, but not limited to, manipulation of target effector cells, e.g., T-cells, including engineered T-cells, in vitro and/or in vivo, isolation of a specific population of effector cells, e.g., a sub-population of CD4+ T-cells and/or CD8+ T-cells, diagnosis and therapy of diseases, and the production of compositions and kits for the diagnosis and therapy of diseases.
  • target effector cells e.g., T-cells, including engineered T-cells
  • isolation of a specific population of effector cells e.g., a sub-population of CD4+ T-cells and/or CD8+ T-cells
  • diagnosis and therapy of diseases e.g., a sub-population of CD4+ T-cells and/or CD8+ T-cells
  • compositions and kits for the diagnosis and therapy of diseases.
  • the instant invention provides a method for manipulating target effector cells or a sub-population thereof (e.g., helper T-cells or cytotoxic T-cells) in vivo.
  • target effector cells e.g., helper T-cells or cytotoxic T-cells
  • the term “manipulation” includes, for example, activation, division, differentiation, growth, expansion, reprogramming, anergy, quiescence, senescence, apoptosis or death of the target effector cells.
  • the target effector cells e.g., T-cells, including engineered T-cells
  • the scaffolds may be implanted or injected at a suitable site in a subject, e.g., subcutaneously or intravenously.
  • the subject is administered the population of target effector cells, e.g., T- cells, including engineered T-cells, prior to, simultaneous with, or after administration of the APC- MS.
  • the subject is administered the population of target effector cells, e.g., T-cells, including engineered T-Cells, prior to administration of the APC-MS.
  • target effector cells may be manipulated, including, fresh samples employed from subjects, primary cultured cells, immortalized cells, cell-lines, hybridomas, etc.
  • the manipulated cells may be used for various immunotherapeutic applications as well as for research.
  • the site of manipulation of target effector cells may be in vivo.
  • the cells are manipulated in vivo (e.g., within the scaffold).
  • the cells need not be physically removed from the scaffold to be manipulated.
  • the cells may be manipulated at or near the implant site.
  • the scaffolds used in manipulating effector cells may be provided with antigen presenting cells (APC) and/or various antigens derived from such APCs.
  • APC antigen presenting cells
  • these secondary agents e.g., APCs or antigens derived from APCs
  • the scaffolds may be provided with various antigens that attract and/or recruit APCs. Representative examples of such attracting and/or recruiting molecules have been provided in the previous sections.
  • the antigen-containing scaffolds may be used to manipulate target effector cells in vivo.
  • the scaffolds may be implanted or injected at a suitable site in a subject, e.g., subcutaneously or intravenously.
  • the scaffolds may be implanted or injected inside a blood vessel, in the lymphatic tissue, at the tumor site, at a disease site (e.g., areas surrounding tissues affected by rheumatoid arthritis) or subcutaneously, such that the target effector cells come into contact with the scaffolds.
  • the scaffolds may be injected in a minimally invasive manner, for example, via needle, catheter or the like.
  • the implanted or injected scaffolds may be allowed to remain at the implant or injection site for about 0.5 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3, months, 6 months, 7 , months, 8 months, 9 months. 1 year, 2 years, or more.
  • the subject is administered the population of target effector cells, e.g., T-cells, including engineered T-cells, prior to, simultaneous with, or after administration of the APC-MS.
  • the scaffolds may be explanted to study, analyze, or even further manipulate the effector cells.
  • the instant invention relates methods of manipulating a population of T- cells, optionally a population of engineered T-cells, in a subject.
  • the instant invention provides methods of manipulating a population of T-cells, in a subject, comprising administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a T-cell ligand (e.g., a T-cell activating ligand or a T-cell inhibiting ligand), wherein the subject was administered a population of T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby manipulating the administered population of T-cells in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the APC-MS comprises a base layer comprising high surface area mes
  • manipulating a population of T-cells can comprise increasing and/or decreasing the level of T-cell activity and/or function as compared to a control level.
  • the method disclosed herein results in a decrease in the level of T-cell activity and/or function in the subject.
  • a decrease in the level of T-cell activity and/or function of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 100% or more as compared to a control level.
  • the method disclosed herein results in an increase in the level of T-cell activity and/or function in the subject.
  • the population of T-cells may be a population of engineered T-cells, such as a population of T-cells comprising a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • a subject administered a sample containing a population of T cells is further administered the scaffolds of the present invention, which activates, co-stimulates and homeostatically maintains the target effector cells in the subject.
  • the sample may be administered and/or associated with the scaffold for about 1 day to 30 days, for about 1 to 15 days or for about 4 to 13 days, e.g., for about 7-8 days, resulting in the selective manipulation of the effector cell population.
  • the antigen-specific effector cells may be additionally manipulated by selecting cells based on the expression of certain gene products, e.g., T-cell receptors (TCR) that recognize the antigen or the antigen- presenting cells of interest.
  • TCR T-cell receptors
  • the effector target cells which are specific to the antigen or APCs may be selectively manipulated over other effector cells (e.g., favoring CD8+ T-cells over CD4+ T-cells).
  • a sample containing CD8+ T-cells (along with CD4+ T-cells) may be administered and/or associated with the scaffolds of the present invention which are mechanically or chemically fabricated to permit infiltration and/or sequestration of CD8+ T-cells.
  • the infiltrated and/or sequestered CD8+ T-cells may be further expanded, activated, proliferated, or grown using techniques known in the art. Representative methods have been described previously.
  • the effector target cells which are specific to the antigen or APCs may be undesired e.g., regulatory/suppressor T-cells) and they are induced to undergo apoptosis, anergy or death following contact with the scaffolds of the instant invention.
  • a population of cells containing regulatory T cells (along with other T-cells) may be administered with the scaffolds of the present invention which are mechanically or chemically fabricated to permit infiltration and/or sequestration of regulatory/suppressor T-cells in vivo.
  • the infiltrated and/or sequestered T-cells may be eliminated using techniques known in the art.
  • the identity of the cells that have infiltrated and/or are sequestered in the scaffolds of the invention may be further determined using art-known techniques.
  • the gene product for identifying or selecting for activated T cells may be a cell surface marker or cytokine, or a combination thereof.
  • Cell surface markers for identifying activated T cells include, but are not limited to, CD69, CD4, CD8, CD25, HLA-DR, CD28, and CD 134.
  • CD69 is an early activation marker found on B and T lymphocytes, NK cells and granulocytes.
  • CD25 is an IL-2 receptor and is a marker for activated T cells and B cells.
  • CD4 is a TCR coreceptor and is marker for thymocytes, TH1- and TH2-type T cells, monocytes, and macrophages.
  • CD8 is also a TCR coreceptor and is marker for cytotoxic T cells.
  • CD134 is expressed only in activated CD4+ T cells.
  • Cell surface markers for selecting for activated T cells include, but are not limited to, CD36, CD40, and CD44.
  • CD28 acts as a stimulatory T-cell activation pathway independent of the T-cell receptor pathway and is expressed on CD4+ and CD8+ cells.
  • CD36 is a membrane glycoprotein and is a marker for platelets, monocytes and endothelial cells.
  • CD40 is a marker for B cells, macrophages and dendritic cells.
  • CD44 is a marker for macrophages and other phagocytic cells.
  • Subsets of T cells may be isolated by using positive selection, negative selection, or a combination thereof for expression of cell surface gene products of helper T cells or cytotoxic T cells (e.g., CD4 vs. CD8).
  • Cytokines for identifying activated T cells of the present invention include, but are not limited to cytokines produced by THl-type T cells (cell-mediated response) and TH2-type T cells (antibody response). Cytokines for identifying activated THl-type T cells include, but are not limited to, IL-2, gamma interferon (ylFN) and tissue necrosis factor alpha (TNFa). Cytokines for identifying activated TH2-type T cells include, but not limited to, IL-4, IL-5, IL- 10 and IL-13. Subsets of T cells may also be isolated by using positive selection, negative selection, or a combination thereof for expression of cytokine gene products of helper T cells or cytotoxic T cells (e.g., ylFN vs. IL4).
  • cytotoxic T cells e.g., ylFN vs. IL4
  • An activated TH 1 -type T cell specific for an antigen of interest may be isolated by identifying cells that express CD69, CD4, CD25, IL-2, IFNy, TNFa, or a combination thereof.
  • An activated TH 1 -type T cell specific for an antigen of interest may also be isolated by identifying cells that express CD69 and CD4 together with IFNy or TNFa.
  • An activated TH2-type T cell specific for an antigen of interest may be isolated by identifying cells that express CD69, CD4, IL-4, IL-5, IL- 10, IL-13, or a combination thereof.
  • a combination of an activated TH 1 -type T cell and a TH2-type T cell specific for an antigen of interest may be isolated by identifying cells that express CD69, CD4, CD25, IL-2, IFNy, TNFa, or a combination thereof and cells that express CD69, CD4, IL-4, IL-5, IL-10, IL-13, or a combination thereof.
  • the gene products used for positive or negative selection of the activated T cells of the present invention may be identified by immunoselection techniques known to those in the art which utilize antibodies including, but not limited to, fluorescence activated cell sorting (FACS), magnetic cell sorting, panning, and chromatography.
  • Immunoselection of two or more markers on activated T cells may be performed in one or more steps, wherein each step positively or negatively selects for one or more markers.
  • FACS fluorescence activated cell sorting
  • the two or more different antibodies may be labeled with different fluorophores.
  • cells may be sorted using microbeads.
  • the antibody may directly bind to the gene product and may be used for cell selection.
  • magnetofluorescent liposomes may be used for cell selection.
  • conventional fluorescently labeled antibodies may not be sensitive enough to detect the presence of the cell surface expressed gene product.
  • Fluorophore-containing liposomes may be conjugated to antibodies with the specificity of interest, thereby allowing detection of the cell surface markers.
  • the antibody may be used after permeabilizing the cells.
  • the intracellular gene product if it is ultimately secreted from the cell may be detected as it is secreted through the cell membrane using a “catch” antibody on the cell surface.
  • the catch antibody may be a double antibody that is specific for two different antigens: (i) the secreted gene product of interest and (ii) a cell surface protein.
  • the cell surface protein may be any surface marker present on T cells, in particular, or lymphocytes, in general, (e.g., CD45).
  • the catch antibody may first bind to the cell surface protein and then bind to the intracellular gene product of interest as it is secreted through the membrane, thereby retaining the gene product on the cell surface.
  • a labeled antibody specific for the captured gene product may then be used to bind to the captured gene product, which allows the selection of the activated T cell.
  • Certain forms of cytokines are also found expressed at low concentration on the cell surface. For example, ylFN is displayed at a low concentration on the cell surface with kinetics similar to those of intracellular ylFN expression (Assenmacher, et al. Ear J. Immunol, 1996, 26:263-267).
  • cytokine of interest For forms of cytokines expressed on the cell surface, conventional fluorescently labeled antibodies or fluorophore containing liposomes may be used for detecting the cytokine of interest.
  • fluorescently labeled antibodies or fluorophore containing liposomes may be used for detecting the cytokine of interest.
  • fluorophore containing liposomes may be used for detecting the cytokine of interest.
  • the T cells isolated by the methods of the present invention may be enriched by at least 40% -90% from whole blood.
  • the T cells may also be enriched by at least 95% from whole blood.
  • the T cells may also be enriched by at least 98% from whole blood.
  • the T cells may also be enriched at least 99.5% from whole blood.
  • Similar methods may be used in the in situ or ex situ manipulation of B-cells.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation.
  • the dry weight ratios of scaffolds to sample cell product may be adjusted.
