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WO2018156649A1 - Compositions de molécules de modulateur cellules t (tcm) et utilisation connexe - Google Patents

Compositions de molécules de modulateur cellules t (tcm) et utilisation connexe Download PDF

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WO2018156649A1
WO2018156649A1 PCT/US2018/019044 US2018019044W WO2018156649A1 WO 2018156649 A1 WO2018156649 A1 WO 2018156649A1 US 2018019044 W US2018019044 W US 2018019044W WO 2018156649 A1 WO2018156649 A1 WO 2018156649A1
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Prior art keywords
cells
tcm
composition
previous
cell
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David Arthur Berry
Douglas Ewen Cameron
Brett Blackman
Chantal KUHN
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Flagship Pioneering Inc
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Flagship Pioneering Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5418—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2046—IL-7
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Cytokines can function in an autocrine, paracrine, or endocrine manner to stimulate or suppress the activity of target cell populations. Signals conveyed by cytokines are essential for generation, survival, and homeostasis of immune cells, as well as for the generation of immune responses upon external stimuli. Cytokines are involved in responses to infection, inflammation, trauma, sepsis, cancer, and in reproduction.
  • the invention features methods and compositions for modifying immune signaling.
  • the aspects as described here may be utilized with any one or more of the embodiments delineated herein.
  • the invention includes a pharmaceutical composition comprising an effective amount of a T cell modulator (TCM) that binds to IL-7Ra, CD 132, or both.
  • TCM T cell modulator
  • the TCM is selected from: a) a polypeptide comprising an amino acid sequence of at least two alpha helices, wherein at least one alpha helix comprises at least one amino acid that directly contacts IL-7R upon TCM binding to IL-7R and activates IL-7R signaling; a nucleic acid (e.g., DNA, RNA, e.g., mRNA) encoding (a); an antibody, or antigen-binding fragment thereof, that binds and activates IL-7R signaling; or a small molecule agonist of IL-7 or thymic stromal lymphopoietin (TSLP) that activates IL-7R signaling.
  • a nucleic acid e.g., DNA, RNA, e.g., mRNA
  • a nucleic acid e.g., DNA, RNA, e.g., mRNA
  • an antibody, or antigen-binding fragment thereof that binds and activates IL-7R
  • the TCM is selected from: a) polypeptide comprising an amino acid sequence of at least one alpha helix, wherein at least one alpha helix comprises at least one amino acid that directly contacts IL-7Ra and/or CD 132 and inhibits IL-7R signaling; a nucleic acid (e.g., DNA, RNA, e.g., mRNA) encoding (a); an antibody, or antigen-binding fragment thereof, that binds and inhibits IL-7R signaling; or a small molecule antagonist of IL-7 or TSLP that inhibits IL-7R signaling.
  • a nucleic acid e.g., DNA, RNA, e.g., mRNA
  • the TCM comprises at least two alpha helices. In some embodiments, at least one alpha helix comprises a ⁇ -helical turn. In some embodiments, the ⁇ -helical turn comprises a hydrophobic ⁇ -branched amino acid that contacts IL-7Ra.
  • At least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to EGKDGKQYESVLMVSIDQLL (SEQ ID NO: 1). In some embodiments, at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • At least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to LCFLKRLLQEIKTCWNKILM (SEQ ID NO: 3).
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to DCDIEGKDGKQYESVLMVSIDQLL (SEQ ID NO: 4). In some embodiments, the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • VKGRK A ALGE AQPT KS LEENKS L (SEQ ID NO: 6).
  • the TCM further comprises at least one alpha helix domain of TSLP; IL-2; IL-4; IL-7; IL-9; IL-15; IL-21; or any variant thereof.
  • the TCM comprises a sequence SxxMxxxD to bind to CD 132, wherein x is any amino acid.
  • the TCM comprises helix A of IL-7, wherein at least one of Serl4, Metl7, and Asp21 of helix A binds to CD132.
  • the TCM binds to at least one of Tyrl03, Asnl28, Pro207, Cys209, Gly210, and Ser211 of CD132.
  • the TCM binds to at least one of EF1, BC2, and FG2 loop of CD 132.
  • the composition further comprises a nanoparticle, liposome, or exosome.
  • the composition further comprises a heterologous moiety.
  • the TCM is a fusion with a heterologous moiety.
  • the heterologous moiety is selected from the group consisting of CD132; Fc domain;
  • antibody e.g., IgGl, anti-OX40, anti-4-lBB, anti-CD45RO, anti-CD45RA, antibodies that block PD-1/CD160/KLRG-1/TIM-3, bispecific Abs, scFvs); MHC; peptide-MHC;
  • the heterologous moiety is a therapeutic (e.g., gamma chain cytokines, PD-1 inhibitors, checkpoint inhibitors, chemotherapy, antivirals, decorin, antibiotic, or cytokines).
  • the TCM comprises a linker.
  • the linker is a cleavable linker.
  • the TCM is multimerized.
  • the TCM modulates (e.g., increased or decreased) solubility, stability, half- life, or bioavailability as compared to wildtype IL-7 or TSLP.
  • the TCM alters binding to extracellular matrix-associated glycosaminoglycan, heparan sulfate, or fibronectin.
  • the effective amount is sufficient to modulate (e.g., sufficient to increase or decrease at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95%) at least one of phosphorylation of STAT5; survival of naive T cells;
  • the composition comprises a cell modified to express the TCM.
  • the invention includes a pharmaceutical composition comprising an effective amount of a T cell modulator (TCM) that binds IL-7, wherein the TCM modulates the solubility, stability, half-life, and/or bioavailability of IL-7.
  • TCM T cell modulator
  • the composition comprises a polypeptide comprising (a) an ectodomain of IL-7Ra, or a fragment or variant thereof, (b) a nucleic acid (e.g., DNA, RNA, e.g., mRNA) encoding (a), or (c) an antibody, or antigen-binding fragment thereof, that binds IL-7.
  • a polypeptide comprising (a) an ectodomain of IL-7Ra, or a fragment or variant thereof, (b) a nucleic acid (e.g., DNA, RNA, e.g., mRNA) encoding (a), or (c) an antibody, or antigen-binding fragment thereof, that binds IL-7.
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising a cell modified to express a nucleic acid encoding a T cell modulator (TCM) that comprises at least two alpha helices from IL-7 or TSLP, or a variant thereof.
  • TCM T cell modulator
  • the cell is an immune cell (e.g., dendritic cell, T cell, B cell, or NK cell).
  • an immune cell e.g., dendritic cell, T cell, B cell, or NK cell.
  • the invention includes a method of modulating (e.g., sufficient to increase or decrease at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95%) at least one of phosphorylation of STAT5; survival of naive T cells;
  • the invention includes a method of increasing or enhancing (e.g., sufficient to increase at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95%) at least one of phosphorylation of STAT5; survival of naive T cells;
  • the invention includes a decreasing or inhibiting (e.g., sufficient to decrease inhibit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95%) at least one of phosphorylation of STAT5; survival of naive T cells; survival of memory CD4+ T cells; survival of memory CD8+ T cells; proliferation of T cells; activation of PI-3K; gene expression of Bcl-2; differentiation of pre-pro B cells; or proliferation of 2E8 cells, comprising contacting the composition described herein to a cell comprising IL-7R.
  • the invention includes a method of increasing thymus cellularity in a subject, comprising administering to the subject an effective amount of the composition described here
  • the administration alters at least one of immune cell localization, activation of immune cells, survival of immune cells, differentiation of immune cells, and proliferation of immune cells.
  • the invention includes a method of decreasing thymus cellularity in a subject, comprising administering to the subject an effective amount of the composition described herein.
  • the administration alters at least one of immune cell localization, activation of immune cells, survival of immune cells, differentiation of immune cells, and proliferation of immune cells.
  • the invention includes a method of increasing or enhancing an immune response in a subject comprising administering to the subject the composition described herein in an amount effective to increase or enhance the immune response in the subject.
  • the administration comprises administering subject-derived cells (e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells) modified to express the TCM.
  • subject-derived cells e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells
  • the immune response is an anti- viral, anti-bacterial, or anti-parasitic response.
  • the invention includes a method of decreasing or inhibiting an immune response in a subject comprising administering to the subject the composition described herein in an amount effective to decrease or inhibit the immune response in the subject.
  • the administration comprises administering subject-derived cells (e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells) modified to express the TCM.
  • subject-derived cells e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells
  • the immune response is an auto-immune, allergic or inflammatory response, e.g., multiple sclerosis, type 1 diabetes, rheumatoid arthritis, lupus, sarcoidosis and inflammatory bowel disease.
  • the invention includes a method of increasing or enhancing an antitumor response in a subject comprising administering to the subject the composition described herein in an amount effective to increase or enhance the anti-tumor response in the subject.
  • the invention includes a method of decreasing or inhibiting an autoimmune, allergic or inflammatory response in a subject comprising administering to the subject the composition described herein in an amount effective to decrease or inhibit the auto-immune, allergic or inflammatory response in the subject.
  • the invention includes a method of screening for a T cell modulator (TCM) that binds to IL-7Ra and modulates at least one of the following: phosphorylation of STAT5; survival of naive T cells; survival of memory CD4+ T cells; survival of memory CD8+ T cells; proliferation of T cells; activation of PI-3K; gene expression of Bcl-2;
  • TCM T cell modulator
  • the invention includes a method of screening for a T cell modulator (TCM) that binds to IL-7Ra and increases or enhances at least one of the following:
  • the invention includes a method of screening for a T cell modulator (TCM) that binds to IL-7Ra and decreases or inhibits at least one of the following:
  • ⁇ -helical turn refers to an amino acid sequence that is capable of hydrogen bond formation between an amino group in the backbone and a carboxyl group in the backbone of the same sequence five residues away from the amino group.
  • alpha helix refers to an amino acid sequence that is capable of hydrogen bond formation between one or more amino groups in the backbone and one or more carboxyl groups in the backbone of the same sequence three-four residues away from the amino group.
  • amino acid linker refers to an amino-acid polypeptide spacer that links two or more polypeptides.
  • the linker can be 2-15 amino acid residues and links two beta strands.
  • the term “combination” or “administered in combination” refers to two (or more) different agents or treatments administered to a subject as part of a defined treatment regimen for a particular disease or condition.
  • the treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap.
  • the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated.
  • the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen.
  • administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic).
  • Sequential or substantially simultaneous administration of each therapeutic agent can be affected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection while a second therapeutic agent of the combination may be administered orally.
  • fragment refers to a nucleic acid or amino acid sequence comprising a portion (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any portion thereof) of the contiguous residues of a nucleotide or amino acid sequence of interest.
  • heterologous moiety refers to any constituent that is linked to (e.g., covalently or non-covalently) or in combination with the TCM, which constituent is different from the TCM.
  • hydrophobic ⁇ -branched amino acid refers to an amino acid having an aliphatic side-chain with a branch (a central carbon atom bound to two or more carbon atoms).
  • examples of hydrophobic ⁇ -branched amino acid include, but are not limited to, valine (val, V), isoleucine (iso, I), leucine (leu, L), threonine (thr, T), and analogs thereof.
  • T cell modulator refers to a molecule that modulates (e.g., increases or decreases) IL-7R signaling.
  • Treatment and “treating,” as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, prevent or cure a disease
  • pathological condition, or disorder This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy).
  • active treatment treatment directed to improve the disease, pathological condition, or disorder
  • causal treatment treatment directed to the cause of the associated disease, pathological condition, or disorder
  • palliative treatment treatment designed for the relief of symptoms
  • preventative treatment treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment treatment employed to supplement another therapy.
  • variant refers to one or more amino acid substitutions, additions, or deletions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, optionally 11-20, 21- 30 or more, for example up to 10% of a polypeptide or nucleic acid), wherein the variant still maintains one or more functions (e.g. completely, partially, minimally) of the starting polypeptide.