  • the scaffold: cell dry weight ratio may range from 1:500 to 500:1 and any integer values in between may be used to manipulate effector cells.
  • the ratio of scaffold to cells may dependent on the scaffold size relative to the target cell.
  • the present invention further relates to methods for expanding T- cells from a population of immune cells, e.g., expanding engineered T-cells in a subject containing a population of engineered B-cells, engineered dendritic cells, engineered macrophages, engineered plasma cells, and the like.
  • the present invention also relates to methods for expanding a specific population of engineered T-cells, e.g., expanding engineered cytotoxic T-cells in a subject containing a population of engineered helper T-cells, engineered natural killer T-cells, engineered regulatory/suppressor T-cells, and the like.
  • the specific sub-population of T-cells may be used downstream in various immunotherapeutic applications.
  • the APC-MS of the instant invention are particularly effective for the expansion of T-cells because the relatively large size and high aspect ratio of the mesoporous silica rods allow for the formation of large clusters of T-cells interacting with each rod which may promote the effective expansion of engineered T-cells by allowing T-cell/T-cell interactions and/or paracrine signaling.
  • the target effector cells e.g., engineered T-cells
  • the scaffolds may be implanted at a suitable site in a subject, e.g., subcutaneously or intravenously.
  • the engineered T cells are derived from primary T-cells obtained from a subject.
  • the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • T-cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, spleen tissue, and tumors. In certain embodiments of the present invention, any number of primary T-cells and/or T-cell lines available in the art, may be used.
  • T-cells in whole blood are very low.
  • the leukocyte population in whole blood is about 0.1-0.2% (due to predominance of erythrocytes), of which T-cells make up about 7-24% of the overall leukocyte population.
  • CD4+ T-cells make up about 4-20% of the overall leukocyte population (translating to less than 0.04% of the overall cell population in whole blood) and CD8+ T- cells make up about 2-11% of the overall leukocyte population (translating to less than 0.022% of the overall cell population in whole blood).
  • methods of the invention may be coupled with other art-known techniques for enrichment of target cells.
  • the enrichment step may be carried out prior to administration of the scaffolds of the instant invention.
  • the enrichment step may be carried out after administration of the scaffolds of the present invention.
  • the engineered effector cell population are derived from effector cells that may be enriched using FICOLL separation.
  • cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. The cells are then washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • a semi-automated “flow-through” centrifuge may also be used according to the manufacturer's instructions.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • peripheral or whole blood T cells may be enriched for use in producing an engineered population of T cells by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
  • a specific subpopulation of T cells such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques.
  • the expanded or manipulated T cell population may be further sorted using a combination of antibodies directed to surface markers unique to the cells.
  • a preferred method is cell sorting and/or selection via magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
  • the concentration of cells and scaffold surface can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which the scaffolds and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and scaffolds. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used.
  • concentrations of 125 or 150 million cells/ml can be used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
  • use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28- negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain.
  • using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • This method selects for cells that express high amounts of desired antigens to be bound to the scaffolds.
  • CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations.
  • the concentration of cells used is 5xl0 6 /ml.
  • the concentration used can be from about lxl0 5 /ml to lxl0 9 /ml, and any integer value in between, e.g., lxl0 5 /ml to lxl0 8 /ml, lxl0 6 /ml to lxl0 7 /ml, lxl0 7 /ml to lxl0 9 /ml.
  • the instant invention may include art-known procedures for sample preparation.
  • T cells may be frozen after the washing step and thawed prior to use. Freezing and subsequent thawing provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media containing for example, HESPAN and PLASMALYTE A, the cells then are frozen to -80° C at a rate of 1 ° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • suitable cell freezing media containing for example, HESPAN and PLASMALYTE A
  • Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.
  • a blood sample or leukapheresis product is taken from a generally healthy subject.
  • a blood sample or a leukapheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or a leukapheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
  • agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, myco
  • the cells are isolated for a patient and frozen for later use in conjunction with (e.g.
  • T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • the cells are isolated prior to and can be frozen for later use for treatment following B- cell ablative therapy such as agents that react with CD20, e.g. Rituxan.
  • T cells may be obtained from a patient directly following treatment for use in preparing a population of engineered T-cells, such as a population of T-cells comprising a chimeric antigen receptor (CAR), to be administered to a subject in accordance with the present methods.
  • CAR chimeric antigen receptor
  • a population of engineered T-cells such as a population of T-cells comprising a chimeric antigen receptor (CAR)
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • Scaffolds containing any ratio of T-cell ligands: T-cell co-stimulatory molecules may be used in accordance with the present methods.
  • a 1:1 ratio of each antibody may be used.
  • the ratio of CD3: CD28 antibody bound to the scaffolds ranges from 100:1 to 1:100 and all integer values there between.
  • more anti-CD28 antibody is bound to the scaffolds than anti-CD3 antibody, i.e. the ratio of CD3: CD28 is less than one.
  • the ratio of anti CD28 antibody to anti CD3 antibody bound to the scaffolds is greater than 2:1.
  • a 1:100 CD3: CD28 ratio of antibody bound to scaffolds is used.
  • a 1:75 CD3: CD28 ratio of antibody bound to scaffolds is used.
  • a 1:50 CD3: CD28 ratio of antibody bound to scaffolds is used.
  • a 1:30 CD3: CD28 ratio of antibody bound to scaffolds is used.
  • a 1:10 CD3: CD28 ratio of antibody bound to scaffolds is used.
  • a 1:3 CD3: CD28 ratio of antibody bound to the scaffolds is used.
  • a 3:1 CD3: CD28 ratio of antibody bound to the scaffolds is used.
  • One aspect of the present invention stems from the surprising finding that wherein the method confers increased expansion of the population of T-cells after about 1 week of contact with the scaffold compared to a control scaffold containing the base layer containing high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell ligands and the T-cell co-stimulatory molecules.
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • a 10-fold to 1000-fold preferably about a 50-fold to 500-fold, or greater, increase in the expansion of the population of T-cells was observed after about 1 week of contact with the scaffold compared to a control scaffold containing the base layer containing high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell ligands and the T-cell co-stimulatory molecules.
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • Another aspect of the present invention stems from the surprising finding that wherein the method confers increased expansion of the population of T-cells after about 1 week of contact with the scaffold as compared to a superparamagnetic spherical polymer particle (DYNABEAD) containing the T-cell ligands and the T-cell co-stimulatory molecules.
  • DYNABEAD superparamagnetic spherical polymer particle
  • a 2-fold to 100-fold preferably about a 5-fold to 20-fold, or greater, increase in the expansion of the population of T-cells was observed after about 1 week of contact with the scaffold compared to a superparamagnetic spherical polymer particle (DYNABEAD) containing the T-cell ligands and the T-cell co-stimulatory molecules.
  • DYNABEAD superparamagnetic spherical polymer particle
  • Yet another aspect of the present invention stems from the surprising finding that manipulating the T-cells in accordance with the aforementioned methods improves the metabolic activity of T-cells.
  • improved metabolic activity of T-cells was observed after 1 week of contact with the scaffold compared to a control scaffold containing the base layer containing high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell ligands and the T-cell co-stimulatory molecules.
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • a 2-fold to 100-fold preferably about a 5-fold to 20-fold, or larger, improvement in the metabolic activity of the population of T-cells was observed after about 1 week of contact with the scaffold compared to a control scaffold comprising the base layer comprising high surface area mesoporous silica micro-rods (MSR) and the fluid supported lipid bilayer (SLB) but not containing the T-cell ligands and the T-cell co-stimulatory molecules.
  • MSR high surface area mesoporous silica micro-rods
  • SLB fluid supported lipid bilayer
  • Another aspect of the present invention stems from the surprising finding that the method confers better metabolic activity of the population of T-cells after about 1 week of contact with the scaffold compared to a superparamagnetic spherical polymer particle (DYNABEAD) containing the T-cell ligands and the T-cell co-stimulatory molecules.
  • DYNABEAD superparamagnetic spherical polymer particle
  • DYNABEAD superparamagnetic spherical polymer particle
  • the expanded T-cells are metabolically active for at least about 7 days post-contact with the scaffold. T-cell metabolic activity was measured via routine techniques, e.g., analyzing levels of cytokine production or monitoring cell doublings. Furthermore, in accordance with the methods of the invention, the expanded T-cells formed larger and more stable aggregates e.g., lasting longer) than control scaffolds. For instance, in one experiment, the expanded T-cells formed stable aggregates for at least about 7 days post-contact with the scaffold whereas the aggregates had considerably disintegrated in samples administered and/or associated with the control scaffold containing only the MSR base layer and the SLB layer.
  • the instant invention relates to methods for obtaining a polyclonal population of CD4+ cells, comprising, contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of CD4+ cells; and isolating a sub-population of detected CD4+ T-cells from the sample.
  • the instant invention relates to methods for obtaining a polyclonal population of CD4+/FOXP3+ or CD4+/FOXP3- cells.
  • the method comprises contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of CD4+ cells; further contacting the T-cells with a reagent for detection of F0XP3+ cells; and isolating a sub-population of detected CD4+/FOXP3+ or CD4+/FOXP3- T-cells from the sample.
  • the reagent for the detection and/or isolation of CD4+ and/or F0XP3+ T-cells is preferably an antibody or antigenbinding fragment thereof which specifically binds to CD4+ and F0XP3 markers.
  • F0XP3 is recognized as a master regulator of the regulatory pathway in the development and function of regulatory T cells (which turn the immune response down)
  • F0XP3- cells for certain applications and F0XP3- cells for other applications.
  • the methods may be adapted to screen for F0XP3- cells.
  • the formulation methods may be modified to positively screen for and include F0XP3+ cells.
  • the instant invention relates to a method for obtaining a polyclonal population of effector memory and/or effector T-cells.
  • the method comprises contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of CD44+ cells; further contacting the T-cells with a reagent for detection of CD62L; and isolating a sub-population of detected CD4+//CD62L+ or CD4+//CD62L- T-cells from the sample.
  • the effector memory and/or effector T-cells are preferably CD4+//CD62L-.
  • the instant invention relates to a method for obtaining a polyclonal population of activated CD8+ T-cells.
  • the method comprises contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of CD8+ cells; further contacting the T-cells with a reagent for detection of CD69+; and isolating a sub-population of detected CD8+//CD69+ or CD8+//CD69- T-cells from the sample.
  • the activated T-cells are preferably CD8+/CD69+.
  • the instant invention relates to a method for obtaining a polyclonal population of cytotoxin-secreting T-cells.
  • the method comprises contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of CD8+ cells; further contacting the T-cells with a reagent for detection of granzyme B; and isolating a sub-population of detected CD8+//granzyme B+ or CD8+//Granzyme B- T-cells from the sample.
  • the cytotoxin-secreting T-cells are preferably CD8+/Granzyme B+.
  • the instant invention relates to a method for obtaining a polyclonal population of activator cytokine-secreting T-cells.
  • the method comprises contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of IFNy+; and isolating a sub-population of detected IFNy+ T-cells from the sample.
  • the T-cells are preferably IFNy-secreting cells.
  • the instant invention relates to a method for obtaining a polyclonal population of memory T-cells.
  • the method comprises contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of CD62L+CCR7+ T-cells; and isolating a subpopulation of detected CD62L+CCR7+ T-cells from the sample.
  • the T-cells are preferably CD62L+CCR7+ CD4+ central memory T-cells.
  • the instant invention relates to a method for obtaining a polyclonal population of memory T-cells comprising contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of CD62L+CCR7+ T-cells; and isolating a sub-population of detected CD62L-CCR7- T-cells from the sample.
  • the CD62L-CCR7- T-cells are effector memory T-cells. See, Sallusto et al., Nature 401: 708-712, 1999.
  • the instant invention relates to a method for detecting and/or removing a polyclonal population of exhausted T-cells from a sample.
  • the method comprises contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, costimulating, homeostatically maintaining and optionally expanding a population of T-cells present in the sample; contacting the T-cells in the sample with a reagent for detection of CD8+ T cells; further contacting the T-cells with a reagent for detection of PD-1+ T-cells; and isolating a sub-population of detected CD8+/ PD-1+ T-cells from the sample.
  • the CD8+/ PD-1+ T-cells, which indicate exhausted cells, may be optionally eliminated from the sample.
  • the instant invention provides a method comprising contacting the scaffolds of the invention with a subject’s biological sample, thereby activating, co-stimulating, homeostatically maintaining and optionally expanding a population of T-cells present within the sample; contacting the T-cells in the sample with a reagent for detection of a co-inhibitory receptor on T-cells; and isolating a sub-population of T-cells expressing the co-inhibitory receptor from the sample.
  • the expression of co-inhibitory receptor generally indicates exhausted cells, which may be optionally eliminated from the sample.
  • the co-inhibitory receptor is a receptor selected from the group consisting of CTLA-4, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. See, Legat et al., Front Immunol., 2013 Dec 19;4:455
  • the reagents for the detection and/or isolation of cells are preferably an antibodies or antigen-binding fragments thereof, e.g., antibodies which specifically bind to the aforementioned markers, e.g., CD8, CD4, FOXP3, CD62L, PD-1, granzyme B, etc.
  • the detection of these cell-surface markers is preferably carried out using FACS analysis.
  • the invention further relates to isolating polyclonal T-cell populations using one or more of the aforementioned methods and further detecting the production of a cytokine selected from the group consisting of interferon gamma (IFNy), tissue necrosis factor alpha (TNFa), IL-2, IL-1, IL -4, IL-5, IL- 10, and IL- 13, or a combination thereof.
  • a cytokine selected from the group consisting of interferon gamma (IFNy), tissue necrosis factor alpha (TNFa), IL-2, IL-1, IL -4, IL-5, IL- 10, and IL- 13, or a combination thereof.
  • IFNy interferon gamma
  • TNFa tissue necrosis factor alpha
  • the methods may comprise detecting the production of a cytokine selected from the group consisting of IL-2, interferon gamma (IFNy) and tissue necrosis factor alpha (TNFa), or a combination thereof.
  • the manipulated T-cells are T-helper 2 (Th2) cells and the method comprises detecting the production of a cytokine selected from the group consisting of IL-4, IL-5, IL- 10 and IL-13, or a combination thereof.
  • the methods may further comprise detecting the production of a cytokine selected from the group consisting of interferon gamma (IFNy) and lymphotoxin alpha (LTa/TNFP), or a combination thereof optionally together with the detection of a secreted cytotoxin selected from the group consisting of a granzyme or a perforin, or a combination thereof.
  • a cytokine selected from the group consisting of interferon gamma (IFNy) and lymphotoxin alpha (LTa/TNFP), or a combination thereof optionally together with the detection of a secreted cytotoxin selected from the group consisting of a granzyme or a perforin, or a combination thereof.
  • IFNy interferon gamma
  • LTa/TNFP lymphotoxin alpha
  • a resting T cell has a mean diameter of about 6.8 microns, and upon initial activation and stimulation, in the presence of the stimulating ligand, the T cell mean diameter will increase to over 12 microns by day 4 and begin to decrease by about day 6.
  • the T cells When the mean T cell diameter decreases to approximately 8 microns, the T cells may be reactivated and re-stimulated to induce further proliferation of the T cells.
  • the rate of T cell proliferation and time for T cell re-stimulation can be monitored by assaying for the presence of cell surface molecules, such as, a cell surface marker selected from the group consisting of CD69, CD4, CD8, CD25, CD62L, F0XP3, HLA-DR, CD28, and CD134, or a combination thereof.
  • the methods may be complemented by assaying for the presence of non T-cell surface molecules, such as, CD36, CD40, and CD44, or a combination thereof.
  • the methods may be complemented by assaying for the presence of non T-cell surface molecules, such as, CD154, CD54, CD25, CD137, CD134, which are induced on activated T cells.
  • Embodiments described herein further relate to methods for treating a disease or a disorder in a subject.
  • the disease is cancer.
  • the instant invention provides methods of treating a cancer in a subject, wherein the cancer is selected from the group consisting of hematological cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, esophageal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, glioblastoma, leukemia, lymphoma, mantle cell lymphoma, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer, optionally, wherein the cancer is a hematological cancer of lymphoid origin expressing a CD 19 surface antigen, optionally, wherein the cancer is an acute lymphoblastic leukemia (ALL), a mature B-cell lymphoma, a chronic lymphocytic leukemia (CLL), a diffuse large B-cell lymphoma (DL
  • the instant invention provides methods of treating a disease in a subject.
  • the instant invention provides methods of treating a cancer in a subject, comprising administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC- MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a T-cell ligand (e.g., a T-cell activating ligand or a T-cell inhibiting ligand), wherein the subject was administered a population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby enhancing the systemic anti-tumor activity of the administered population of engineered T-cells in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the APC- MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MS
  • the instant invention provides methods of enhancing anti-tumor activity of an engineered T-cell therapy in a subject, comprising administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a T-cell ligand (e.g., a T-cell activating ligand or a T-cell inhibiting ligand), wherein the subject was administered an engineered T-cell therapy comprising a population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby enhancing the systemic anti-tumor activity of an engineered T-cell therapy in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MS
  • a subject with a disease may be treated by contacting the administered population of T-cell, e.g., engineered T-cells, with the antigen presenting cell-mimetic scaffold (APC-MS) of the invention, thereby activating, co-stimulating and homeostatically maintaining the population of T-cells; optionally expanding and/or re-stimulating the population of T- cells; and administering the activated, co-stimulated, maintained and optionally expanded T-cells into the subject, thereby treating the disease in the subject.
  • T-cell e.g., engineered T-cells
  • APC-MS antigen presenting cell-mimetic scaffold
  • the T-cell population is contacted with the scaffold for a period, e.g., 0.5 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 38 days, 45 days, 50 days, 60 days, or more, and the cells contained therein are manipulated using one or more of the aforementioned techniques. Examples of manipulation include, for example, activation, division, differentiation, growth, expansion, reprogramming, anergy, quiescence, senescence, apoptosis, death, etc. The cells need not be physically removed from the scaffold to be manipulated.
  • the scaffolds are contacted with the subject’s sample in situ (e.g., by implanting or by injecting the scaffold into the subject).
  • a subject with a disease may be treated by contacting the subject’s sample comprising a T-cell population with the antigen presenting cell-mimetic scaffold (APC-MS) of the invention, thereby activating, co-stimulating and homeostatically maintaining the population of T-cells; optionally expanding the population of T-cells; and administering the activated, costimulated, maintained and optionally expanded T-cells into the subject, thereby treating the disease in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the T-cell population is contacted with the scaffold for a period, e.g., 0.5 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 38 days, 45 days, 50 days, 60 days, or more, and the cells contained therein are manipulated using one or more of the aforementioned techniques. Examples of manipulation include, for example, activation, division, differentiation, growth, expansion, reprogramming, anergy, quiescence, senescence, apoptosis, death, etc.
  • the scaffolds are contacted with the subject’s sample in situ (e.g., by implanting the scaffold into the subject).
  • the cells are manipulated ex situ (e.g., administrating and/or associating the scaffold and the subject’s withdrawn blood sample).
  • the T cells administered to the mammal are about 4 to about 35 days old, whereupon the regression of the disease in the mammal is promoted.
  • the administered T cells are less than about 14 days old, e.g., about 7 to about 21 days old.
  • the inventive methods provide numerous advantages. For example, T cells that are about 4 to about 14 days old are believed to provide improved in vivo proliferation, survival, and activity as compared to T cells that are about 60 days old or older.
  • the period of time required to generate T cells for adoptive cell therapy (ACT) may be shortened from an average of about 44 days to a range of about 4 to about 15 days (or less than about 35 days, e.g., about 7 to about 15 days).
  • inventive methods may advantageously administer T cells that are pooled from bulk cultures instead of those derived from microcultures.
  • T cells that could be falsely predicted to be unreactive in vivo by in vitro testing of specific antigen reactivity.
  • T cell cultures generated from a single tumor specimen have diverse specific reactivities
  • the lack of in vitro antigen reactivity testing advantageously avoids having to choose only a few T cell cultures to expand, and therefore provides a more diverse repertoire of tumor reactivities to be administered to the patient.
  • T cells that are about 4 to about 30 days old also contain a greater diversity of cells and a higher frequency of active/healthy cells than T cells.
  • one or more aspects (e.g., but not limited to, culturing and/or expanding) of the inventive methods may be automatable.
  • An embodiment of the method comprises culturing autologous T cells.
  • Tumor samples are obtained from patients and a single cell suspension is obtained.
  • the single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a GENTLEMACSTM Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase).
  • Single -cell suspensions of tumor enzymatic digests are cultured in scaffolds or scaffolds of the invention.
  • the cells are cultured until confluence (e.g., about 2xl0 6 lymphocytes), e.g., from about 2 to about 21 days, preferably from about 4 to about 14 days.
  • the cells may be cultured from 5 days, 5.5 days, or 5.8 days, 6.0 days, 6.5 days, 7.0 days to 21 days, 21.5 days, or 21.8 days, preferably from 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days, or 14.8 days.
  • An embodiment of the method comprises expanding cultured T cells.
  • the cultured T cells are pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T-cells of at least about 10-fold (e.g., 10-, 20-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 7 to about 14 days, preferably about 14 days.
  • rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 7 to about 14 days, preferably about 14 days. Most preferably, rapid expansion provides an increase of at least about 400-fold or greater over a period of about 10 to about 14 days, preferably about 14 days.
  • the cells may undergo initial expansion in the scaffolds, upon which they are subject to rapid expansion. Under this two-step expansion protocol, an increase of about 1000-fold over a period of about 7 to 14 days may be achieved.
  • T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin- 15 (IL- 15), with IL -2 being preferred.
  • the non-specific T-cell receptor stimulus can include around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.).
  • T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 pM MART-1 :26-35 (27 L) or gpl00:209-217 (210M), in the presence of a T-cell growth factor, such as 300 lU/ml IL-2 or IL- 15, with IL-2 being preferred.
  • a vector such as an human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 pM MART-1 :26-35 (27 L) or gpl00:209-217 (210M)
  • a T-cell growth factor such as 300 lU/ml IL-2 or IL- 15, with
  • the in vztro-induced T-cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.
  • the T-cells can be restimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL -2, for example.
  • An embodiment of the method comprises administering to the subject, the expanded T cells, wherein the T cells administered to the mammal are about 4 to about 35 days old.
  • the administered cells may be 6, 7, or 8 to 14, 15, or 16 days old.
  • the T cells administered to the mammal are about 4 to about 29 or about 7 to about 15 days old, or about 10 days old.
  • the T cells that are administered to the mammal according to an embodiment of the invention are “young” T cells, i.e., minimally cultured T cells.
  • Young T cell cultures that are administered to the mammal in accordance with an embodiment of the invention advantageously have features associated with in vivo persistence, proliferation, and antitumor activity.
  • young T cell cultures have a higher expression of CD27 and/or CD28 than T cells that are about 44 days old.
  • CD27 and CD28 are associated with proliferation, in vivo persistence, and a less differentiated state of T cells (the increased differentiation of T cells is believed to negatively affect the capacity of T cells to function in vivo).