  • amino acid substitutions for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, optionally 11-20, 21- 30 or more, for example up to 10% of a polypeptide or nucleic acid
  • functions e.g. completely, partially, minimally
  • Percent (%) sequence identity with respect to a reference polypeptide sequence (or nucleic acid sequence) is the percentage of amino acid residues (or nucleotides in case of nucleic acid sequence) in a candidate sequence that are identical with the amino acid residues (or nucleotides) in the reference polypeptide sequence (or nucleic acid sequence), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Figure 1 is a schematic drawing of a TCM fused to the N-terminus of the Fc domain, the C-terminus of the Fc domain or to a side group in the Fc domain.
  • FIG. 2 is a schematic drawing of a TCM-antibody fusion.
  • FIG. 3 is a schematic drawing of a TCM useful for the treatment of cancer.
  • Figure 4A is a panel of histograms showing the mean fluorescence intensity (MFI) of live Streptavidin-Alexa 647 (SA-A647) stained CD4+ T cells that were previously incubated with recombinant human IL-7 (rhuIL-7; control) or the indicated TCM alone (upper curve), biotinylated recombinant human IL-7 alone (middle line) or consecutively with non- biotinylated IL-7 or the respective TCM followed by biotinylated recombinant human IL-7 (lower line).
  • MFI mean fluorescence intensity
  • Figure 4B is a panel of histograms showing the mean fluorescence intensity (MFI) of live Streptavidin-Alexa 647 (SA-A647) stained CD8+ T cells that were previously incubated with recombinant human IL-7 (rhuIL-7; control) or the indicated TCM alone (upper curve), biotinylated recombinant human IL-7 alone (middle line) or consecutively with non- biotinylated IL-7 or the respective TCM followed by biotinylated recombinant human IL-7 (lower line).
  • MFI mean fluorescence intensity
  • Figure 5A is a panel of histograms showing the mean fluorescence intensity (MFI) and percentage of CD4+ cells positive (% pos.) for STAT5 pY694 (P-STAT5) after stimulation with the indicated concentrations of rhuIL-7 (first panel) or the respective indicated TCM for 15 minutes.
  • MFI mean fluorescence intensity
  • % pos. percentage of CD4+ cells positive
  • Figure 5B is a panel of histograms showing the mean fluorescence intensity (MFI) and percentage of CD8+ T cells positive (% pos.) for P-STAT5 after stimulation with the indicated concentrations of rhuIL-7 (first panel) or the respective indicated TCM for 15 minutes.
  • MFI mean fluorescence intensity
  • % pos. percentage of CD8+ T cells positive
  • Figure 6A is a panel of histograms showing the mean fluorescence intensity (MFI) and percentage of CD4+ cells T cells positive (% pos.) for P-STAT5 after stimulation with rhuIL-7or the respective indicated TCM (lOng/ml) for the indicated time spans.
  • Figure 6B is a panel of histograms showing the mean fluorescence intensity (MFI) and percentage of CD8+ T cells positive (% pos.) for P-STAT5 after stimulation with rhuIL-
  • Figure 7A is a graph showing the percentage of CD4+ T cells that proliferated after stimulation of primary human PBMCs with the indicated concentrations (i.e. 0.01 ng/ml, 0.1 ng/ml or 1 ng/ml) of rhuIL-7 or the indicated TCM for 6 days as compared to samples that were cultured in the absence of rhuIL-7 or TCM (w/o; black bars).
  • Figure 7B is a graph showing the percentage of CD8+ T cells that proliferated after stimulation of primary human PBMCs with the indicated concentrations (i.e. 0.01 ng/ml, 0.1 ng/ml or 1 ng/ml) of rhuIL-7 or the indicated TCM for 6 days as compared to samples that were cultured in the absence of rhuIL-7 or TCM (w/o; black bars).
  • Figure 8 is a graph showing the percentage of live of human T cells after 6 days of culture in the presence or absence of rhuIL-7 or different TCMs at the indicated
  • Figure 9A is a graph showing the percentage of CD8+ T cells that express IL-7Ra (CD 127) after stimulation of primary human PBMCs with the indicated concentrations of IL- 7 or TCM2 for 6 days as compared to samples that were cultured in the absence of rhuIL-7 or TCM (w/o; black bars).
  • Figure 9B is a graph showing the percentage of CD8+ T cells that express IL-7Ra (CD 127) after stimulation of primary human PBMCs with the indicated concentrations of IL- 7 or TCM2 for 6 days as compared to samples that were cultured in the absence of rhuIL-7 or TCM (w/o; black bars).
  • Figure 10A is a graph showing the percentage of CD8+ T cells that express IL-7Ra (CD 127) after stimulation of primary human PBMCs with the indicated concentrations of IL- 7 or different TCMs for 6 days as compared to samples that were cultured in the absence of rhuIL-7 or TCM (w/o; black bars).
  • Figure 10B is a graph showing the percentage of CD8+ T cells that express IL-7Ra (CD 127) after stimulation of primary human PBMCs with the indicated concentrations of IL- 7 or different TCMs for 6 days as compared to samples that were cultured in the absence of rhuIL-7 or TCM (w/o; black bars).
  • the invention describes T cell modulator molecules and pharmaceutical compositions that have beneficial characteristics suitable for administration to a target tissue or cell (e.g., ex vivo or in vivo), useful in methods to modify (e.g., modify the activation inhibition of) a target signaling pathway (e.g., IL-7R pathway) in a tissue or cell (e.g., ex vivo or in vivo), and/or to treat a subject (e.g., a mammal such as a human).
  • a target tissue or cell e.g., ex vivo or in vivo
  • a target signaling pathway e.g., IL-7R pathway
  • compositions described herein may also be modified, e.g., variants, fusions, or combinations.
  • T Cell Modulator T Cell Modulator
  • IL-7 is a pleiotropic cytokine with central roles in modulating T- and B-cell development and T-cell homeostasis by inducing signaling through the IL-7 receptor (IL-7R) that consists of the IL-7R alpha chain (CD127 or IL-7Ra; NM_002185 and NP_002176) paired with the common gamma chain (CD132; NM_000206 and NP_000197).
  • IL-7 signaling involves a number of non-receptor tyrosine kinase pathways that associate with the cytoplasmic tail of the receptor.
  • Jak/STAT Janus kinase/signal transducer and activator of transcription
  • PI3-kinase phosphatidylinositol 3- kinase
  • Src family tyrosine kinases Interactions between IL-7 and the IL- 7Ra ectodomain is described, for example, in McElroy et al., Structure, 2009, 17:54-65.
  • Applicants have developed novel agents to modify IL-7R receptor activity to allow the modulation of immune function in subjects in need thereof, e.g., to increase or decrease the immune response in a patient in need thereof.
  • TCM T cell modulator
  • the invention includes a pharmaceutical composition comprising an effective amount of a T cell modulator (TCM) that binds IL-7, wherein the TCM modulates the solubility, stability, half-life, and/or bioavailability of IL-7.
  • TCM T cell modulator
  • the composition comprises a polypeptide comprising (a) an ectodomain of IL-7Ra, or a fragment or variant thereof, (b) a nucleic acid (e.g., DNA, RNA, e.g., mRNA) encoding (a), or (c) an antibody, or antigen-binding fragment thereof, that binds IL-7.
  • a polypeptide comprising (a) an ectodomain of IL-7Ra, or a fragment or variant thereof, (b) a nucleic acid (e.g., DNA, RNA, e.g., mRNA) encoding (a), or (c) an antibody, or antigen-binding fragment thereof, that binds IL-7.
  • a TCM is a polypeptide comprising an amino acid sequence that forms a plurality of alpha helices, wherein at least one of the plurality comprises at least one amino acid that directly contacts IL-7Ra upon TCM binding to IL-7R (e.g., IL-7Ra and CD132), and activates IL-7R (e.g., IL-7Ra) signaling.
  • TCM embodiments are combined.
  • the TCM comprises at least 2 alpha helices and activates IL- 7R signaling.
  • the TCM includes at least one alpha helix comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to EGKDGKQYESVLMVSIDQLL (SEQ ID NO: 1); at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • LCFLKRLLQEIKTCWNKILM (SEQ ID NO: 3); and/or at least one alpha helix comprises a sequence at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to LCFLKRLLQEIKTCWNKILM (SEQ ID NO: 3).
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to DCDIEGKDGKQYESVLMSIDQLL (SEQ ID NO: 7); the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to KEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 5); and/or the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • VKGRK A ALGE AQPT KS LEENKS L (SEQ ID NO: 6).
  • the TCM comprises a sequence SxxMxxxD to bind CD 132 subunit and activates IL-7R signaling, wherein x is any amino acid.
  • the TCM comprises a sequence
  • K/TV/FS/AEGTxILLNxx (SEQ ID NO: 9) and activates IL-7R signaling, wherein x is any amino acid.
  • the TCM comprises at least 3 alpha helices and activates IL- 7R signaling.
  • the alpha helices are connected by a linker region (e.g. GS linker) and/or a helix B and/or connecting peptide sequences.
  • the TCM comprises at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to xxKxxK/AQ/A/IYESVLMVSI/LxQLL (SEQ ID NO: 8),wherein x is any amino acid, K/TV/FS/AEGTxILLNxx (SEQ ID NO: 9),wherein x is any amino acid, and at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% identical to
  • At least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% identical to KVSEGTTILLNCT (SEQ ID NO: 2), and at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises at least 4 alpha helices and activates IL- 7R signaling.
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises one or more glycosylation sites and activates IL-7R signaling.
  • the TCM is glycosylated at sites Asn70, Asn91, Asnl l6, and O(Thrl lO).
  • the TCM comprises at least one or more of Cys2, Cys34, Cys47, Cys92, Cysl 11, and Cysl23 that form one or more disulfide bridges and activates IL- 7R signaling.
  • the TCM comprises one or more disulfide bridges, e.g., Cys: 1-4 (Cys2-Cys92); 2-5 (Cys34-Cysl l l); and 3-6 (Cys47-Cysl23).
  • the TCM comprises at least 4 alpha helices, wherein at least one or more amino acids in the alpha helices interact with CD 132 that induces
  • the TCM comprises at least one alpha helix comprising a ⁇ - helical turn and activates IL-7R signaling.
  • the ⁇ -helical turn comprises a hydrophobic ⁇ -branched amino acid residue projecting from the ⁇ -helical turn of the alpha- helix.
  • the hydrophobic ⁇ -branched amino acid residue interacts with a hydrophobic pocket at the D1-D2 junction of IL-7Ra.
  • Hydrophobic amino acids include amino acids having hydrophobic side chains and include, but are not limited to, alanine (ala, A), valine (val, V), isoleucine (iso, I), leucine (leu, L), methionine (met, M), phenylalanine (phe, F), tyrosine (tyr, Y), tryptophan (trp, W), and analogs thereof.
  • Amino acid analogs include, but are not limited to, D-amino acids, amino acids lacking a hydrogen on the a-carbon such as dehydroalanine, metabolic intermediates such as ornithine and citrulline, non-alpha amino acids such as ⁇ -alanine, ⁇ -aminobutyric acid, and 4- aminobenzoic acid, twin a-carbon amino acids such as cystathionine, lanthionine, djenkolic acid and diaminopimelic acid, and any others known in the art.
  • TCM comprises at least one alpha helix having at least 85% identity to the Helix A of TSLP.
  • TCM comprises at least one alpha helix having at least 85% identity to the Helix D from of IL-2.
  • TCM comprises CD132 binding region having at least 85% identity to the gamma chain binding region of IL-2.
  • TCM comprises at least one alpha helix with a sequence having at least 85% identity to Helix A from TSLP.
  • TCM comprises a sequence having at least 85% identity to Helix D from IL-4.
  • TCM comprises a sequence having at least 85% identity to the gamma chain binding region of IL-4.
  • TCM comprises a sequence having at least 85% identity to Helix D from IL-9.
  • TCM comprises a sequence having at least 85% identity to the gamma chain binding region of IL-9.
  • TCM comprises a sequence having at least 85% identity to Helix D from IL-15
  • TCM comprises a sequence having at least 85% identity to the gamma chain binding region of IL-15.
  • TCM comprises a sequence having at least 85% identity to Helix D from IL-21.
  • the TCM interacts with IL-7Ra (CD127) and CD132 to activate IL-7R signaling.
  • IL-7Ra CD127
  • CD132 CD132
  • the TCM comprises helix A of IL-7, wherein at least one of Serl4, Metl7, and Asp21 of helix A binds to CD132.
  • such a TCM induces IL-7R dependent phosphorylation of STAT5.
  • the TCM binds to at least one of Tyrl03, Asnl28, Pro207, Cys209, Gly210, and Ser211 of CD132.
  • the TCM interacts with both CD127 and CD132 and thereby increases survival of naive T cells [0099] In some embodiments, the TCM interacts with both CD127 and CD132 to increase proliferation of T cells.
  • TCM binds IL-7R (e.g., the IL-7R alpha chain (IL-7Ra) and/or CD132).
  • TCM may bind to one or more of IL-7Ra and CD 132.
  • the IL-7Ra binding domain of TCM may include one or more domains of TSLP or fragments thereof. See for example, Verstraete et al. Nature structural & molecular biology. 2014.
  • TCM may include one or more CD132 binding domains, or fragments thereof. See Olosz, et al. J Biol Chem, 2000, 275:30100-30105, for CD132 loop domains.
  • one or more IL-7Ra binding domains can be exchanged with CD132 binding domains of IL-2, IL-4, IL-7, IL-9, IL-15 or IL-21. See for example, Walsh. Immunol Rev. 2012 November; 250(1): 303-316. doi: 10.1111/j. l600-065X.2012.01160.x, for cytokine domains that bind to CD132.
  • TCM comprises a sequence having at least 85% identity to the gamma chain binding region of IL-21.
  • TCM comprises at least one of exon 2 and exon 4 of IL-7 with at least one of the following amino acid substitutions or deletions to improve hydrogen bonding and/or affinity to IL-7Ra.
  • TCM comprises exon 6 of IL-7 with at least one of the following substitutions: Lysl21A, Leul36A, Lysl40A, or Trpl43A.
  • a TCM is a polypeptide comprising an amino acid sequence that forms a plurality of alpha helices, wherein at least one of the plurality comprises at least one amino acid that directly contacts either a IL-7Ra or CD132 upon TCM binding to IL-7R, and inhibits IL-7R signaling.
  • TCM embodiments are combined.
  • the TCM comprises at least 2 alpha helices and inhibits IL-7R signaling.
  • the TCM includes at least one alpha helix comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to EGKDGKQYESVLMVSIDQLL (SEQ ID NO: 1); at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • LCFLKRLLQEIKTCWNKILM (SEQ ID NO: 3); and/or at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to LCFLKRLLQEIKTCWNKILM (SEQ ID NO: 3).
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to KEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 5); and/or the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • VKGRKP A ALGE AQPT KS LEENKS L (SEQ ID NO: 6).
  • the TCM comprises a sequence SxxMxxxD to bind to CD 132 and inhibits IL-7R signaling, wherein x can be any amino acid.
  • the TCM comprises a sequence
  • K/TV/FS/AEGTxILLNxx (SEQ ID NO: 9) and inhibits IL-7R signaling, wherein x can be any amino acid.
  • the TCM comprises at least 3 alpha helices and inhibits IL-7R signaling.
  • the alpha helices are connected by a linker region (e.g. GS linker) and/or a helix B and/or connecting peptide sequences.
  • the TCM comprises at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to xxKxxK/AQ/A/IYESVLMVSI/LxQLL (SEQ ID NO: 8),wherein x is any amino acid, K/TV/FS/AEGTxILLNxx (SEQ ID NO: 9), wherein x is any amino acid, and at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • At least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to KVSEGTTILLNCT (SEQ ID NO: 2), and at least one alpha helix comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • LCFLKRLLQEIKTCWNKxLx (SEQ ID NO: 10), wherein x is any amino acid.
  • the TCM comprises at least 4 alpha helices and inhibits IL-7R signaling.
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLF RAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQEENKSLKEQKKLNDLCFLK RLLQEIKTCWNKILMGTKEH (SEQ ID NO: 11).
  • the TCM comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to
  • the TCM comprises one or more glycosylation sites and inhibits IL-7R signaling.
  • the TCM is glycosylated at sites Asn70, Asn91, Asnl l6, and O(Thrl lO).
  • the TCM comprises at least one or more of Cys2, Cys34, Cys47, Cys92, Cysl 11, and Cysl23 that form one or more disulfide bridges and inhibits IL- 7R signaling.
  • the TCM comprises one or more disulfide bridges, e.g., Cys: 1-4 (Cys2-Cys92); 2-5 (Cys34-Cysl l l); and 3-6 (Cys47-Cysl23).
  • the TCM comprises at least 4 alpha helices, wherein at least one or more amino acids in the alpha helices interact with CD 132 that inhibits
  • the TCM comprises at least one alpha helix comprises a ⁇ - helical turn and inhibits IL-7R signaling.
  • the ⁇ -helical turn comprises a hydrophobic ⁇ -branched amino acid residue projecting from the ⁇ -helical turn of the alpha- helix.
  • the hydrophobic ⁇ -branched amino acid residue interacts with a hydrophobic pocket at the D1-D2 junction of IL-7Ra.
  • the TCM interacts with IL-7Ra (CD127) or CD132, and inhibits IL-7R signaling.
  • the TCM comprises helix A of IL-7, wherein at least one of Serl4, Metl7, and Asp21 of helix A binds to CD132 and inhibits IL-7R signaling. In some embodiments, such a TCM inhibits IL-7R dependent phosphorylation of STAT5. In some embodiments, the TCM binds to at least one of Tyrl03, Asnl28, Pro207, Cys209, Gly210, and Ser211 of CD 132 and inhibits IL-7R signaling.
  • the TCM interacts with either CD127 or CD132 decrease survival of naive T cells.
  • the TCM interacts with either CD127 or CD132 to inhibit proliferation of T cells.
  • TCM binds either the IL-7R alpha chain (IL-7Ra) or CD 132 and inhibits IL-7R signaling.
  • the IL-7Ra binding domain of TCM may include one or more domains of TSLP or fragments thereof.
  • the TCM may include one or more CD 132 binding domains, or fragments thereof.
  • one or more IL-7Ra binding domains can be exchanged with CD132 binding domains of IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21.
  • the TCM is an antibody, or antigen-binding fragment thereof, that binds and activates IL-7R signaling.
  • the antibody, or antigen-binding fragment thereof interacts with IL-7Ra (CD127) and CD132. [0124] In some embodiments, the antibody, or antigen-binding fragment thereof interacts with CD132. In some embodiments, such a TCM induces IL-7R dependent phosphorylation of STAT5. In some embodiments, the antibody, or antigen-binding fragment thereof interacts with at least one of Tyrl03, Asnl28, Pro207, Cys209, Gly210, and Ser211 of CD132.
  • the antibody, or antigen-binding fragment thereof interacts with both CD127 and CD132 and thereby increases survival of naive T cells
  • the antibody, or antigen-binding fragment thereof interacts with both CD127 and CD132 to increase proliferation of T cells.
  • the antibody or antigen-binding fragment thereof binds IL-7Ra and/or CD 132.
  • antibodies are screened for binding affinity to IL-7R (e.g., the IL-7R alpha chain (IL-7Ra) and/or CD 132) and an antibody, or antigen-binding fragment thereof is selected based on its binding characteristics.
  • IL-7R e.g., the IL-7R alpha chain (IL-7Ra) and/or CD 132
  • an antibody, or antigen-binding fragment thereof is selected based on its binding characteristics.
  • the TCM is an antibody, or antigen-binding fragment thereof, that inhibits IL-7R signaling.
  • the antibody, or antigen-binding fragment thereof interacts with either IL-7Ra (CD127) or CD132.
  • the antibody, or antigen-binding fragment thereof interacts with CD132 and inhibits IL-7R signaling. In some embodiments, such a TCM inhibits IL-7R dependent phosphorylation of STAT5. In some embodiments, the antibody, or antigen- binding fragment thereof interacts with at least one of Tyrl03, Asnl28, Pro207, Cys209, Gly210, and Ser211 of CD132, and inhibits IL-7R signaling.
  • the antibody, or antigen-binding fragment thereof interacts with either CD127 or CD132 and thereby decreases survival of naive T cells
  • the antibody, or antigen-binding fragment thereof interacts with either CD127 or CD132 to inhibit proliferation of T cells.
  • the antibody or antigen-binding fragment thereof binds only one of the IL-7Ra or CD132.
  • antibodies are screened for binding affinity to IL-7Ra or
  • CD 132 and an antibody or antigen-binding fragment thereof is selected based on its selective binding to one and not the other.
  • human antibodies For in vivo use of antibodies in humans, it may be preferable to use human antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,
  • a human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non- functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Antibodies directed against the target of choice can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • IgG, IgA, IgM and IgE antibodies including, but not limited to, IgGl (gamma 1) and IgG3.
  • IgGl gamma 1
  • IgG3 IgG3
  • PCT Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735 See, e.g., PCT Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625, 126; 5,633,425;
  • Human antibodies can also be derived from phage-display libraries (Hoogenboom et al, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581- 597 (1991); Vaughan et al, Nature Biotech., 14:309 (1996)).
  • Phage display technology McCafferty et al, Nature, 348:552-553 (1990)
  • V immunoglobulin variable
  • antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
  • phage mimics some of the properties of the B cell.
  • Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).
  • V-gene segments can be used for phage display.
  • Human antibodies may also be generated by in vitro activated B cells (see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety). Human antibodies may also be generated in vitro using hybridoma techniques such as, but not limited to, that described by Roder et al. (Methods Enzymol, 121: 140-167 (1986)).
  • a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human.
  • the antigen binding domain is humanized.
  • a humanized antibody retains a similar antigenic specificity as the original antibody.
  • affinity and/or specificity of binding of the antibody for human CD3 antigen may be increased using methods of directed evolution, as described by Wu et al, J. Mol. Biol, 294: 151 (1999), the contents of which are
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human.
  • humanized chimeric antibodies substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592, 106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al, Protein Engineering, 7(6):805-814 (1994); and Roguska et al, PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference herein in their entirety. [0143]
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be to reduce antigenicity.
  • the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al, J. Immunol, 151:2296 (1993); Chothia et al, J. Mol. Biol, 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al, J. Immunol, 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).
  • Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Bispecific antibodies are useful in a number of biomedical applications. For instance, methods comprising an activating bispecific TCM with at least one binding site for IL-7R (IL-7Ra and CD 132) and at least one binding site for another molecule, such as a tumor- associated antigen (TAA) (e.g., CEACAM 5 and/or CEACAM6) may be of therapeutic use to activate an immune response.
  • TAA tumor-associated antigen
  • Activating bispecific TCM comprising antigen-binding variable region sequences of any known anti-tumor-associated antigen antibody may be utilized, including but not limited to hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No.
  • antibodies of use may be commercially obtained from a wide variety of known sources.
  • a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, Va.).
  • a large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published and are available for use in the claimed methods and compositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;
  • methods with inhibitory bispecific TCM comprising at least one binding site for IL-7Ra and/or CD 132 and at least one binding site for another molecule, such as an allergen, autoimmune antigen, and tissue specific antigen (see, e.g., U.S. Pat. Nos. 7,300,644; 7,138,103; 7,074,405; 7,052,872; 6,962,702; 6,458,933, the Examples section of each of which is incorporated herein by reference) may be of therapeutic use to inhibit an immune response.
  • antibody sequences or antibody- secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest.
  • the antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art.
  • Bispecific antibodies can be produced by the quadroma method, which involves the fusion of two different hybridomas, each producing a monoclonal antibody recognizing a different antigenic site (Milstein and Cuello, Nature, 1983; 305:537- 540).