  • T cells expressing higher levels of CD27 are believed to have better antitumor activity than CD27-low cells.
  • young T cell cultures have a higher frequency of CD4+ cells than T cells that are about 44 days old.
  • young T cell cultures have a mean telomere length that is longer than that of T cells that are about 44 days old. Without being bound to a particular theory, it is believed that T cells lose an estimated telomere length of 0.8 kb per week in culture, and that young T cell cultures have telomeres that are about 1.4 kb longer than T cells that are about 44 days old. Without being bound to a particular theory, it is believed that longer telomere lengths are associated with positive objective clinical responses in patients, and persistence of the cells in vivo.
  • the T-cells can be administered by any suitable route as known in the art.
  • the T- cells are administered as an intra-arterial or intravenous infusion, which preferably lasts about 30 to about 60 minutes.
  • routes of administration include subcutaneous, intraperitoneal, intrathecal and intralymphatic.
  • embodiments of the instant invention provide for various modes of administering the therapeutic compositions comprising the expanded cells.
  • the expanded cells are first purified and then administered into a subject.
  • the expanded cells may be mixed with the scaffolds of the invention prior to administration into the subject.
  • the scaffolds may continue to stimulate cells in vivo and may also function to selectively manipulate target whole blood cells in the in vivo setting.
  • the therapeutic methods of the invention may involve re-stimulating the population of T-cells prior to the administration step.
  • the re-stimulation step may be carried out using art-known techniques.
  • the re-stimulation step is carried out by re-administering and/or reassociating the cells with the scaffold composition.
  • re-stimulation is carried out by addition of phorbol 12-myristate 13-acetate (PMA, 10 ng/ml, Sigma-Aldrich, Inc.), ionomycin (0.5 pg/ml, Sigma-Aldrich, Inc.) and Brefeldin A (eBiosciences, Inc.).
  • the re-stimulation step is carried out by including an antigen (e.g., a pathogenic antigen or a cancer antigen) in the scaffold or extrinsically in the culture.
  • the therapeutic methods are conducted by manipulating T-cells that are obtained from a blood sample, a bone marrow sample, a lymphatic sample or a splenic sample of a subject.
  • embodiments of the instant invention provide for methods for treating hematological malignancies, such as hematological cancer, in a subject.
  • hematological malignancies such as hematological cancer
  • Such cancers can arise from the malignant transformation of either a hematopoietic precursor in the bone marrow or a mature cell of hematopoietic origin in the blood.
  • the former subtype called leukemias
  • leukemias are classified based on their myeloid or lymphoid origin, and their acute or chronic stage.
  • lymphomas the transformed cell is a mature immune cell, of B, T, or NK cell origin, with B-cell dyscrasias being the most common subtypes.
  • CAR-T cells may be used in the treatment of various hematological cancers, including those of lymphoid origin expressing the CD19 surface antigen, which can include pediatric and adult acute lymphoblastic leukemia (ALL), and mature B-cell lymphomas, such as chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and follicle cell lymphoma (FCL).
  • ALL pediatric and adult acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • FCL follicle cell lymphoma
  • inventions provide for methods for treating cancer in a subject.
  • the method comprises contacting the subject’s sample comprising a T-cell population with the antigen presenting cell-mimetic scaffold (APC-MS) of the invention, thereby activating, costimulating and homeostatically maintaining the population of T-cells; optionally expanding the population of T-cells; and administering the activated, co-stimulated, maintained and optionally expanded T-cells into the subject, thereby treating the cancer in the subject.
  • the scaffolds may be provided with a cancer antigen.
  • the cancer antigen is presented, e.g., for recognition by T-cells, in an MHC molecule or a fragment thereof. In certain instances, whole cell products may be provided.
  • the method of treating a cancer in a subject comprises administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; and a functional molecule selected from the group consisting of a T-cell ligand, a T-cell co-stimulatory molecule, a T-cell homeostatic agent, and a combination thereof, wherein the subject is administered a population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby enhancing the systemic anti-tumor activity of the administered population of engineered T-cells in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid
  • the method of method of enhancing anti-tumor activity of an engineered T-cell therapy in a subject comprises administering to the subject an antigen presenting cell-mimetic scaffold (APC-MS), wherein the APC-MS comprises a base layer comprising high surface area mesoporous silica micro-rods (MSR); a fluid supported lipid bilayer (SLB) layered on the base layer; a functional molecule selected from the group consisting of a T-cell ligand, a T-cell co-stimulatory molecule, a T-cell homeostatic agent, and a combination thereof, wherein the subject is administered an engineered T-cell therapy comprising a population of engineered T-cells prior to, simultaneous with, or after administration of the APC-MS, thereby enhancing the systemic anti-tumor activity of an engineered T-cell therapy in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the APC-MS comprises a base layer comprising high surface area mesop
  • cancer antigens include, but are not limited to, MAGE-1, MAGE- 2, MAGE-3, CEA, Tyrosinase, midkin, BAGE, CASP-8, P-catenin, - catenin, y-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gplOO, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf 112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L, CYNL2, TPR, SOX2, GOL
  • the cancer antigen is a neoantigen identified in a patient.
  • a neoantigenic determinant is an epitope on a neoantigen, which is a newly formed antigen that has not been previously recognized by the immune system.
  • Neoantigens are often associated with tumor antigens and are found in oncogenic cells.
  • Neoantigens and, by extension, neoantigenic determinants can be formed when a protein undergoes further modification within a biochemical pathway such as glycosylation, phosphorylation or proteolysis, leading to the generation of new epitopes.
  • These epitopes can be recognized by separate, specific antibodies. See, Schumacher et al., Science 348 (6230): 69-74, 2015.
  • the neoantigen may be detected in a patient-specific manner. Methods for detecting neoantigens from a patient sample, e.g., blood sample, are described in US 9,115,402, which is incorporated by reference herein,
  • the neoantigen is a peptide derived from SF3B1, MYD88, TP53, ATM, Abl, A FBXW7, a DDX3X, MAPK1, GNB1, CDK4, MUM1, CTNNB1, CDC27, TRAPPCI, TPI, ASCC3, HHAT, FN1, OS-9, PTPRK, CDKN2A, HLA-A11, GAS7, GAPDH, SIRT2, GPNMB, SNRP116, RBAF600, SNRPD1, Prdx5, CLPP, PPP1R3B, EF2, ACTN4, MEI, NF-YC, HLA-A2, HSP70-2, KIAA1440, CASP
  • Embodiments of the instant invention provide for methods for treating acute lymphoblastic leukemia in a subject.
  • Acute lymphoblastic leukemia is the most common pediatric malignancy (i.e., nearly 1 out of every 3 cancers; WHO), with relative good and often curative outcomes following intensive therapy. ALL can also occur in adults, although in such cases, the probability of a curative outcome is greatly reduced. ALL arises from B-cell precursors - immune cells responsible for antibody production and humoral immunity. Specific, well -documented genetic abnormalities halt normal B-cell development, resulting in simultaneous developmental arrest and oncogenic transformation.
  • B-ALL/LBL lymphoblastic leukemia
  • Treatment for ALL relies primarily on combination chemotherapy.
  • These chemotherapy regimens have evolved into complex schemes that employ numerous agents in various doses, combinations, and treatment schedules. With these regimens, >80% of treated patients achieve sustained complete remissions.
  • r/r refractory/relapsing
  • r/r refractory/relapsing
  • HSCT allogeneic bone marrow transplants
  • AML acute myeloid leukemia
  • Tumor cells that persistent due to incomplete eradication are often resistant to conventional therapies, emphasizing the importance of highly-effective and safe treatments that can completely eliminate the primary tumor to minimize the risk for development of therapy-resistant secondary tumors.
  • Embodiments of the instant invention provide for methods for treating chronic lymphocytic leukemia in a subject.
  • Chronic lymphocytic leukemia is a chronic, lymphoproliferative neoplasm characterized by the progressive accumulation of typically monoclonal, and functionally-incompetent B cells in the peripheral blood, lymph nodes, and spleen. CLL occurs later in life and is generally less aggressive than ALL.
  • the pathogenesis of CLL/SLL small lymphocytic lymphoma
  • CLL/SLL small lymphocytic lymphoma
  • CLL leukemogenesis could be conceptualized as a series of cancer transforming events that accumulate over time, resulting in structural changes in B-cell regulatory genes or epigenetic changes in the regulation of structural genes. These pre-leukemic cells are nurtured by signals in their microenvironment. Specific genetic alterations have been described, with certain CLL alteration profiles being associated with more aggressive disease and subsequently, lower median survival.
  • the presentation and diagnosis of CLL is similar to ALL, but highly variable; ranging from asymptomatic disease that never require therapy (i.e., approximately 1/3 of patients) to active disease that can result in symptoms similar to ALL.
  • Standard treatment of CLL involves chemo-immunotherapy; the use of various chemotherapeutic agents in combination with anti-CD20 antibodies.
  • new agents such as kinase inhibitors and BCL-2 inhibitors
  • BTK B-cell receptor signaling kinases
  • BCL-2 inhibitors such as Venetoclax
  • T-cell immunotherapies are actively being pursued to treat ALL and CLL due to their multiple targetable tumor-associated antigens (TAAs) expressed only on B-cells, including CD19, CD20, CD22, CD23, CD40, and others.
  • TAAs tumor-associated antigens
  • Monoclonal antibody therapies which are highly specific for a tumor antigen, but lack cell-mediated effector responses, can be adapted into chimeric antigen receptors (CARs).
  • CARs link the single chain antibody domain responsible for tumor antigen recognition and intracellular T-cell activation domains, resulting in potent and durable T-cell immunotherapy.
  • cytotoxic T-cell-specific ligands and cytotoxic T- cell-specific co-stimulatory molecules optionally together with one or more additional agents that confer activation, division, differentiation, growth, expansion, or reprogramming of cytotoxic T cells.
  • additional agents that confer activation, division, differentiation, growth, expansion, or reprogramming of cytotoxic T cells. Representative examples of such molecules and agents have been provided above.
  • the sequestered and/or isolated cells may be genetically modified.
  • the effector cells are genetically modified to express a chimeric antigen receptor (CAR) specific for CD 19 (CD 19 CAR-T cells).
  • CAR chimeric antigen receptor
  • This particular type of T-cells has produced a high rate of complete remission (CR) in adult and pediatric patients with relapsed and refractory B cell acute lymphoblastic leukemia (B-ALL) in small phase I clinical trials. See, Turtle et al. J Clin Invest., 126, 2123-38, 2016) and the references cited therein.
  • the invention relates to methods for further formulating T-cell compositions for cancer therapy, comprising, further genetically modifying the T-cells obtained from the scaffolds.
  • the genetic modification may be mediated ex situ or in situ.
  • any technique may be used to genetically modify T-cells, including, but not relating to, using viral vectors, plasmids, transposon/transposase systems, shRNA, siRNA, antisense RNA, and the like.
  • the T-cell has been genetically-modified using a gene editing system (e.g., a CRISPR/Cas9 system).
  • the isolated T-cells are genetically modified using a viral delivery system.
  • the isolated T-cells are genetically modified using a lentiviral system.
  • the isolated T-cells are genetically modified using a retroviral system.
  • the isolated T-cells are genetically modified using an adenoviral system.
  • the isolated T-cells are contacted with an agent that promotes interaction with the viral delivery system or viral sequestration (e.g., an agent that promotes receptor-mediated interactions with the viral delivery system or agents that promote electrostatic interactions with the viral delivery system).
  • the isolated T-cells are genetically modified using a viral delivery system in situ.