  • bispecific antibodies uses heterobifunctional cross- linkers to chemically tether two different monoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez, et al. Nature. 1985; 316:354-356). Bispecific antibodies can also be produced by reduction of each of two parental monoclonal antibodies to the respective half molecules, which are then mixed and allowed to reoxidize to obtain the hybrid structure (Staerz and Bevan. Proc Natl Acad Sci USA. 1986; 83: 1453-1457). Another alternative involves chemically cross-linking two or three separately purified Fab' fragments using appropriate linkers. (See, e.g., European Patent Application 0453082).
  • Other methods include improving the efficiency of generating hybrid hybridomas by gene transfer of distinct selectable markers via retrovirus-derived shuttle vectors into respective parental hybridomas, which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of a hybridoma cell line with expression plasmids containing the heavy and light chain genes of a different antibody.
  • Cognate VH and VL domains can be joined with a peptide linker of appropriate composition and length (usually consisting of more than 12 amino acid residues) to form a single-chain Fv (scFv) with binding activity.
  • a peptide linker of appropriate composition and length usually consisting of more than 12 amino acid residues
  • Methods of manufacturing scFvs are disclosed in U.S. Pat. Nos. 4,946,778 and 5,132,405, the Examples section of each of which is incorporated herein by reference. Reduction of the peptide linker length to less than 12 amino acid residues prevents pairing of VH and VL domains on the same chain and forces pairing of VH and VL domains with complementary domains on other chains, resulting in the formation of functional multimers.
  • Polypeptide chains of VH and VL domains that are joined with linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed triabodies) and tetramers (termed tetrabodies) are favored, but the exact patterns of oligomerization appear to depend on the composition as well as the orientation of V-domains (VH-linker-VL or VL-linker- VH), in addition to the linker length.
  • the TCM is a nucleic acid (e.g., DNA, RNA, e.g., mRNA).
  • the nucleic acid encodes any one of the TCMs described herein.
  • a nucleic acid may include, but is not limited to, DNA, RNA, and artificial nucleic acids.
  • the nucleic acid may include, but is not limited to, genomic DNA, cDNA, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNAi molecule.
  • the nucleic acid is an siRNA to target a gene expression product.
  • the nucleic acid includes one or more nucleoside analogs as described herein.
  • a vector may comprise a nucleic acid encoding a TCM described herein.
  • nucleic acid sequences coding for a desired target can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • a gene of interest can be produced synthetically, rather than cloned.
  • Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding the gene of interest to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration in eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence.
  • Additional promoter elements can regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor- la (EF- la).
  • EF- la Elongation Growth Factor- la
  • other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus
  • MMTV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV avian leukemia virus promoter
  • ELR Epstein-Barr virus immediate early promoter
  • Rous sarcoma virus promoter as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
  • an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes can be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene can be assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the TCM includes oligonucleotides, especially antisense oligonucleotides that are directed against, e.g., IL-7, TSLP, and/or IL-7R.
  • antisense oligonucleotides include those known as siRNA or RNAi.
  • Nucleic acids can have a length from about 2 nucleotides (nts) to about 5000 nts, about 10 nts to about 100 nts, about 50 nts to about 150 nts, about 100 nts to about 200 nts, about 150 nts to about 250 nts, about 200 nts to about 300 nts, about 250 nts to about 350 nts, about 300 nts to about 500 nts, about 10 nts to about 1000 nts, about 50 nts to about 1000 nts, about 100 nts to about 1000 nts, about 1000 nts to about 2000 nts, about 2000 nts to about 3000 nts, about 3000 nts to about 4000 nts, about 4000 nts to about 5000 nts, or any range therebetween.
  • nts nucleotides
  • Nucleic acids can have a length from 2 nts to 5000 nts, 10 nts to 100 nts, 50 nts to 150 nts, 100 nts to 200 nts, 150 nts to 250 nts, 200 nts to 300 nts, 250 nts to350 nts, 300 nts to 500 nts, 10 nts to 1000 nts, 50 nts to 1000 nts, 100 nts to 1000 nts, 1000 nts to 2000 nts, 2000 nts to 3000 nts, 3000 nts to 4000 nts, 4000 nts to 5000 nts, or any range therebetween.
  • RNA therapeutics e.g., modified RNAs
  • TCMs useful in the compositions described herein.
  • modified mRNA encoding a protein of interest may be linked to the TCM described herein and expressed in vivo in a subject.
  • the modified RNA linked to a polypeptide described herein has modified nucleosides or nucleotides.
  • modifications are known and are described, e.g., in WO 2012/019168. Additional modifications are described, e.g., in WO2015038892; WO2015038892; WO2015089511 ; WO2015196130; WO2015196118 and
  • the modified RNA linked to the polypeptide described herein has one or more terminal modifications, e.g., a 5'Cap structure and/or a poly-A tail (e.g., of between 100-200 nucleotides in length).
  • the 5' cap structure may be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7- deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.
  • the modified RNAs also contain a 5 ' UTR comprising at least one Kozak sequence, and a 3 ' UTR.
  • modifications are known and are described, e.g., in WO2012135805 and WO2013052523. Additional terminal modifications are described, e.g., in WO2014164253 and WO2016011306. WO2012045075 and WO2014093924.
  • Chimeric enzymes for synthesizing capped RNA molecules (e.g., modified mRNA) which may include at least one chemical modification are described in WO2014028429.
  • a modified mRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 '-end binding proteins.
  • the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
  • the newly formed 5'-/3'- linkage may be intramolecular or intermolecular.
  • modifications are described, e.g., in WO2013151736.
  • modified RNAs are made using only in vitro transcription (IVT) enzymatic synthesis.
  • IVT in vitro transcription
  • Methods of making IVT polynucleotides are known in the art and are described in WO2013151666, WO2013151668, WO2013151663, WO2013151669, WO2013151670, WO2013151664, WO2013151665, WO2013151671, WO2013151672, WO2013151667 and WO2013151736.
  • S Methods of purification include purifying an RNA transcript comprising a polyA tail by contacting the sample with a surface linked to a plurality of thymidines or derivatives thereof and/or a plurality of uracils or derivatives thereof (polyT/U) under conditions such that the RNA transcript binds to the surface and eluting the purified RNA transcript from the surface (WO2014152031); using ion (e.g.
  • RNAs encoding proteins in the fields of human disease, antibodies, viruses, and a variety of in vivo settings are known and are disclosed in for example, Table 6 of International Publication Nos. WO2013151666, WO2013151668, WO2013151663,
  • the TCM comprises a CRISPR component that modulates (e.g., activates or inhibits) IL-7R signaling.
  • a CRISPR component that modulates (e.g., activates or inhibits) IL-7R signaling.
  • One method for modulating IL-7R signaling uses clustered regulatory interspaced short palindromic repeat (CRISPR) system for gene editing.
  • CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea.
  • CRISPR systems use RNA-guided nucleases termed CRISPR-associated or "Cas"
  • endonucleases e.g., Cas9 or Cpfl
  • Cas9 or Cpfl endonucleases
  • an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding "guide RNAs" that target single- or double- stranded DNA sequences.
  • target nucleotide sequence e. g., a site in the genome that is to be sequence-edited
  • guide RNAs sequence-specific, non-coding "guide RNAs" that target single- or double- stranded DNA sequences.
  • Three classes (I-III) of CRISPR systems have been identified.
  • the class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins).
  • One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA ("tracrRNA”).
  • the crRNA contains a "guide RNA", typically an about 20-nucleotide RNA sequence that corresponds to a target DNA sequence.
  • the crRNA also contains a region that binds to the tracrRNA to form a partially double- stranded structure which is cleaved by RNase III, resulting in a
  • the crRNA/tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave the target DNA sequence.
  • the target DNA sequence must generally be adjacent to a "protospacer adjacent motif ("PAM”) that is specific for a given Cas endonuclease; however, PAM sequences appear throughout a given genome.
  • PAM protospacer adjacent motif
  • CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'- NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis).
  • 5'-NGG Streptococcus pyogenes
  • 5'- NNAGAA Streptococcus thermophilus CRISPR1
  • 5'-NGGNG Streptococcus thermophilus CRISPR3
  • 5'-NNNGATT Neisseria meningiditis
  • Cas9 endonucleases are associated with G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5' from) the PAM site.
  • Another class II CRISPR system includes the type V endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from
  • Cpfl-associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words a Cpfl system requires only the Cpfl nuclease and a crRNA to cleave the target DNA sequence.
  • Cpfl endonucleases are associated with T-rich PAM sites, e.g., 5'-TTN. Cpfl can also recognize a 5'-CTA PAM motif.
  • Cpfl cleaves the target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5' overhang, for example, cleaving a target DNA with a 5- nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759 - 771.
  • CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281 - 2308. At least about 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavage to occur; for Cpfl at least about 16 nucleotides of gRNA sequence is needed to achieve detectable DNA cleavage.
  • guide RNA sequences are generally designed to have a length of between 17 - 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and complementarity to the targeted gene or nucleic acid sequence.
  • Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs.
  • Gene editing has also been achieved using a chimeric "single guide RNA" (“sgRNA”), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA- tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing).
  • sgRNA single guide RNA
  • the invention includes a composition comprising a sgRNA.
  • dCas9 can further be fused with an effector to repress (CRISPRi) or activate (CRISPRa) expression of a target gene.
  • CRISPRi is described in U.S. Publication No.: 2014/0068797.
  • CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations.
  • a catalytically dead Cas9 lacks endonuclease activity.
  • a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.
  • CRISPRi can work independently of host cellular machineries.
  • CRISPRi is when dCas9 is fused to Kruppel associated box (KRAB), a transcriptional repressor domain, transcription is repressed.
  • KRAB Kruppel associated box
  • CRISPRi is inducible, reversible, and no n- toxic; it also enables knockdown of non-coding RNAs.
  • CRISPRa activation of transcription is achieved by use of dCas9 protein containing a fused C-terminal end transcriptional activator.
  • activators may include, but are not limited to, VP64 (4X VP16), AtERF98 activation domain, or AtERF98x4
  • dCas9 is fused to the SunTag, a sequence containing multiple copies of the activator recruitment domain of general control protein (GCN4). This dCas9 fusion activates transcription.
  • GCN4 general control protein
  • This dCas9 fusion activates transcription.
  • the SunTag recruits multiple copies of various proteins, such as a tandem array of the transcriptional activator virus protein 16 (VP 16) to activate transcription in a robust manner.
  • Cas9 can be fused to a transcriptional repressor (e.g., a KRAB domain) or a transcriptional activator (e.g., a dCas9-VP64 fusion).
  • a transcriptional repressor e.g., a KRAB domain
  • a transcriptional activator e.g., a dCas9-VP64 fusion
  • a catalytically inactive Cas9 (dCas9) fused to Fokl nuclease (“dCas9-FokI”) can be used to generate DSBs at target sequences homologous to two gRNAs. See, e.
  • the invention includes a composition comprising a CRISPR endonuclease.
  • CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016/0138008 A 1 and US2015/0344912A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616.
  • Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 Al.
  • the desired genome modification involves homologous recombination, wherein one or more double- stranded DNA breaks in the target nucleotide sequence is generated by the RNA-guided nuclease and guide RNA(s), followed by repair of the break(s) using a homologous recombination mechanism ("ho mo logy-directed repair").
  • a donor template that encodes the desired nucleotide sequence to be inserted or knocked-in at the double- stranded break is provided to the cell or subject;
  • suitable templates include single- stranded DNA templates and double- stranded DNA templates (e.g., linked to the polypeptide described herein).
  • a donor template encoding a nucleotide change over a region of less than about 50 nucleotides is provided in the form of single- stranded DNA; larger donor templates (e.g., more than 100 nucleotides) are often provided as double- stranded DNA plasmids.
  • the donor template is provided to the cell or subject in a quantity that is sufficient to achieve the desired homo logy-directed repair but that does not persist in the cell or subject after a given period of time (e. g., after one or more cell division cycles).
  • a donor template has a core nucleotide sequence that differs from the target nucleotide sequence (e.g., a homologous endogenous genomic region) by at least 1 nucleotide, at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more than 50 nucleotides.
  • This core sequence is flanked by
  • the regions of high identity include at least 10 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 750 nucleotides, or at least 1000 nucleotides on each side of the core sequence.
  • the core sequence is flanked by homology arms including at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, or at least 100 nucleotides on each side of the core sequence.
  • the core sequence is flanked by homology arms including at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, or at least 1000 nucleotides on each side of the core sequence.
  • two separate double-strand breaks are introduced into the cell or subject's target nucleotide sequence with a "double nickase" Cas9 (see Ran et al. (2013) Cell, 154: 1380 - 1389), followed by delivery of the donor template.
  • the composition comprising a gRNA and a targeted nuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A), a dead Cas9 (dCas9), eSpCas9, Cpfl, C2C1, or C2C3, or a nucleic acid encoding such a nuclease, are used to modulate gene expression.
  • a Cas9 e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A), a dead Cas9 (dCas9), eSpCas9, Cpfl, C2C1, or C2C3, or a nucleic acid encoding such a nuclease
  • nuclease and gRNA(s) are determined by whether the targeted mutation is a deletion, substitution, or addition of nucleotides, e.g., a deletion, substitution, or addition of nucleotides to a targeted sequence.
  • Fusions of a catalytically inactive endonuclease e.g., a dead Cas9 (dCas9, e.g., D10A; H840A) tethered with all or a portion of (e.g., biologically active portion of) an (one or more) effector domain create chimeric proteins that can be linked to the polypeptide to guide the composition to specific DNA sites by one or more RNA sequences (sgRNA) to modulate activity and/or expression of one or more target nucleic acids sequences (e.g., to methylate or demethylate a DNA sequence).
  • sgRNA RNA sequences
  • the composition comprises one or more components of a CRISPR system described hereinabove.
  • the methods described herein include a method of delivering one or more CRISPR system component described hereinabove to a source.
  • a zinc finger protein is engineered to bind a predetermined DNA sequence. Fusing a zinc finger protein to a nuclease domain creates a zinc-finger nuclease (ZFN) that can cleave DNA adjacent to the specific ZFP-binding site. By designing a single chain quasi-dimeric ZFN with a predetermined DNA binding domain, the ZFN can recognize a pathogenic point mutation in the DNA, selectively cleave and eliminate the mutant DNA and thereby increase the proportion of wild type DNA.
  • the invention includes a composition comprising ZFN cleaved DNA.
  • the CRISPR components target a gene that results in activation of IL-7R as described herein. In some embodiments, the CRISPRa components activate an IL-7R gene as described herein.
  • the CRISPR components target a gene that results in inhibition of IL-7Ra or CD132 as described herein. In some embodiments, the CRISPRi components inhibit IL-7Ra or CD 132 gene as described herein.
  • the TCM may be an oligonucleotide aptamer that activates IL- 7R signaling.
  • the oligonucleotide aptamers (pieces of DNA that can create complex tertiary structures that mimic a protein) bind to IL-7Ra and CD 132.
  • the oligonucleotide aptamer induces phosphorylation of STAT5.
  • the TCM may be an oligonucleotide aptamer that inhibits IL- 7R signaling.
  • the oligonucleotide aptamers bind to IL-7Ra and/or CD132.
  • the oligonucleotide aptamer inhibits phosphorylation of STAT5.
  • Oligonucleotide aptamers are single- stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind to pre-selected targets including proteins and peptides with high affinity and specificity.
  • Oligonucleotide aptamers are nucleic acid species that may be engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers provide discriminate molecular recognition, and can be produced by chemical synthesis. In addition, aptamers possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • DNA and RNA aptamers show robust binding affinities for various targets.
  • DNA and RNA aptamers have been selected for lysozyme, thrombin, human immunodeficiency virus trans-acting responsive element (HIV TAR), hemin, interferon ⁇ , vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), dopamine, and the non-classical oncogene, heat shock factor 1 (HSF1).
  • Diagnostic techniques for aptamer based plasma protein profiling include aptamer plasma proteomics. This technology will enable future multi-biomarker protein
  • the TCM may be may be a peptide aptamer that activates or inhibits IL-7R signaling.
  • Peptide aptamers have one (or more) short variable peptide domains, including peptides having low molecular weight, 12-14 kDa. Peptide aptamers may be designed to specifically bind to and interfere with protein-protein interactions inside cells.
  • Peptide aptamers are artificial proteins selected or engineered to bind specific target molecules. These proteins include of one or more peptide loops of variable sequence. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind cellular protein targets and exert biological effects, including interference with the normal protein interactions of their targeted molecules with other proteins. In particular, a variable peptide aptamer loop attached to a transcription factor binding domain is screened against the target protein attached to a transcription factor activating domain. In vivo binding of the peptide aptamer to its target via this selection strategy is detected as expression of a downstream yeast marker gene.
  • Peptide aptamers can also recognize targets in vitro. They have found use in lieu of antibodies in biosensors and used to detect active isoforms of proteins from populations containing both inactive and active protein forms. Derivatives known as tadpoles, in which peptide aptamer "heads" are covalently linked to unique sequence double- stranded DNA "tails”, allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative real-time polymerase chain reaction) of their DNA tails.
  • Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system. Peptide aptamers can also be selected from
  • combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers. All the peptides panned from combinatorial peptide libraries have been stored in a special database with the name MimoDB.
  • the TCM is a small molecule agonist of IL-7 or TSLP that activates IL-7R signaling.
  • the TCM is a small molecule antagonist of IL-7 or TSLP that inhibits IL-7R signaling.
  • Small molecule moieties include, but are not limited to, small peptides,
  • pep tido mimetic s e.g., peptoids
  • amino acids amino acid analogs
  • synthetic polynucleotides polynucleotide analogs
  • nucleotides nucleotide analogs
  • organic and inorganic compounds including heterorganic and organometallic compounds
  • molecular weight less than about 5,000 grams per mole e.g., organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • Small molecules may include, but are not limited to, a neurotransmitter, a hormone, a drug, a toxin, a viral or microbial particle, a synthetic molecule, and agonists or antagonists that modulate IL-7R signaling.
  • the small molecule is a pharmaceutically active agent.
  • the small molecule is an inhibitor of a metabolic activity or component.
  • Useful classes of pharmaceutically active agents include, but are not limited to, antibiotics, antiinflammatory drugs, angiogenic or vasoactive agents, growth factors, and chemotherapeutic (anti-neoplastic) agents (e.g., tumor suppressers).
  • antibiotics antibiotics
  • antiinflammatory drugs angiogenic or vasoactive agents
  • growth factors e.g., tumor suppressers
  • chemotherapeutic (anti-neoplastic) agents e.g., tumor suppressers.
  • the invention includes a composition comprising an antibiotic, ant i- inflammatory drug, angiogenic or vasoactive agent, growth factor, or chemotherapeutic agent.
  • TCM is optimized for IL-7R activation by insertion, deletion, substitution, or alternative exon usage. These modifications can increase half-life, affinity, activity as well as specificity.
  • the TCM variant induces phosphorylation of STAT5 in a IL- 7R dependent manner.
  • the TCM variant induces activation of PI-3K in an IL-7R dependent manner. [0203] In some embodiments, the TCM variant induces gene expression of Bcl-2 in an IL-7R dependent manner.
  • the TCM variant stimulates differentiation of pre-pro B cells by engagement of the IL-7 receptor.
  • the TCM variant is a non-protein molecule that binds to IL-7R (CD127 and CD132) and induces conformation changes leading to downstream
  • the TCM variant is a molecule that enhances survival and proliferation of naive and memory CD4+ and CD8+ T cells dependent on engagement of the IL-7R.
  • the TCM variant is a molecule that stimulates proliferation of 2E8 cells (ATCC: TIB-239) in an IL-7Ra dependent manner.
  • the TCM variant is a molecule that increases thymus cellularity in an IL-7R dependent manner.
  • the TCM variant is a molecule that engages CD 127 via a ⁇ - helical turn within an alpha helix and shows surface complementarity with the hydrophobic platform presented by IL-7Ra that engages CD132 simultaneously.
  • the TCM variant interacts with IL-7RaDl by van der Waals contacts mediated by hydrophobic residues protruding from the CC'l and EF1 loops of IL- 7RaDl and hydrogen-bond interactions of main chain carbonyl oxygen atoms in the CCl and EF1 loops in IL-7RaDl with long side chains out from helix C. See, for example, Olosz and Malek, J Biol Chem, 275:30100-30105 (2000) for CD132 domains.
  • the TCM induces phosphorylation of STAT5 dependent on concomitant engagement of CD132.
  • the binding affinity of the TCM to IL-7Ra and/or CD132 may be optimized using display technology (phage display, yeast display, mammalian display). For example, specific amino acids in a helix may be altered, substituted, added, or deleted, through targeted or random alteration. DNA encoding a library of these altered TCMs may be cloned into a DNA vector and transformed into a yeast strain that enables surface display of the altered TCMs.
  • display technology phage display, yeast display, mammalian display.
  • specific amino acids in a helix may be altered, substituted, added, or deleted, through targeted or random alteration.
  • DNA encoding a library of these altered TCMs may be cloned into a DNA vector and transformed into a yeast strain that enables surface display of the altered TCMs.
  • a molecule containing portions of the extracellular region IL-7Ra and/or CD 132 may be used to identify yeast cells that express TCMs with altered binding affinity to IL-7Ra and/or CD 132, and differential isolation by flow cytometry or other selection methods to enrich yeast expressing TCMs with optimized binding characteristics.
  • the DNA from these TCMs may then be isolated and sequenced to identify the specific amino acid changes. These altered amino acids are identified as increased binding affinity to IL-7Ra and/or CD 132.
  • Similar methods such as phage display, mammalian display and mRNA display may also be used to identify TCM variants with increased binding affinity and/or increased receptor activation.
  • a method for screening for TCMs with increased binding affinity to IL-7Ra and/or CD 132.
  • the TCM variant induces IL-7R dependent and TSLP-Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant induces IL- 7R dependent and IL-2Ra and/or IL-2RP independent phosphorylation of STAT5. In some embodiments, the TCM variant induces IL-7R dependent but IL-2Ra and/or IL-2RP independent phosphorylation of STAT5. In some embodiments, the TCM variant induces IL- 7R dependent and IL-4Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant induces IL-7R dependent and IL-15Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant induces IL-7R dependent and IL-21Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant induces IL- 7R dependent and IL-21Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant induces IL- 7R dependent and IL-21Ra independent
  • TCM is optimized for IL-7R inhibition by insertion, deletion, substitution, or alternative exon usage. These modifications can decrease affinity, activity as well as specificity.
  • the TCM variant inhibits phosphorylation of STAT5 in an IL- 7R dependent manner.
  • the TCM variant inhibits activation of PI-3K in an IL-7R dependent manner.
  • the TCM variant inhibits gene expression of Bcl-2 in an IL-7R dependent manner.
  • the TCM variant inhibits differentiation of pre-pro B cells by engagement of the IL-7 receptor.
  • the TCM variant is a non-protein molecule that binds IL-7Ra and/or CD132 and inhibits downstream phosphorylation of STAT5.
  • the TCM variant is a molecule that decreases survival and proliferation of naive and memory CD4+ and CD8+ T cells dependent on engagement of the IL-7R.
  • the TCM variant is a molecule that inhibits proliferation of 2E8 cells (ATCC: TIB-239) in an IL-7Ra dependent manner.
  • the TCM variant is a molecule that inhibits thymus cellularity in an IL-7R dependent manner.
  • the TCM variant is a molecule that engages IL-7Ra via a ⁇ - helical turn within an alpha helix, while inhibiting engagement with CD 132.
  • the TCM inhibits phosphorylation of STAT5 by failing to simultaneously engage CD 132.