  • the scaffold may comprise an agent that promotes viral sequestration.
  • the agent(s) that promote viral sequestration may be present on the surface of the lipid bilayer of the MSR-SLB either through adsorption or by attachment to a lipid headgroup.
  • the agent that promotes viral sequestration is a fibronectin peptide, such as RetroNectin®.
  • the agent that promotes viral sequestration is an amphipathic peptide, such as Vectofusin-1®.
  • the scaffold may further comprise one or more T-cell ligands, one or more T-cell co-stimulatory molecules and/or one or more T-cell homeostatic agents.
  • T-cell ligands one or more T-cell co-stimulatory molecules and/or one or more T-cell homeostatic agents.
  • the scaffold may facilitate the activation and expansion of T-cells which may lead to cell clustering and allow for a viral delivery system to be in close proximity with the T-cells thereby promoting more efficient transduction of the cells.
  • the one or more T-cell ligands, one or more T-cell co-stimulatory molecules and/or one or more T-cell homeostatic agents present on the scaffold may be selected to result in the desired T-cell phenotype which may enhance the therapeutic efficacy of the resulting T-cell (see, e.g., Sommermeyer et al, Leukemia 30(2): 492-500 (2016)).
  • the isolated T-cells are genetically modified to express a chimeric antigen receptor (CAR).
  • CD4+ and CD8+ T cells are lentivirally transduced to express the CD 19 CAR and a truncated human epidermal growth factor receptor (EGFRt) that enables identification of transduced cells by flow cytometry using the anti-EGFR monoclonal antibody cetuximab.
  • EGFRt-i- CD4+ and CD8+ T cells are enriched during culture by a single stimulation with irradiated CD 19+ lymphoblastoid cell line (LCL).
  • the median frequency of EGFRt+ CAR-T cells within the CD3+CD4+ and CD3+CD8+ subsets in the products at release for infusion, which confers good therapeutic outcome, is about 80% (range 50.0%-95.9%) and about 85% (range 13.0%— 95.6%), respectively. See, Turtle et al. (J Clin Invest., 126, 2123-38, 2016).
  • the genetically modified T-cells may be further expanded by administrating and/or associating the T-cell product with the scaffolds of the invention.
  • scaffolds containing CAR T-cell-specific antigens e.g., CD19, CD22 or a fragment thereof or a variant thereof, may be employed to selectively expand the desired CAR T-cells.
  • the scaffolds are provided with products that are useful in practicing the cancer therapy methods.
  • Representative examples include, for example, hybridomas of B-cells, stable lineages of T-cells, antibodies derived from B-cells or hybridomas thereof, receptors which bind to the cancer antigens (receptors which bind to MHC molecules presenting the antigens), including fragments thereof, nucleic acids encoding the receptors or antigen-binding domains thereof, nucleic acids encoding antibodies, including whole cells.
  • Embodiments of the instant invention provide for methods for treating an immunodeficiency disorder in a subject comprising contacting the subject’s sample comprising a T- cell population with the antigen presenting cell-mimetic scaffold (APC-MS) of the invention, thereby activating, co-stimulating and homeostatically maintaining the population of T-cells; optionally expanding the population of T-cells; and administering the activated, co-stimulated, maintained and optionally expanded T-cells into the subject, thereby treating the immunodeficiency disorder in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • an immunodeficiency disorder selected from the group consisting of primary immunodeficiency disorder and acquired immunodeficiency disorder, comprising contacting the subject’s sample comprising a T-cell population with the APC-MS of the invention, thereby activating, co-stimulating and homeostatically maintaining the population of T-cells; optionally expanding the population of T-cells; and administering the activated, co-stimulated, maintained and optionally expanded T-cells into the subject, thereby treating the immunodeficiency disorder in the subject.
  • the immunodeficiency disorder may be an acquired immunodeficiency disorder, e.g., acquired immunodeficiency syndrome (AIDS) or a hereditary disorder, e.g., DiGeorge syndrome (DGS), chromosomal breakage syndrome (CBS), ataxia telangiectasia (AT) and Wiskott-Aldrich syndrome (WAS), or a combination thereof.
  • AIDS acquired immunodeficiency syndrome
  • a hereditary disorder e.g., DiGeorge syndrome (DGS), chromosomal breakage syndrome (CBS), ataxia telangiectasia (AT) and Wiskott-Aldrich syndrome (WAS), or a combination thereof.
  • DFS DiGeorge syndrome
  • CBS chromosomal breakage syndrome
  • AT ataxia telangiectasia
  • WAS Wiskott-Aldrich syndrome
  • helper T-cell-specific ligands and helper T-cell-specific co-stimulatory molecules optionally together with one or more additional agents that confer activation, division, differentiation, growth, expansion, or reprogramming of helper T cells.
  • additional agents that confer activation, division, differentiation, growth, expansion, or reprogramming of helper T cells. Representative examples of such molecules and agents have been provided above.
  • Embodiments of the instant invention provide for methods for treating a pathogenic disease in a subject comprising contacting the subject’s sample comprising a T-cell population with the antigen presenting cell-mimetic scaffold (APC-MS) of the invention, thereby activating, costimulating and homeostatically maintaining the population of T-cells; optionally expanding the population of T-cells; and administering the activated, co-stimulated, maintained and optionally expanded T-cells into the subject, thereby treating the pathogenic disease in the subject.
  • the immune cells or compositions derived from the manipulation step may be administered prophylactically, e.g., before the onset of the disease symptoms in the subject.
  • Pathogenic diseases that may be treated in accordance with the aforementioned embodiment include, bacterial diseases, viral diseases, fungal diseases, or a combination thereof.
  • Embodiments of the instant invention provide for methods for treating an autoimmune disease in a subject.
  • the method comprises contacting the subject’s sample comprising a T-cell population with the antigen presenting cell-mimetic scaffold (APC-MS) of the invention, thereby activating, co-stimulating and homeostatically maintaining the population of T-cells; optionally expanding the population of T-cells; and administering the activated, co-stimulated, maintained and optionally expanded T-cells into the subject, thereby treating the autoimmune disease in the subject.
  • APC-MS antigen presenting cell-mimetic scaffold
  • the immune cells are T-cells.
  • regulators of immune cells e.g., regulatory T cells or suppressor T cells, may be administered.
  • the scaffolds/devices may be fabricated for the manipulation of Ts/Treg cell sub-populations, which, are then administered into subjects.
  • the invention provides for a method for treating an autoimmune disease by administering to subject in need thereof, the scaffold of the invention, wherein the plurality of antigens in the scaffold are specific for the autoimmune disease, collecting a plurality of regulatory or suppressor T-cells in the scaffold/device, wherein the plurality of regulatory or suppressor T-cells are specific to the autoimmune antigens, and administering the plurality of regulatory T-cells or suppressor T-cells or products derived therefrom into the subject, thereby treating the autoimmune disease.
  • Cell products that are useful in practicing the therapy of autoimmune diseases include, for example, antibodies and receptors which bind to autoreactive cells, regulatory proteins located in suppressor or regulatory T-cells, including nucleic acid sequences which encode such molecules.
  • cells may be formulated at total cell concentrations including from about 5xl0 2 cells/ml to about IxlO 9 cells/ml.
  • Preferred doses of T cells range from about 2xl0 6 cells to about 9xl0 7 cells.
  • Embodiments of the instant invention further relate to therapy of diseases by administering one or more of the aforementioned compositions.
  • the composition may be a pharmaceutical composition, which is administered by any means that achieve their intended purpose.
  • administration may be by parenteral, subcutaneous, intravenous, intraarterial, intradermal, intramuscular, intraperitoneal, transdermal, transmucosal, intracerebral, intrathecal, or intraventricular routes.
  • administration may be by the oral route.
  • the pharmaceutical compositions may be administered parenterally by bolus injection or by gradual perfusion over time.
  • the dosage administered may be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the dose ranges for the administration of the pharmaceutical compositions may be large enough to produce the desired effect, whereby, for example, autoreactive T cells are depleted and/or the autoimmune disease is significantly prevented, suppressed, or treated.
  • the doses may not be so large as to cause adverse side effects, such as unwanted cross reactions, generalized immunosuppression, anaphylactic reactions and the like.
  • Embodiments described herein further relate to methods for detecting or diagnosing a disease or a disorder in a subject. Any disease or disorder may be detected or diagnosed using the aforementioned methods.
  • the disease is an autoimmune disease selected from the group consisting of rheumatoid arthritis , lupus, celiac disease, inflammatory bowel disease or Crohn’s disease, sjdgren’s syndrome polymyalgia rheumatic, multiple sclerosis, ankylosing spondylitis, Type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, etc.
  • the disease is a cancer which is selected from the group consisting of head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, esophageal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, glioblastoma, leukemia, lymphoma, mantle cell lymphoma, pre -neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.
  • Pathogenic diseases that may be diagnosed in accordance with the aforementioned embodiment include, bacterial diseases, viral diseases, fungal diseases, or a combination thereof.
  • a subject with a disease may be diagnosed by first contacting a subject’s sample containing the immune cell of interest with a scaffold of the invention, wherein the antigens in the scaffold are specific to the disease.
  • the sample contains T- cells and the scaffold/device is contacted with the sample for a period, e.g., 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, or more, and the cells in the scaffold are analyzed using one or more of the aforementioned techniques.
  • the cells that are analyzed may include activated T-cells.
  • the cells that are analyzed my include tumor-antigen specific T-cells.
  • the cells that are analyzed may include T-cells which specifically eliminate the pathogens (e.g., by analyzing Thl cells in case of intracellular pathogens and Th2 cells in case of extracellular pathogens).
  • the subject is an animal, preferably a mammal or a bird. Particularly preferably, the subject is selected from the group consisting of humans, dogs, cats, pigs, cows, buffalo and horses. Most preferably, the subject is a human. Any immune cell may be used in the diagnosis of the disease or disorder. Preferably, diagnosis is performed with a lymphocyte, e.g., T-cells.
  • lymphocyte e.g., T-cells.
  • the analytical step may be carried out using any routine methods. Accordingly, in one embodiment, the analytical step may involve determining the number of immune cells that are specific to the autoimmune disease. Any routine technique may be used to determine antigen-binding specificity of immune cells, e.g., loading cell samples onto antigen-coated surfaces, washing away non-specifically bound cells, and quantitating the number of antigen-specific cells (either in free form by releasing the bound cells or in bound form) using a detection agent e.g., an antibody that binds to a cell-surface epitope located on the antigen-specific cells).
  • the analytical step may involve determining the physical or biological characteristics of the antigen-specific immune cells. Examples of physical characteristics include, for example, size, shape, reflectivity, morphology, density. Examples of biological characteristics include, for example, expression of particular cell surface markers, secretion of cytokines, reactivity to particular antigens or agents, patterns of gene expression.
  • the analytical step may be tied to a correlation step, wherein, the results of the analytical step are correlated to the parameter of interest.
  • Representative types of parameters include, presence (or absence of disease), type of disease (e.g., aggressive vs. non-aggressive autoimmune disorder; druggable vs. non-druggable disease, e.g., antibiotic susceptible vs. antibiotic resistant bacterial infection, immunotherapy-resistant vs. immunotherapy-sensitive cancer), stage of disease, progression/regression of disease (over time), etc.
  • the parameter relates to presence or absence of disease (which can be expressed in binary terms).
  • the parameter relates to staging of disease (which can be expressed in a nominal scale, e.g., stage I-IV, with stage IV being the highest). Yet in another embodiment, the parameter relates to odds or likelihood of occurrence of the disease, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 2-fold, 3 -fold, 5 -fold, 10-fold, 20-fold or more.