  • the binding affinity of TCM to either the IL-7Ra or CD 132, but not the other, may be optimized using display technology (phage display, yeast display, mammalian display). For example, specific amino acids in a helix may be altered, substituted, added, or deleted, through targeted or random alteration.
  • DNA encoding a library of these altered TCMs are cloned into a DNA vector and transformed into a yeast strain that enables surface display of the altered TCMs.
  • a molecule containing portions of the extracellular region IL-7Ra and/or CD 132 is used to identify yeast cells that express TCMs with binding affinity to either the IL- 7Ra or CD 132, but not the other, and differential isolation by flow cytometry or other selection methods to enrich yeast expressing TCMs with optimized binding characteristics.
  • the DNA from these TCMs may then be isolated and sequenced to identify the specific amino acid changes. These altered amino acids are identified as decreasing binding affinity to IL-7Ra or CD 132.
  • Similar methods such as phage display, mammalian display and mRNA display may also be used to identify TCM variants with decreased binding affinity and/or decreased receptor activation.
  • a method for screening for TCMs with decreased binding affinity to IL-7Ra or CD 132.
  • the TCM variant inhibits IL-7R dependent and TSLP-Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant inhibits IL- 7R dependent and IL-2Ra and/or IL-2RP independent phosphorylation of STAT5. In some embodiments, the TCM variant inhibits IL-7R dependent and IL-2Ra and/or IL-2RP independent phosphorylation of STAT5. In some embodiments, the TCM variant inhibits IL- 7R dependent and IL-4Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant inhibits IL-7R dependent and IL-15Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant inhibits IL-7R dependent and IL-21Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant inhibits IL- 7R dependent and IL-21Ra independent phosphorylation of STAT5. In some embodiments, the TCM variant inhibits IL- 7R dependent and IL-21Ra independent
  • inhibitory TCM blocks signaling through the IL-7R which can be of interest in autoimmune diseases that are associated with increased IL-7R signaling, such as multiple sclerosis or type 1 diabetes.
  • partial agonists may lead to weak activation of the IL-7R and downstream signaling.
  • TCM is modified to modulate stability (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95%) and thus interaction with its receptor.
  • stabilizing mutations are introduced within one or more a-helices.
  • TCM may be modified by substituting residues with low a- helical propensity (e.g., proline, glycine) to residues with high a-helical propensity (e.g., alanine).
  • TCM stability is modulated using display technology (phage display, yeast display, mammalian display) followed by binding assays such as surface plasmon resonance (Biacore).
  • display technology phage display, yeast display, mammalian display
  • binding assays such as surface plasmon resonance (Biacore).
  • one or more cysteines in the TCM are substituted with serine to reduce or decrease disulfide bond formation.
  • one or more serines in the TCM are substituted with cysteine to create or increase new disulfide bonds.
  • TCM solubility is modified (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95%), such as through the introduction of glycosylation sites.
  • TCM glycosylation
  • Solubility of TCM can be increased by artificially creation of at least one glycosylation site.
  • TCM solubility is modified by removing at least one glycosylation site.
  • the TCM comprises a linker comprising a Glu and Lys rich sequence to improve solubility.
  • TCM half-life or bioavailability is modified (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95%).
  • TCM is linked to a carrier molecule, such as an Fc receptor, albumin, polyethylene glycol (PEG) or SUMO.
  • a carrier molecule such as an Fc receptor, albumin, polyethylene glycol (PEG) or SUMO.
  • the linked molecule evades the host's immune system and reduces renal clearance and degradation.
  • TCM is linked to an antibody, such as anti-TCM monoclonal antibody to increase the half-life of TCM.
  • TCM is linked to at least one of: anti-IL-7 monoclonal antibody, soluble IL-7Ra (CD127), soluble CD132, and anti-CD132 monoclonal antibody.
  • glycosaminoglycan, heparan sulfate, or fibronectin may be modulated to increase serum half- life.
  • TCM linked to an immunoglobulin may modulate serum half-life.
  • an immunoglobulin e.g., a glycosylated human IgGl
  • TCM may be conjugated to one or more targeting agents.
  • TCM can be attached to an antibody, or antibody fragment, or targeting peptide directly or via a linker to reduced SH groups and/or to carbohydrate side chains.
  • Many methods for making covalent or non-covalent conjugates of therapeutic or diagnostic agents with antibodies or fusion proteins are known in the art and any such known method may be utilized.
  • TCM can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
  • TCM can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio propionate (SPDP). Yu et al., Int. J.
  • TCM can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
  • the carbohydrate group can be used to increase the loading of the same agent that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different therapeutic or diagnostic agent.
  • the Fc region may be absent if the antibody used as the antibody component of the immunoconjugate is an antibody fragment. However, it is possible to introduce a
  • carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, each incorporated herein by reference.
  • the engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
  • MMPs matrix metalloproteinases
  • composition described herein also includes one or more
  • heterologous moiety may be linked to the TCM described herein.
  • a TCM described herein is linked to one or more heterologous moieties.
  • a heterologous moiety may be an effector (e.g., a drug, small molecule), a targeting agent (e.g., a DNA targeting agent, antibody, receptor ligand), a tag (e.g., fluorophore, marker), or any of the TCMs described herein.
  • a targeting agent e.g., a DNA targeting agent, antibody, receptor ligand
  • a tag e.g., fluorophore, marker
  • the heterologous moiety is at least one selected from the group consisting of a domain of IL-7, TSLP, IL-2, IL- 4, IL-9, IL-15, IL-2; Fc domain; antibody (e.g., adalimumab); drug; small molecule; carrier molecule (e.g., increase half-life, stability, PEG, albumin); and targeting domain (e.g., receptor specificity or cell/tissue specificity).
  • the heterologous moiety is a small molecule (e.g., a
  • pep tido mimetic or a small organic molecule with a molecular weight of less than 2000 daltons
  • a peptide or polypeptide e.g., an antibody or antigen-binding fragment thereof
  • a nucleic acid e.g., siRNA, mRNA, RNA, DNA, an antisense RNA, a ribozyme, a therapeutic mRNA encoding a protein
  • a nanoparticle e.g., aptamer.
  • the heterologous moiety may cleaved from the TCM (e.g., after administration) by specific proteolysis or enzymatic cleavage (e.g., by TEV
  • protease Thrombin, Factor Xa, or Enteropeptidase
  • TCM is linked an antibody (bispecific or antibody fragment), a receptor, or a ligand that binds to a protein expressed in an organ specific manner.
  • TCM is linked to a cytokine, growth factor, or signaling molecule to confer additional characteristics to TCM.
  • HGF-beta fusion proteins can increase the expression of IL-7R on the cell surface and thus enhance IL-7R signaling.
  • TCM is linked to a molecule that engages the receptor c-met.
  • TCM is linked to a molecule that engages the GM-CSF-R.
  • TCM is linked to a molecule that increases phosphorylation of STAT5 in an IL-7R dependent manner.
  • TCM is linked to a molecule that increases concomitant phosphorylation of STAT5 and STAT-3.
  • TCM is linked to a molecule that increases concomitant phosphorylation of STAT5 and STAT-1 [0259] In some embodiments, TCM is linked to a molecule that inhibits PD-1 upregulation. PD-1 upregulation normally occurs as result of IL-7R signaling.
  • TCM is linked to a molecule that increases or stabilizes CD 127 on the cell surface.
  • B cell growth- stimulating factor PBSF
  • PBSF B cell growth- stimulating factor
  • TCM is linked to a molecule that enhances recycling of CD127.
  • TCM is linked to a molecule that enhances thymus cellularity.
  • TCM is linked to a molecule that enhances T cell activation and/or proliferation.
  • TCM is linked to soluble OX40L, soluble 4-1BBL or monoclonal antibodies against OX40 or 4- IBB to enhance T cell activation, and/or increase survival and memory T cell generation.
  • TCM is linked to a targeting antibody (e.g., for tumor specific surface antigen), and optionally, linked with a cleavable or non-cleavable linker.
  • a targeting antibody e.g., for tumor specific surface antigen
  • a heterologous moiety may be an effector moiety that possesses effector activity.
  • the effector moiety may modulate a biological activity, for example increasing or decreasing enzymatic activity, gene expression, cell signaling, and cellular or organ function.
  • Effector activities may also include binding regulatory proteins to modulate activity of the regulator, such as transcription or translation.
  • Effector activities also may include activator or inhibitor functions.
  • Effector activities may also include modulating protein stability/degradation and/or transcript stability/degradation. For example, proteins may be targeted for degradation by the polypeptide co-factor, ubiquitin, that marks proteins for degradation.
  • the heterologous moiety inhibits enzymatic activity by blocking the enzyme's active site, e.g., methotrexate is a structural analog of tetrahydrofolate, a coenzyme for the enzyme dihydrofolate reductase that binds to dihydrofolate reductase 1000-fold more tightly than the natural substrate and inhibits nucleotide base synthesis.
  • methotrexate is a structural analog of tetrahydrofolate, a coenzyme for the enzyme dihydrofolate reductase that binds to dihydrofolate reductase 1000-fold more tightly than the natural substrate and inhibits nucleotide base synthesis.
  • the composition comprises TCM that is operably linked to an effector moiety.
  • the effector moiety is a chemical, e.g., a chemical that modulates a cytosine (C) or an adenine(A) (e.g., Na bisulfite, ammonium bisulfite).
  • the effector moiety has enzymatic activity (nuclease (e.g., Cas9)).
  • a heterologous moiety may be a targeting moiety with targeted function.
  • the targeted moiety may modulate a specific function, modulate a specific molecule (e.g., enzyme, protein, or nucleic acid), and specifically bind for localization.
  • the targeted function may act on a specific molecule, e.g., a molecular target.
  • a heterologous moiety may include a targeted therapeutic that interacts with a specific molecular target to increase, decrease, or otherwise modulate its function.
  • the composition comprises a targeting moiety (e.g., gRNA or membrane translocating polypeptide) that is operably linked to TCM.
  • the targeting moiety may bind a target sequence (e.g., alters affinity for IL-7R, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95%).
  • a targeting moiety e.g., gRNA or membrane translocating polypeptide
  • the targeting moiety may bind a target sequence (e.g., alters affinity for IL-7R, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 5
  • TCM is linked to a molecule that targets a specific cell or tissue.
  • a non-exhaustive list of target proteins overexpressed in specific tissue types or their related vasculature includes:
  • Immune organs spleen, lymph nodes, thymus: CCL19, CCL21
  • Thymus CCL25/CCR9, E-cadherin/CD103
  • Mucus producing glandular cells e.g., Villin-1, MUC5B and serine proteases, such as SPINK4
  • GFAP GFAP, EN02, SNCB, and S-100 proteins, proteins involved in neurotransmission and neurological development, e.g., Gap43, OMG, INA, SLC1A3, and MAP2, other very significantly up-regulated proteins are Gap43 and SLC1A3
  • Endothelial cells CD 133
  • BBB Blood brain barrier
  • beta-endorphin or (D-Ala ) beta-endorphin or (D-Ala ), b-endorphin fusion (for transcytosis of cargo); high transferrin receptor, high insulin receptor
  • Kidney Organic Anion Transporting Polypeptide 4C1 (OATP4C1)
  • Liver Endothelial cells: CD44, Mannose/N- acetyl glucose amine receptor
  • Hepatocytes 60-70% of liver cells: Scavenger receptor class B type I, Glycyrrhizin receptors, asialoglycoprotein receptor
  • Sinusoidal Endothelial cells (20%): scavenger receptors: hyaluronan receptor, (pro)collagen receptor recognizing N-terminal propeptides of types I and III procollagen ... but all lead to receptor mediated endocytosis and degradation
  • Kupffer Cells Mannose/N-acetyl glucose amine R, Galactose particle receptor, Galactose specific receptor
  • Hepatic Stellate Cells (5-8%): Mannose - 6 -phosphate receptor, Type VI collagen receptor, PDGF receptor, Scavenger receptor class A
  • Endothelial cells PECAM-1 (the pulmonary vasculature contains roughly one-third of the endothelial cells in the body), CD36
  • Bone marrow
  • Endothelial cells CD37
  • GLP-1R Dipeptidyl peptidase IV (DPPIV)-resistant glucagon- like peptide 1 (GLP-1) analogues (e.g., exenatide) and DPPIV inhibitors (e.g., sitagliptin)
  • DPPIV Dipeptidyl peptidase IV
  • GLP-1-1 analogues e.g., exenatide
  • DPPIV inhibitors e.g., sitagliptin
  • SUR1 hyperglycaemia in type 2 diabetes can be alleviated by sulfonylureas such as glibenclamide that bind and block the sulfonylurea receptor 1 (SUR1) subunits of the inwardly rectifying ATP-sensitive potassium channel (KATP channel), closure of which is essential for membrane depolarisation that initiates insulin secretion
  • sulfonylureas such as glibenclamide that bind and block the sulfonylurea receptor 1 (SUR1) subunits of the inwardly rectifying ATP-sensitive potassium channel (KATP channel), closure of which is essential for membrane depolarisation that initiates insulin secretion
  • Antibodies targeting pancreas specific antigen (R2D6, DTPA-IC2, K14D10).
  • TCM is linked to a molecule, such as an antibody, ligand, receptor, that targets or specifically binds to a molecule associated with a specific disease cell/tissue or state.
  • a molecule such as an antibody, ligand, receptor
  • targets or specifically binds to a molecule associated with a specific disease cell/tissue or state include: Cancer targets:
  • Kidney Cancer Carbohydrate antigen 50 (CA 50), mucin-like cancer associated antigen
  • MCA renal cell carcinoma
  • Tissue polypeptide antigen TAA
  • CA 19.9 Tissue polypeptide antigen
  • SCC Squamous cell carcinoma antigen
  • TPA tissue polypeptide antigen
  • EGFR EGFR
  • IGFIR IGFIR
  • FAP FAP
  • EpCAM EpCAM
  • Lung cancer small cell cancer
  • NSE Neuron specific enolase
  • Lung cancer (Epidermoid cancer): Cytokeratin fragment 21.1 (Cyfra21.1)
  • Lung cancer (Adenocarcinoma): Carcinoembryonic antigen (CEA)
  • Ovarian cancer Chocarcinoma: Alpha fetoprotein (AFP), b human chorionic gonadotropin (b-hCG)
  • AFP Alpha fetoprotein
  • b-hCG human chorionic gonadotropin
  • Ovarian cancer (Serous cancer): CA 125
  • Esophageal cancer SCC, TPA, CEA
  • Testicular cancer AFP, ⁇ -hCG
  • Colorectal cancer CEA, CA 19.9, TPA, gpA33, Mucins, TAG-72, Le y , EGFR, ERBB2, ERBB3, EPHA3, TRAILR1, TRAILR2, FAP
  • EpCAM EpCAM
  • Prostate cancer Prostate specific antigen (PSA), prostatic acid phosphatase (PAP), RANKL, PSMA (prostate carcinoma), Tenascin, IGFIR, Le y , ERBB2, ERBB3, IGFIR, Tenascin Pancreatic cancer: CA 19.9, CEA, AFP, TRAILR1, TRAILR2, FAP
  • liver cancer AFP, Ferritin Melanoma: S-100, NSE, EPHA3
  • EpCAM EpCAM
  • AFP AFP
  • GPC3 glypcian-3
  • Thyroid cancer (Iodine as targeting agent), IGF1R
  • VEGFR Epithelial derived solid tumors
  • Gangliosides such as GD2, GD3 and GM2
  • Glioma EGFR, IGF1R, EPHA3, Tenascin
  • CD20 Non Hodgkin's lymphoma
  • CD52 Chronic lymphocytic leukemia
  • Tumor vasculature VEGF, Integrin ⁇ 3, Integrin ⁇ 5 ⁇ 1
  • CARSKNKDC SEQ ID NO: 21
  • CAR targets injured tissue undergoing early stages of regeneration; homologous to heparin-binding sites in various proteins (e.g., BMP4); utilizes surface heparan sulfate proteoglycans (HSPGs) for binding and efficient cell and tissue penetration
  • HSPGs surface heparan sulfate proteoglycans
  • CRKDKC Peptide CRKDKC (SEQ ID NO: 22) (CRK) targets injured tissue undergoing late stages of regeneration; structural similarity to segments in thrombospondin type 1 and 3 repeats
  • NRG CNGRC (SEQ ID NO: 23)
  • CD13 aminopeptidase N (CD13) isoform selectively expressed by endothelial cells in tumor vessels and increases tumor- homing of fusion partners
  • Antibodies that selectively target injured/ inflamed tissues are provided.
  • Antibody scFv F8 recognizes a domain of fibronectin expressed exclusively on inflammatory neo vasculature and would facilitate transport of TCM to inflamed joints in rheumatoid arthritis and other autoimmune sites
  • Antibody LI 9 recognizes a similar motif as antibody F8.
  • a targeting agent may be a viral peptide cell fusion inhibitor.
  • This may include the T-20 HIV-1 gp41 fusion inhibitor which targets fusion receptors on HIV infected cells (for T-20, see U.S. Pat. Nos. 6,281,331 and 6,015,881 to Kang et al.; Nagashima et al. J. Infectious Diseases 183: 1121, 2001; for other HIV inhibitors see U.S. Pat. No.
  • Targeting agents also include peptide hormones or peptide hormone analogues such as LHRH, bombesin/gastrin releasing peptide, somatastatin (e.g., RC-121 octapeptide), and the like, which may be used to target any of a variety of cancers ovarian, mammary, prostate small cell of the lung, colorectal, gastric, and pancreatic. See, e.g., Schally et al., Eur. J. Endocrinology, 141: 1-14, 1999.
  • peptide hormones or peptide hormone analogues such as LHRH, bombesin/gastrin releasing peptide, somatastatin (e.g., RC-121 octapeptide), and the like, which may be used to target any of a variety of cancers ovarian, mammary, prostate small cell of the lung, colorectal, gastric, and pancreatic. See, e.g
  • Peptide targeting agents suitable for use in targeting compounds of the invention also may be identified using in vivo targeting of phage libraries that display a random library of peptide sequences (see, e.g., Arap et al., Nature Medicine, 2002 8(2): 121-7; Arap et al., Proc. Natl. Acad. Sci. USA 2002 99(3): 1527- 1531; Trepel et al. Curr. Opin. Chem. Biol. 2002 6(3):399-404).
  • the targeting agent is specific for an integrin.
  • Integrins are heterodimeric transmembrane glycoprotein complexes that function in cellular adhesion events and signal transduction processes. Integrin ⁇ 3 is expressed on numerous cells and has been shown to mediate biologically relevant processes, including adhesion of osteoclasts to bone matrix, migration of vascular smooth muscle cells, and angiogenesis. Integrin ⁇ 3 antagonists likely have use in the treatment of several human diseases, including diseases involving neovascularization, such as rheumatoid arthritis, cancer, and ocular diseases.
  • Suitable targeting agents for integrins include RGD peptides or peptido mimetic s or non-RGD peptides or peptido mimetic s.
  • RGD peptides or peptido mimetic s or non-RGD peptides or peptido mimetic s.
  • Arg-Gly-Asp peptide or “RGD peptide” is intended to refer to a peptide having one or more Arg-Gly-Asp containing sequence which may function as a binding site for a receptor of the "Arg-Gly-Asp family of receptors", e.g., an integrin.
  • Integrins which comprise and alpha and abeta subunit, include numerous types including ⁇ , ⁇ 2 ⁇ 1, ⁇ 3 ⁇ 1, ⁇ 4 ⁇ 1, ⁇ 5 ⁇ 1, ⁇ 6 ⁇ 1, ⁇ 7 ⁇ 1, ⁇ 8 ⁇ 1, ⁇ 9 ⁇ 1, ⁇ , ⁇ 6 ⁇ 4, ⁇ 4 ⁇ 7, ⁇ 2, ⁇ 2, ⁇ 2, ⁇ 2, ⁇ , ⁇ 3, ⁇ 5, ⁇ , ⁇ 8, ⁇ 2, ⁇ 3 ⁇ 4 ⁇ 3, ⁇ 7, and the like.
  • the sequence RGD is present in several matrix proteins and is the target for cell binding to matrix by integrins.
  • Platelets contain a large amount of RGD-cell surface receptors of the protein GP Ilb/IIIa, which is primarily responsible, through interaction with other platelets and with the endothelial surface of injured blood vessels, for the development of coronary artery thrombosis.
  • RGD peptide also includes amino acids that are functional equivalents (e.g., RLD or KGD) thereof provided they interact with the same RGD receptor.
  • Peptides containing RGD sequences can be synthesized from amino acids by means well known in the art, using, for example, an automated peptide synthesizer, such as those manufactured by Applied Biosystems, Inc., Foster City, Calif.
  • non-RGD peptide refers to a peptide that is an antagonist or agonist of integrin binding to its ligand (e.g. fibronectin, vitronectin, laminin, collagen etc.) but does not involve an RGD binding site.
  • ligand e.g. fibronectin, vitronectin, laminin, collagen etc.
  • Non-RGD integrin peptides are known for ⁇ 3 (see, e.g., U.S. Pat. Nos. 5,767,071 and 5,780,426) as well as for other integrins such as ⁇ 4 ⁇ 1 (VLA-4), ⁇ 4 ⁇ 7 (see, e.g., U.S. Pat. No.
  • An integrin targeting agent may be a pep tido mimetic agonist or antagonist, which preferably is a pep tido mimetic agonist or antagonist of an RGD peptide or non-RGD peptide.
  • peptido mimetic is a compound containing non-peptidic structural elements that are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide.
  • a peptido mimetic of an RGD peptide is an organic molecule that retains similar peptide chain pharmacophore groups of the RGD amino acid sequence but lacks amino acids or peptide bonds in the binding site sequence.
  • a peptidomimetic of a non-RGD peptide is an organic molecule that retains similar peptide chain pharmacophore groups of the non-RGD binding site sequence but lacks amino acids or peptide bonds in the binding site sequence.
  • a "pharmacophore” is a particular three-dimensional arrangement of functional groups that are required for a compound to produce a particular response or have a desired activity.
  • the term "RGD peptidomimetic" is intended to refer to a compound that comprises a molecule containing the RGD pharmacophores supported by an organic/non- peptide structure. It is understood that an RGD peptidomimetic (or non-RGD
  • peptidomimetic may be part of a larger molecule that itself includes conventional or modified amino acids linked by peptide bonds.
  • RGD pe tido mimetic s are well known in the art, and have been described with respect to integrins such as GPIIb/IIIa, ⁇ 3 and ⁇ 5 (See, e.g., Miller et al., J. Med. Chem. 2000, 43:22-26; and International Patent Publications WO 0110867, WO 9915178, WO 9915170, WO 9815278, WO 9814192, WO 0035887, WO 9906049, WO 9724119 and WO 9600730; see also Kumar et al., Cancer Res.
  • RGD peptidomimetics are generally based on a core or template (also referred to as "fibrinogen receptor antagonist template"), to which are linked by way of spacers to an acidic group at one end and a basic group at the other end of the core.
  • the acidic group is generally a carboxylic acid functionality while the basic group is generally a N-containing moiety such as an amidine or guanidine.
  • the core structure adds a form of rigid spacing between the acidic moiety and the basic nitrogen moiety, and contains one or more ring structures (e.g., pyridine, indazole, etc.) or amide bonds for this purpose.
  • a fibrinogen receptor antagonist generally, about twelve to fifteen, more preferably thirteen or fourteen, intervening covalent bonds are present (via the shortest intramolecular path) between the acidic group of the RGD pep tido mimetic and a nitrogen of the basic group.
  • the number of intervening covalent bonds between the acidic and basic moiety is generally shorter, two to five, preferably three or four, for a vitronectin receptor antagonist.