  • the parameters may be compared to a baseline value.
  • the baseline value may be a value that is pre-determined, e.g., in a population of healthy subjects.
  • RA rheumatoid arthritis
  • a baseline level of RA-specific antibodies (or T-cells) in healthy subjects may be used in the correlation step.
  • the baseline value may be experimentally identified using suitable controls. The skilled worker can use routine techniques to correlate and/or draw inferences between various subject groups.
  • embodiments of the invention relate to detecting or diagnosing autoimmune disease, cancer, or a pathogenic disease in a subject by contacting a subject’s sample with the scaffolds of the invention containing antigens which are specific to the autoimmune disease, cancer disease, or pathogenic disease, and analyzing the immune cells contained therein.
  • the contacting step may be performed in vivo (e.g., by implanting the scaffold in a subject) .
  • the analytical step may be performed by first removing the immune cells from the scaffolds using routine techniques, i.e. , via ex situ analysis. For instance, mild detergents and enzymes may be used to dislodge the cells from the scaffolds.
  • the detection/analytical steps may be carried out without removing the cells from the scaffolds, i.e. , via in situ analysis.
  • the method comprises contacting a subject’s sample with the scaffolds of the invention containing antigens that are specific to the disease and analyzing the immune cells contained therein.
  • the number/types of immune cells contained in the device may offer valuable cues as to the progression of the disease.
  • analogous methods may be used to monitor the therapy of disease and/or disease management.
  • the aforementioned methods may be used to monitor the progression/therapy of autoimmune disorders, cancers, pathogenic diseases, and the like.
  • the immune cells that are used in the diagnostic methods are T-cells.
  • the progression of the disease may be monitored by analyzing the number and/or type of autoreactive T cells.
  • methods of intervention and/therapy may be designed to minimize the severity of the symptoms.
  • preventive methods may be undertaken, including providing recommendations to subjects on dietary, nutritional and/or other lifestyle changes.
  • Embodiments described herein further relate to methods for devising and producing novel compositions for treating a disease.
  • the method comprises administering the scaffolds of the invention containing disease specific antigens to a subject, which then manipulate immune cells that are specific to the disease, optionally isolating, enriching, and expanding the immune cells manipulated in the device, and then administering the immune cells back to the subject.
  • products derived from such immune cells may be administered to the subjects. Examples of products derived from the immune cells include, nucleic acids (including vectors and cells containing such nucleic acids), peptides, proteins, antibodies, cytokines, etc.
  • the disease is an autoimmune disease.
  • autoreactive T cells which have been isolated (and optionally expanded in culture as described herein) by the aforementioned methods may be inactivated in situ or ex situ. Methods of inactivating T cells are known in the art. Examples include, but not limited to, chemical inactivation or irradiation.
  • the autoreactive T cells may be preserved either before or after inactivation using a number of techniques known to those skilled in the art including, but not limited to, cryopreservation.
  • the composition may be used as a vaccine to deplete autoreactive T cells in autoimmune patients.
  • Embodiments described herein further relate to compositions and vaccines produced by the aforementioned methods.
  • the composition may be a pharmaceutical composition, which may be produced using methods well known in the art.
  • Pharmaceutical compositions used as preclinical and clinical therapeutics in the treatment of disease or disorders may be produced by those of skill, employing accepted principles of diagnosis and treatment.
  • the vaccine may comprise autoreactive T cells comprising homogeneous (“monoclonal”) or heterogeneous (“polyclonal”) patterns of VP-DP-jp gene usage.
  • autoreactive T cells comprising homogeneous (“monoclonal”) or heterogeneous (“polyclonal”) patterns of VP-DP-jp gene usage.
  • Clinical studies indicate that autoimmune patients receiving autologous monoclonal T cell vaccination may show a gradual decline in the immunity against autoreactive T cells. In some cases, the reappearing autoreactive T cells may originate from different clonal populations, suggesting that the T cells may undergo clonal shift or epitope spreading potentially associated with the ongoing disease process. Clonal shift or epitope spreading may be a problem in autoimmune diseases mediated by autoreactive T cells.
  • a vaccine comprising polyclonal autoreactive T cells capable of depleting multiple populations of autoreactive T cells may avoid problems with clonal shift or epitope spreading.
  • the compositions/vaccines of the invention containing desired T-cells may be provided with a pharmaceutically acceptable carrier. Lyophilized preparations of T-cells may be provided as well. IV. Kits/De vices
  • kits comprising, in one or separate compartments, the scaffolds of the instant invention.
  • the kits may further comprise additional ingredients.
  • the kits may optionally comprise instructions for formulating the scaffolds for diagnostic or therapeutic applications.
  • the kits may also comprise instructions for using the kit components, either individually or together, in the therapy or diagnosis of various disorders and/or diseases.
  • kits comprising the scaffolds of the invention along with reagents for selecting, culturing, expanding, sustaining, and/or transplanting the manipulated cells of interest.
  • Representative examples of cell selection kits, culture kits, expansion kits, transplantation kits for T-cells, B-cells and antigen presenting cells are known in the art.
  • EXAMPLE 1 Local in vivo stimulation of therapeutic T cells to enhance systemic anti-tumor responses.
  • This example describes the application of the system described herein as a subcutaneously injectable scaffold microenvironment to directly manipulate and “boost” T-cell responses in vivo.
  • T cells genetically engineered to express chimeric antigen receptors have demonstrated highly efficacious outcomes in patients with B-cell malignancies 6,84 .
  • CARs chimeric antigen receptors
  • Several factors, including poor T-cell persistence and aggravated T-cell exhaustion have been implicated in reduced clinical benefit.
  • various genetic tools are being used to develop next generation therapies to improve antigen recognition and signaling 74,246 , maintain sternness 247,248 , and minimize exhaustion 249 , there remain limited cell-extrinsic tools to manipulate CAR-T cell activity, particularly after they are infused.
  • cell-extrinsic approaches are subject to fewer manufacturing limitations and could result in fewer T cells of lower toxicity as these approaches do not involve extensive modification of homeostatic T-cell signaling networks.
  • cell-extrinsic approaches have been previously developed - these strategies are broadly based on vaccination, which has been used to create de novo T-cell responses or boost pre-existing T-cell immunity in the context of infectious disease and cancer.
  • exogenous CAR-T cell responses can be improved by targeting the CAR antigen to lymphoid organs via CAR antigen- conjugated amphiphile ligands 96 or CAR antigen-encoding RNA nanovaccines 97 .
  • APCs antigen-presenting cells
  • T cells 138,250 T cells 138,250 .
  • whole body distribution and presentation of a CAR antigen by APCs in major lymphoid organs could result in unpredictable and uncontrollable toxicity in the clinical setting.
  • CAR adoptive chimeric antigen receptor
  • This study shows that subcutaneously injected materials, which form a scaffold microenvironment for local T-cell simulation, boosts the systemic anti-tumor activity of prior administered CAR T-cells.
  • These scaffolds emulate key features of physiological T cell activation and enable the continuous infiltration and stimulation of specific T-cell subpopulations.
  • Presentation of T-cell activating ligands and costimulatory molecules drives selective T-cell expansion, differentiation, and cytotoxic function, depending on the dose, type, and context in which the ligands are delivered.
  • animals administered scaffolds following subtherapeutic CAR T-cell dosing promotes improved anti-tumor responses accompanied by increased therapeutic cell output and persistent, memory-like differentiation profiles.
  • This example utilizes antigen-presenting cell mimetic scaffold (APC-ms) to create a subcutaneously injectable material scaffold for local stimulation of T cells in vzvo 145,208 .
  • APC-ms antigen-presenting cell mimetic scaffold
  • MSRs biodegradable mesoporous silica rods
  • FIG. 2A biodegradable mesoporous silica rods
  • POPC palmitoyl-2- oleoyl-glycero-3- phosphocholine
  • T-cell activating ligands By attaching T-cell activating ligands via biotin-streptavidin intermediates, these materials present ligands for T-cell activation via a fluid lipid bilayer while slowly releasing mitogenic factors to interacting T cells, mimicking key features of antigen presentation, including physical TCR rearrangement and paracrine signaling (FIG. 2B).
  • 2 nd generation anti-CD19 CAR-T i.e., FMC63 anti-CD19 scFv-CD8a- 41BB- CD3Q cells were generated as model cells to investigate the restimulation potential of the materials.
  • CD19.BBz CAR-T cells were restimulated with various APC-ms presenting polyclonal anti-CD3/anti-CD28 across a range of mol% and anti-CD3:anti-CD28 ratios (FIG. 1, FIGS. 2A-2B, FIGS. 6A-6C).
  • IL -2 was supplemented into the culture media.
  • CAR-T cells interacted robustly with the materials and physically rearranged them in culture (FIGS. 2B-2C, FIG. 6A).
  • Materials presenting 0.1 mol% anti-CD3/anti-CD28 at a 1:1 ratio promoted robust T-cell restimulation and expansion without any substantial loss in CAR+ T-cell frequency (FIGS. 2E-2D, FIGS. 6B-6C).
  • the anti-CD3/anti-CD28 restimulated CAR-T cells had similar CD4-to-CD8 ratios, but exhibited elevated expression of CD25 in both CD4+ and CD8+ T cells (FIG. 2F). Moreover, these cells were relatively more CD45RA CCR7 , suggesting an effector-memory phenotype (Fig. 2G). These cells responded robustly to tumor antigen stimulation provided by CD19-expressing Raji cells and elicited potent cell killing (FIGS. 2H-2I). Mock- stimulated CAR-T cells represent cells cultured with 100 lU/mL human IL-2 alone.
  • Subcutaneously-injected material scaffolds are biodegradable and biofunctional
  • the scaffolds were well-vascularized, retained the Cy5-lipid signal (FIG. 3D) and lipid bilayer fluidity as measured by fluorescence recovery after photobleaching (FIG. 3E).
  • FIG. 3D Cy5-lipid signal
  • FIG. 3E fluorescence recovery after photobleaching
  • material scaffolds were prepared with anti-CD3/anti-CD28 ligands were subcutaneously administered into mice and excised 7, 14, and 21 days thereafter, and cultured ex vivo with CFSE-labeled CAR-T cells in the absence of IL-2 to eliminate the effects of cytokine -driven proliferation.
  • 21 days only 66.6% of subcutaneous scaffolds were detected (FIGS. 4A-4F).
  • CFSE and CD25 expression were observed, suggesting that the subcutaneous scaffolds are capable of restimulating CAR-T cells for at least 7 days following administration.
  • Subcutaneous scaffolds facilitate local T-cell infiltration, activation, and egress
  • the T cells that remained in scaffolds containing surface i.e., anti-CD3/anti-CD28
  • soluble ligands i.e., IL -2
  • CFSE staining compared to cells harvested from scaffolds containing no ligands, suggesting that the scaffolds activated and expanded the T-cells (FIGS.5A-5B, FIG. 9C).
  • this example next evaluated the ability of T cells to infiltrate into the scaffolds (FIGS. 5C-5D).
  • Animals were used that had been previously inoculated with CD19-expressing luciferized Raji cells (a well-established xenograft model of Burkitt’s lymphoma), then cured with CD19 CAR-T cells (i.e., tumor free for at least 8 weeks) to establish a baseline frequency of relevant CAR-T cells. Circulating T-cell frequencies were on average around 1% of live cells prior to subcutaneous scaffold injection (range: 0.85-1.85%).
  • CD19 CAR-T cells materials were prepared to present 0.1 mol% human CD 19 protein (pCD19) or an anti-CD19 idiotype antibody (aCD19) designed to activate the FMC63 scFv-CAR specifically (FIGS. 8A-8J).
  • Materials presenting polyclonal 0.1 mol% anti-CD3/anti-CD28 (aCD3/aCD28, duplicated from FIG. 2) are included here for reference.
  • CAR-T cells interacted robustly with the materials, with more robust interactions observed in materials presenting aCD19 as compared to pCD19.
  • FIG. 8A The presence of aCD28 costimulation increased T-cell interactions with the materials, forming large T cell-material clusters (FIG. 8A).
  • CAR-T cells restimulated with materials presenting aCD19/aCD28 promoted robust T-cell expansion, similar to levels with aCD3/aCD28 materials (FIG. 8B).
  • aCD19 or aCD19/aCD28 -stimulated cultures were observed to be enriched in CAR + T cells (FIG. 8C).
  • pCD19 materials failed to strongly promote T-cell expansion or CAR-enrichment, despite promoting similar levels of intracellular IL-2 expression compared to 0.1 mol% aCD19 materials, albeit only when formulated at 0.5 mol%, suggesting a density-dependency requirement for pCD19 but not aCD19 (FIG. 11).
  • This example next characterized the phenotype of CAR-T cells restimulated with aCD19- containing materials in vitro. Restimulation with aCD19 materials produced CAR-T cells with lower CD4-to-CD8 ratios, while aCD19/aCD28 or aCD3/aCD28 material co-cultures increased the CD4-to- CD8 ratio relative to mock (i.e., IL-2) -stimulated cultures (FIG. 8D).
  • aCD19- and aCD3-containing materials strongly biased the T cells towards a CD45RA CCR7 effector memory phenotype
  • mock and pCD19 material cultures contained more CD45RA + CCR7 effector T cells and CCR7 + CD45RA + stem memory cells
  • CAR-T cells cocultured with aCD19- and aCD3 -containing materials expressed higher levels of CD25 relative to mock-stimulated cultures (FIG. 8G).
  • the restimulated cells were co-cultured with CD19-expressing Raji cells at various effector-to-target ratios (FIGS. 8H-8J).
  • CAR-T cells restimulated with aCD19-materials generated cells that responded with the highest level of IFN-y and IL -2 co-expression following Raji cell co-culture (FIGS. 8H-8I).
  • aCD19- and aCD19/aCD28-restimulated CAR-T cells having a lower proportion of effector CD8+ T cells, which are the predominant T-cell subpopulation responsible for in vitro tumor cell killing they exhibited the greatest cytotoxic activity, suggesting that CAR-specific restimulation can improve the function of CAR-T cells (FIG. 8J).
  • this example showed the application of an injectable, subcutaneous material scaffold based on APC-ms to boost the activity of prior-administered CAR-T cells.
  • polyclonal anti-CD3/anti-CD28 stimulation these materials restimulated CD19 CAR-T cells in vitro to promote their proliferation and differentiation without compromising any cytotoxic functions. Little stimulation was required - CAR-T cells were observed to form large lymphoblasts quickly after 2 days of culture (as compared to 3-4 days in naive T cells), suggesting that residual T- cell signaling from the initial polyclonal T-cell activation may be a supporting component in driving CAR-T cell responses 232 .
  • the individual micro-rods self-assembled into a 3D, macro- porous scaffold microenvironment, consistent with past applications of high-aspect ratio mesoporous silica rods in the cancer vaccine setting 111 1 13 .
  • These subcutaneous scaffolds were permissive to T-cell infiltration, activation, and egress. It was observed that the materials degraded linearly over a period of three weeks; however, the T-cell activating ligands presented on the scaffolds progressively loss their ability to restimulate CAR-T cells after approximately one week. This could represent two separate therapeutic windows, one for the surface-bound T-cell ligands and another for the material- loaded soluble factors.
  • cytokines from MSR materials are dependent on their biodegradation 111 112 189 ’ 211 .
  • pre-loaded IL -2 was released from APC-ms over the course of several days, but the MSRs had degraded completely 145 .
  • the preloaded IL-2 used in these scaffolds were still present in the material following the loss of the surface ligand’ s activating potential. Studies are ongoing to confirm the kinetics of IL -2 release from subcutaneous scaffolds in vivo.
  • CD 19 CAR-T cells receive tumor antigen stimulation from malignant B cells in the blood and lymphoid organs, resulting in massive in vivo proliferation. Since there are no chemoattractants in the subcutaneous scaffolds, T-cell infiltration likely occurred passively, driven by inflammation and leaky blood vessels caused by the MSRs and local tissue damage from the injection itself 252 254 . It was observed that subcutaneous scaffolds delivering surface anti-CD3/anti-CD28 and soluble IL-2 promoted modest increases in the concentration of circulating CAR-T cells compared to scaffolds delivering IL-2 only.
  • a single injection of the material scaffolds was able to improve animal survival in a therapeutic xenograft setting.
  • This example co-delivered the subcutaneous scaffolds with collagen to improve T-cell adhesion but did not observe any differences in animal survival in the same scaffolds without collagen, suggesting that any collagen-dependent changes in T-cell infiltration, activation, and egress kinetics do not impact overall therapeutic outcomes in the high tumor burden setting.
  • subcutaneous scaffolds loaded with IL -2 led to a moderate improvement in animal survival initially, we observed a sharp drop in animal survival starting from day 34. This could be due to the lack of surface ligands and subsequent CD3 A signaling, resulting in decreased CAR-T cell proliferation.
  • CAR-T cells including Treg subsets 255 , which could have contributed to impaired CAR+ CD8+ T-cell responses through different mechanisms 47,255 .
  • Elevated numbers of CAR-T cells were found in scaffolds delivering both surface and soluble ligands which was accompanied by more circulating effector T cells. These observations demonstrated the importance of delivering both surface and soluble activating T-cell ligands to boost CAR-T cell responses. Additionally, the persistence of CAR-T cells in the scaffolds several weeks following subcutaneous injection suggested that CAR-T cells could continuously infiltrate and egress from the scaffolds, which could have important implications in the solid tumor setting.
  • a local subcutaneous scaffold injected close to a tumor lesion could provide more optimal tumor-reactive T-cell stimulation while providing desirable survival and proliferation signals. Studies are ongoing to evaluate this concept.
  • MSR mesoporous silica rod
  • Millipore-Sigma e.g., concentrated hydrochloric acid; Pluronic P123, Mn -5800; tetraethyl orthosilicate, TEOS, 98%). All lipid materials were obtained from Avanti Polar lipids (e.g., 16:0-18:1 PC, 18:1 biotinyl cap PE).
  • Cell culture reagents were obtained from Lonza (e.g., RPMI 1640, X- VIVO 15), ThermoFisher (e.g., HI-FBS, EDTA), Gibco (e.g., penicillin-streptomycin), MilliporeSigma (e.g., bovine serum albumin; BSA), and GeminiBio (e.g., Human serum AB).
  • Recombinant human interleukin-2 (IL-2) was obtained from Biolegend and Akron Biotech and carefully titrated before use where indicated. Streptavidin was obtained from VWR.
  • Ligands for T cell activation were obtained from Biolegend (e.g., biotinylated anti-CD3, anti- CD28). Biotinylated human CD 19 protein and biotinylated anti-CD19 idiotype was obtained from Acrobiosy stems.
  • Human CD3/CD28 T cell expansion Dynabeads were purchased from ThermoFisher Scientific.
  • MSR mesoporous silica rod
  • MilliporeSigma e.g., concentrated hydrochloric acid; Pluronic P123, Mn -5800; tetraethyl orthosilicate, TEOS, 98%).
  • 16:018:1 PC (POPC) and 18:1 biotinyl cap PE were obtained from Avanti Polar Lipids.
  • Cell culture-related reagents were obtained from Lonza (e.g., RPMI 1640, X-VIVO 15), ThermoFisher (e.g., HI-FBS, EDTA), Gibco (e.g., penicillin-streptomycin), MilliporeSigma (e.g., bovine serum albumin; BSA), and GeminiBio (e.g., Human serum AB).
  • Recombinant human IL-2 was obtained from Akron Biotech and titrated before use.
  • Streptavidin was obtained from VWR.
  • Ligands for T cell activation were obtained from Biolegend (i.e., biotinylated anti-CD3, anti-CD28).
  • Human CD3/CD28 T-cell expansion Dynabeads were obtained from ThermoFisher. Fixable blue dead stain was obtained from ThermoFisher.
  • Anti-human antibodies for flow cytometry were obtained from BioLegend: CD3-PerCP/Cy5.5 (HIT3a), CD4-BV510 (SK3), CD8-APC/Fire750 (SKI), PD-1- PE (EH12.2H7), TIM-3-BV421 (F38-2E2), CD25-BV711 (M- A251), CD45RA-PE/Cy7 (HI100), CCR7-APC (G043H7), truncated EGFR-AF488 (AY13), CD95- PE/Dazzle (DX2), CD137-PE/Cy5 (4B4-1), CD3-PE/Dazzle (HIT3a), and CD25-PE/Cy5 (M-A251). Anti-mouse CD45-PerCP/Cy5.5 (30-F11)
  • MSR Mesoporous silica rod
  • High-aspect ratio mesoporous silica rods (MSRs) were synthesized via the sol-gel method by dissolving 4 g Pluronic P123 in 150 g of 0.6 M HC1 solution and stirred with 8.6 g of tetraethylorthosilicate at 40 C for at 20 h. The suspension was transferred to a 100 C oven and aged for 24 hr. The synthesized MSRs were sieved using a 63 pm test sieve (VWR) and calcined at 550 C for 4 h to remove leftover Pluronic, contaminants, and endotoxin.
  • VWR 63 pm test sieve
  • the as-synthesized, dried MSRs were sieved through 63 pm test sieves (VWR), then calcinated at 550 °C for 4 h to remove excess surfactant and contaminants.
  • VWR test sieves
  • the materials were resuspended in cell culture media to 1.5 mg/mL and added to CAR-T cell cultures.
  • materials were prepared under aseptic conditions and used immediately after assembly. b. Material characterization. All MSR materials were analyzed before in vitro T-cell activation.
  • Thermogravimetric analysis was conducted to confirm removal of contaminants and the individual MSRs were processed for sizing via bright field microscopy (EVOS FL Cell Imaging System) and scanning electron microscopy (SEM) to determine rod dimensions and ultrastructure. Nitrogen physisorption (3Flex) was performed to determine the pore size, volume, and surface area of the MSRs. Furthermore, MSRs were also evaluated for their ability to coat liposomes and release IL -2, relevant biological proxies to confirm consistent physical structure between batches. MSRs were further confirmed to be endotoxin free via the Limulus Amebocyte Lysate test (Charles River). c. APC-ms preparation and assembly. Liposomes were prepared as previously described.
  • Lipid films consisting of l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) doped with 0.02 to 0.3 mol% l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (biotinyl-cap-PE) were prepared and dried under nitrogen. The films were rehydrated in IX PBS, incubated for 1 hr with periodic vortexing, and sized (Mini-Extruder, Avanti Polar Lipids). The resulting liposomes were -120 nm in diameter and used within one week.
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • biotinyl-cap-PE biotinyl-cap-PE
  • APC-ms were prepared by mixing monodisperse liposomes with MSRs at a 1:4 w/w (lipid:MSR) ratio for 1 hr at room temperature with gentle periodic mixing.
  • the lipid- coated MSRs were washed and blocked using 0.25% w/v bovine serum albumin.
  • Anti-CD3 (OKT3) and anti-CD28 (CD28.2; Biolegend) were premixed at a 1:1 ratio in appropriate quantities.
  • streptavidin was added prior to the addition of the anti-CD3/anti-CD28 cocktail, and mixed periodically for 7 min.
  • the assembled APC-ms were washed in IX PBS and resuspended in X-VIVO 15 supplemented with 10% FBS and 1% P/S prior to T-cell activation and CART cell production.
  • CAR-T cell production a. Lentivirus construction and production.
  • the 2 nd generation CD 19 CAR construct was composed of the scFv fragment from the FMC63 antibody (GenBank: ADM64594.1) fused to the human CD8a hinge and transmembrane region (Gene bank number NP_001759.3, aa 138-206) and linked to human 4-1BB (Gene bank number NP_001552.2, aa 214-255) and human CD3Z (Gene bank number NP_000725, aa 52-163) intracellular signaling domains.
  • tEGFR cleavable truncated EGFR
  • Lentiviral supernatants were produced using the HEK 293T packaging line. Briefly, 70% confluent HEK 293T cells in a well of a 6-well plate was cotransfected with 0.2 ug CAR-vector plasmid, 0.9 ug pMD2.G, 1.9 ug psPAX2 using lipofectamine 2000 (Life Technologies). The cultures were grown for 60 hr, after which the supernatants were collected, filtered to remove debris, and frozen at -80 C before use. b. Primary T-cell isolation, transduction, and expansion.
  • T cells were isolated from either healthy or diseased apheresis samples using a negative human pan-T cell isolation kit (Miltenyi Biotec) to obtain CD3 + T cells for CAR-T cell production.
  • Isolated T cells were co-cultured with various APC-ms formulations or Dynabeads (either 1:1, 3:1, or 5:1 bead:cell ratio) at a density of 3.13 x 105cells/cm 2 in X-VIVO 15 supplemented with 10% FBS and 1% P/S.
  • APC-ms was seeded at a material density of 93.75 pg/cm 2 (e.g., 30 pg in a 96-well plate with 1 x 105 isolated T cells).
  • the activated T cells were transduced by adding 140 pL of pre-warmed lenti viral supernatant containing the CD19 CAR construct.
  • the media containing T cells and any remaining material were transferred to a 6-well G-Rex plate (Wilson Wolf) containing pre-warmed X-VIVO 15 supplemented with 5% human AB serum (HABS) and 100 lU/mL IL-2 (Akron Biotech) and expanded for 5 days (total 8-day culture), then cryopreserved using Bambanker (Wako/Fujifilm).
  • APC-ms were not pre-loaded with IL-2.
  • the T-cell concentration was maintained between 0.1 - 2 x 10 6 cells/mL by adding fresh media containing 100 lU/mL IL-2 throughout the duration of culture.
  • the beads were magnetically separated prior to cryopreservation.
  • One CLL sample (CLL9) failed to proliferate following T-cell activation regardless of stimulation type (i.e., APC-ms or Dynabeads).
  • the CAR transduction efficiency was consistently -30-50%.
  • T-cell analysis Following CAR-T cell production, cells were enumerated using an automatic cell counter (MUSE).
  • Fold expansion was calculated by dividing the number of live cells by the number of seeded cells at the beginning of culture.
  • Phenotyping At indicated time points, apheresis samples or CAR-T cell products were processed for flow cytometry. All anti-human antibodies were used at the manufacturer’s recommended concentration.
  • Flow cytometry using a BD LSRFortessa instrument. Gates were set using fluorescence minus one (FMO) controls. Laser intensities were standardized using Rainbow beads (Biolegend) to minimize variability between runs. Data was analyzed using FlowJo vlO (TreeStar). All CAR-T cell products were phenotyped to determine the frequency of CAR+ cells prior to the following analyses. e.
  • CD19-expressing Raji cells were luciferized as previously described and positively-selected using puromycin antibiotic selection.
  • Luciferized Raji cells Raji-luc were washed and were co-cultured with CAR-T cell products at a 5:1, 2:1, 1:1, 1:2, or 1:5 effector:target (CAR+ T-cell:Raji-luc) ratio in 96-well U-bottom plates (VWR) in a total volume of 100 pL RPMI supplemented with 10% HI-FBS.
  • the absolute number of CAR+T cells was fixed at 5 x 10 4 .
  • cytokine secretion After 1 hr of co-culture, Brefeldin A (BD Biosciences) was added to inhibit cytokine secretion, and the cells were cultured for an additional 3 h prior to flow cytometry analysis to evaluate the expression of intracellular cytokines (i.e., granzyme B, IFN-y, TNF-a, and IL -2).
  • cytokines i.e., granzyme B, IFN-y, TNF-a, and IL -2).
  • 1% triton X-100 (VWR) was used as a positive control.
  • 2 x 105 CAR+ T cells and 1 x 104 target cells were used as the highest effector-to-target ratio in a total volume of 100 pL RPMI supplemented with 10% HI-FBS.
  • CAR-T cells and Raji-luc cocultures were lysed using Bright-GLO reagent (Promega) and the luminescence signal of the solution was quantified using a plate reader (Biotek Hl). Percent cytotoxicity was calculated as l-(sample - ctrl A x,100)/(ctrlM - ctrLTM x-TM) %.
  • CAR-T cells were cultured with material scaffolds presenting anti-CD3/anti-CD28 at 0.1 - 0.3 mol% biotinyl-cap-PE across a range of streptavidin densities and cocultured.
  • IL-2 was supplemented into the cultures at 100 lU/mL.
  • images of the cultures were taken, and live cells were manually enumerated using a hemocytometer with Trypan blue exclusion or via an automated cell counter (MUSE).
  • MUSE automated cell counter
  • Cell phenotype was assessed using flow cytometry (BD LSRFortessa).
  • T cell fold expansion was calculated by dividing the number of live T cells by the number of cells seeded at the start of culture (i.e., 1 x 10 5 cells).
  • the materials were mixed using a 1 mL pipette until uniform before injection.
  • a 1 mL syringe with a 16 G needle was loaded with the 200 pL material: collagen (or PBS) mixture.
  • a small subcutaneous pocket was made in the dorsal flank by moving the needle from side to side using forceps, then the mixture was injected.
  • Materials were typically subcutaneously administered 5 days following CAR-T cell infusion, unless otherwise indicated.
  • the material-collagen mixture was mixed with 5 x 10 5 CAR + 19.BBz cells prior to injection (total -300 pL volume).
  • the animals were left under anesthesia for 10 min on a warmly heated pad following injection to allow for collagen crosslinking, as needed. Following injection, animals were carefully monitored for signs of discomfort.
  • mice Female, NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ mice (NSG) mice, between 6-7 weeks of age and ⁇ 20g in weight (Jackson Laboratories), were used for in vivo therapeutic studies. Animals were maintained on 1012 h light cycles and fed chow and water ad libitum. Procedures were approved by Harvard University’s Institutional Animal Care and Use Committee and in compliance with National Institutes of Health guidelines. a. Tumor inoculation and CAR-T cell treatment. NSG mice were inoculated with a high dose of 5x105 luciferized Raji cells (Raji-luc) intravenously on day 0.
  • 5x105 luciferized Raji cells Raji-luc
  • mock RPMI-1640
  • 5xl0 5 CAR + T cells made using APC-ms presenting anti- CD3 and anti-CD28 at a 0.1, 0.15, or 0.3 mol% biotin stimulation dose (abbreviated A0.1, A0.15, and A0.3 products).
  • cryopreserved CAR-T cells were thawed in pre -warmed X- VIVO 15 supplemented with 5% human AB serum and enumerated via hemocytometer with trypan blue exclusion.
  • mice were monitored daily for signs of discomfort and euthanized upon the development of hind-limb paralysis, when immediate and significant graft-versus-host disease (hair loss, behavior changes, health decline) was observed, or when more than 25% of the initial body weight was lost.
  • c Longitudinal CAR-T cell analysis. At the indicated time points, animals were bled via the tail vein and ⁇ 50 pL blood was collected in K2-EDTA-coated collection tubes (BD). The samples were treated with ACK lysis buffer (Lonza), washed, and processed for flow cytometry as described above (BD LSRFortessa). A combination of anti-human and anti-mouse antibodies were used.
  • BD LSRFortessa ACK lysis buffer
  • a combination of anti-human and anti-mouse antibodies were used.
  • d Blood collection and analysis. A FACS panel was conducted.
  • Anti-human CD3- PerCP/Cy5.5 was used instead of anti-human CD3-PE/Cy5 in some instances.
  • the Anti-mouse CD45-PE/Cy5 was correspondingly switched to anti-mouse CM5-PerCP/Cy5.5 where appropriate.
  • mice were sacrificed and the material scaffolds and surrounding subcutaneous tissue were separated from the skin and muscle layers. Tissues were weighed, then minced and digested using a combination of Collagenase IV (Worthington) and DNase I (ThermoFisher). The tissues were digested for 30 min at 37 °C with occasional pipetting. The remaining cell/material suspension was filtered using a 40 pm cell strainer (Fisher Scientific) to separate MSR debris and cells. The remaining cells were washed in PBS supplemented with 0.5% BSA and 2 mM EDTA, pelleted and processed for FACS analysis.
  • Collagenase IV Worthington
  • DNase I ThermoFisher
  • BTK tyrosine kinase
  • Keskin, D. B. et al. Neoantigen vaccine generates intratumoral T cell responses in phase lb glioblastoma trial. Nature 565, 234-239 (2019).

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Abstract

Des modes de réalisation de la présente divulgation concernent des échafaudages mimétiques de cellules présentatrices d'antigène (APC-MS) et l'utilisation de tels échafaudages pour manipuler des lymphocytes T. Plus spécifiquement, les échafaudages sont utiles pour favoriser la croissance, la division, la différenciation, l'expansion, la prolifération, l'activité, la viabilité, l'épuisement, l'anergie, la quiescence, l'apoptose ou la mort de lymphocytes T dans divers modes, par exemple, in vivo. En particulier, les échafaudages sont utiles pour améliorer l'efficacité d'une population administrée de lymphocytes T modifiés, tels que des lymphocytes T CAR. Des modes de réalisation divulgués ici concernent en outre des compositions pharmaceutiques, des kits et des conditionnements contenant de tels échafaudages. D'autres modes de réalisation concernent des procédés de fabrication des échafaudages, des compositions et des kits/conditionnements. La présente divulgation concerne également des procédés d'utilisation des échafaudages, des compositions et/ou des kits dans le traitement de maladies telles que des cancers.
PCT/US2023/012027 2022-01-31 2023-01-31 Procédés de fabrication et d'utilisation d'échafaudages mimétiques de cellules présentatrices d'antigène pour améliorer des thérapies par lymphocytes t WO2023147185A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190292517A1 (en) * 2016-07-13 2019-09-26 President And Fellows Of Harvard College Antigen-presenting cell-mimetic scaffolds and methods for making and using the same
WO2019222290A1 (fr) * 2018-05-14 2019-11-21 Torque Therapeutics Inc. Présentation de peptides à des cellules présentatrices d'antigène à l'aide d'un véhicule lipidique
WO2020237134A1 (fr) * 2019-05-22 2020-11-26 Baylor Research Institute Vaccins à base de cellules présentant un antigène artificiel à une étape

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190292517A1 (en) * 2016-07-13 2019-09-26 President And Fellows Of Harvard College Antigen-presenting cell-mimetic scaffolds and methods for making and using the same
WO2019222290A1 (fr) * 2018-05-14 2019-11-21 Torque Therapeutics Inc. Présentation de peptides à des cellules présentatrices d'antigène à l'aide d'un véhicule lipidique
WO2020237134A1 (fr) * 2019-05-22 2020-11-26 Baylor Research Institute Vaccins à base de cellules présentant un antigène artificiel à une étape

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