  • the particular core may be chosen to obtain the proper spacing between the acidic moiety of the fibrinogen antagonist template and the nitrogen atom of the pyridine.
  • a fibrinogen antagonist will have an intramolecular distance of about 16 angstroms (1.6 nm) between the acidic moiety (e.g., the atom which gives up the proton or accepts the electron pair) and the basic moiety (e.g., which accepts a proton or donates an electron pair), while a vitronectin antagonist will have about 14 angstroms (1.4 nm) between the respective acidic and basic centers. Further description for converting from a fibrinogen receptor mimetic to a vitronectin receptor mimetic can be found in U.S. Pat. No. 6,159,964.
  • the pep tido mimetic RGD core can comprise a 5-11 membered aromatic or nonaromatic mono- or polycyclic ring system containing 0 to 6 double bonds, and containing 0 to 6 heteroatoms chosen from N, O and S.
  • the ring system may be unsubstituted or may be substituted on a carbon or nitrogen atom.
  • Preferred core structures with suitable substituents useful for vitronectin binding include monocyclic and bicyclic groups, such as benzazapine described in WO 98/14192, benzdiazapine described in U.S. Pat. No. 6,239,168, and fused tricyclics described in U.S. Pat. No. 6,008,213.
  • U.S. Pat. No. 6,159,964 contains an extensive list of references in Table 1 of that document which disclose RGD peptido mimetic cores structures (referred to as fibrinogen templates) which can be used for prepraring RGD pep tido mimetic s.
  • Preferred vitronectin RGD and fibronectin RGD peptido mimetic s are disclosed in U.S. Pat. Nos. 6,335,330;
  • TCM is linked to ligand of the CD 13 receptor.
  • the ligand can be an antibody or a fragment thereof such as Fab, Fv, single-chain Fv, a peptide or a peptido- mimetic, namely a peptido-like molecule capable to bind the aminopeptidase-N receptor (CD 13), optionally containing modified, not naturally occurring amino acids.
  • CD 13 receptor is also known as "NGR" receptor, in that its peptide ligands share the amino acidic "NGR" motif.
  • a chelating agent may be attached to TCM or to its carrier, such as an antibody, antibody fragment or fusion protein and used to chelate TCM.
  • Exemplary chelators include but are not limited to DTPA (such as Mx-DTPA), DOT A, TETA, NETA or NOTA.
  • DTPA such as Mx-DTPA
  • radioactive metals or paramagnetic ions may be attached to TCM or its carrier by reaction with a reagent having a long tail, to which may be attached a multiplicity of chelating groups for binding ions.
  • a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chains having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • porphyrins polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this
  • Chelates may be directly linked to polypeptides or antibodies, for example as disclosed in U.S. Pat. No. 4,824,659, incorporated herein by reference.
  • Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 1251, 1311, 1231, 1241, 62Cu, 64Cu, 18F, l l lln, 67Ga, 68Ga, 99mTc, 94mTc, 11C, 13N, 150, 76Br, for radio-imaging.
  • MRL Macrocyclic chelates such as NOT A, DOT A, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper, respectively.
  • metal- chelate complexes can be made very stable by tailoring the ring size to the metal of interest.
  • Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223Ra for RAIT are encompassed.
  • TCM is linked to a molecule that specifically binds to a target cell subset of the innate or adaptive immunity.
  • TCM is linked a peptide-MHC complex (monomer, dimer, oligomer or polymer) to target T cells that are specific for the used peptide-MHC complex.
  • a fusion would specifically activate or inactivate T cells according to their antigen specificity.
  • TCM may be linked to a MHC I molecule carrying a virus-antigen or cancer-neoantigen and activate CD8+ T cells that are specific for the antigen to enhance clearance of the virus or attack of the tumor.
  • TCM is linked to LAP (latency associated peptide) via a cleavable linker sensitive to proteases enriched in cancer (e.g. MMP2 and MMP9 in prostate cancer), infection (e.g., HIV-PR (HIV-1 protease), NS3 protease (HCV protease) or autoimmunity (e.g. MTl-MMP, MMP-13, stromelysin- 1 (MMP-3) and coUagenase- 1 (MMP- 1) in rheumatoid diseases).
  • cancer e.g. MMP2 and MMP9 in prostate cancer
  • infection e.g., HIV-PR (HIV-1 protease), NS3 protease (HCV protease) or autoimmunity (e.g. MTl-MMP, MMP-13, stromelysin- 1 (MMP-3) and coUagenase- 1 (MMP- 1) in rheum
  • TCM is linked to CCR9 to retain TCM in the gut and the thymus.
  • TCM is linked to CCR7 to retain TCM in lymphatic organs.
  • a heterologous moiety may be a tag to label or monitor the TCM described herein or another heterologous moiety linked to the TCM.
  • the tagging or monitoring moiety may be removable by chemical agents or enzymatic cleavage, such as proteolysis.
  • An affinity tag may be useful to purify the tagged TCM using an affinity technique.
  • Some examples include, chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S- transferase (GST), and poly(His) tag.
  • CBP chitin binding protein
  • MBP maltose binding protein
  • GST glutathione-S- transferase
  • poly(His) tag may be useful to aid recombinant proteins expressed in chaperone-deficient species such as E. coli to assist in the proper folding in proteins and keep them from precipitating.
  • the tagging or monitoring moiety may include thioredoxin (TRX) and poly(NANP).
  • TRX thioredoxin
  • the tagging or monitoring moiety may include a light sensitive tag, e.g., fluorescence. Fluorescent tags are useful for visualization. GFP and its variants are some examples commonly used as fluorescent tags. Protein tags may allow specific enzymatic modifications (such as biotinylation by biotin ligase) or chemical modifications (such as reaction with FlAsH-EDT2 for fluorescence imaging) to occur. Often tagging and monitoring moiety are combined, in order to connect proteins to multiple other components. The tagging or monitoring moiety may also be removed by specific proteolysis or enzymatic cleavage (e.g., by TEV protease, Thrombin, Factor Xa or Enteropeptidase).
  • a heterologous moiety may be a nucleic acid.
  • a nucleic acid heterologous moiety may include, but is not limited to, DNA, RNA, and artificial nucleic acids.
  • the nucleic acid may include, but is not limited to, genomic DNA, cDNA, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNAi molecule.
  • the nucleic acid is an siRNA to target a gene expression product.
  • the nucleic acid includes one or more nucleoside analogs as described herein.
  • heterologous moiety includes oligonucleotides, especially antisense oligonucleotides that are directed against oncogenes and oncogene products of B- cell malignancies, such as bcl-2.
  • antisense oligonucleotides include those known as siRNA or RNAi.
  • Nucleic acids can have a length from about 2 nts to about 5000 nts, about 10 nts to about 100 nts, about 50 nts to about 150 nts, about 100 nts to about 200 nts, about 150 nts to about 250 nts, about 200 nts to about 300 nts, about 250 nts to about 350 nts, about 300 nts to about 500 nts, about 10 nts to about 1000 nts, about 50 nts to about 1000 nts, about 100 nts to about 1000 nts, about 1000 nts to about 2000 nts, about 2000 nts to about 3000 nts, about 3000 nts to about 4000 nts, about 4000 nts to about 5000 nts, or any range therebetween.
  • Nucleic acids can have a length from 2 nts to 5000 nts, 10 nts to 100 nts, 50 nts to 150 nts, 100 nts to 200 nts, 150 nts to 250 nts, 200 nts to 300 nts, 250 nts to 350 nts, 300 nts to 500 nts, 10 nts to 1000 nts, 50 nts to 1000 nts, 100 nts to 1000 nts, 1000 nts to 2000 nts, 2000 nts to 3000 nts, 3000 nts to 4000 nts, 4000 nts to 5000 nts, or any range therebetween.
  • RNA therapeutics e.g., modified RNAs
  • heterologous moieties useful in the compositions described herein.
  • a modified mRNA encoding a protein of interest may be linked to the TCM described herein and expressed in vivo in a subject.
  • the modified RNA linked to a TCM described herein has modified nucleosides or nucleotides. Such modifications are known and are described, e.g., in WO 2012/019168. Additional modifications are described, e.g., in WO2015038892;
  • the modified RNA linked to the TCM described herein has one or more terminal modifications, e.g., a 5'Cap structure and/or a poly-A tail (e.g., of between 100-200 nucleotides in length).
  • the 5' cap structure may be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7- deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.
  • the modified RNAs also contains a 5 ' UTR comprising at least one Kozak sequence, and a 3 ' UTR.
  • modifications are known and are described, e.g., in WO2012135805 and WO2013052523. Additional terminal modifications are described, e.g., in WO2014164253 and WO2016011306. WO2012045075 and WO2014093924.
  • capped RNA molecules e.g., modified mRNA
  • capped RNA molecules e.g., modified mRNA
  • WO2014028429 Chimeric enzymes for synthesizing capped RNA molecules (e.g., modified mRNA) which may include at least one chemical modification are described in WO2014028429.
  • a modified mRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 '-end binding proteins.
  • the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
  • the newly formed 5'-/3'- linkage may be intramolecular or intermolecular.
  • modifications are described, e.g., in WO2013151736.
  • modified RNAs are made using only in vitro transcription (IVT) enzymatic synthesis.
  • IVT in vitro transcription
  • Methods of making IVT polynucleotides are known in the art and are described in WO2013151666, WO2013151668, WO2013151663, WO2013151669, WO2013151670, WO2013151664, WO2013151665, WO2013151671, WO2013151672, WO2013151667 and WO2013151736.
  • S Methods of purification include purifying an RNA transcript comprising a polyA tail by contacting the sample with a surface linked to a plurality of thymidines or derivatives thereof and/or a plurality of uracils or derivatives thereof (polyT/U) under conditions such that the RNA transcript binds to the surface and eluting the purified RNA transcript from the surface (WO2014152031); using ion (e.g.
  • RNAs encoding proteins in the fields of human disease, antibodies, viruses, and a variety of in vivo settings are known and are disclosed in for example, Table 6 of International Publication Nos. WO2013151666, WO2013151668, WO2013151663,
  • a heterologous moiety may be a nanoparticle.
  • Nanoparticles include inorganic materials with a size from about 1 nanometers (nm) and about 1000 nm, from about 1 nm and about 500 nm in size, from about 1 nm and about 100 nm, from about 50 nm and about 300 nm, from about 75 nm and about 200 nm, from about 100 nm and about 200 nm, and any range therebetween.
  • a nanoparticle has a composite structure of nanoscale dimensions.
  • nanoparticles are typically spherical although different morphologies are possible depending on the nanoparticle composition.
  • the portion of the nanoparticle contacting an environment external to the nanoparticle is generally identified as the surface of the nanoparticle.
  • the size limitation can be restricted to two dimensions and so that nanoparticles include composite structure having a diameter from about 1 nm to about 1000 nm, where the specific diameter depends on the nanoparticle composition and on the intended use of the nanoparticle according to the experimental design.
  • nanoparticles used in therapeutic applications typically have a size of about 200 nm or below.
  • Nanoparticle dimensions and properties can be detected by techniques known in the art. Exemplary techniques to detect particles dimensions include but are not limited to dynamic light scattering (DLS) and a variety of microscopies such at transmission electron microscopy (TEM) and atomic force microscopy (AFM).
  • DLS dynamic light scattering
  • TEM transmission electron microscopy
  • AFM atomic force microscopy
  • Exemplary techniques to detect particle morphology include but are not limited to TEM and AFM.
  • Exemplary techniques to detect surface charges of the nanoparticle include but are not limited to zeta potential method.
  • Additional techniques suitable to detect other chemical properties comprise by 1 H, U B, and 13 C and 19 F NMR, UV/Vis and infrared/Raman spectroscopies and fluorescence spectroscopy (when nanoparticle is used in combination with fluorescent labels) and additional techniques identifiable by a skilled person.
  • a heterologous moiety may be a small molecule.
  • Small molecule moieties include, but are not limited to, small peptides, pep tido mimetic s (e.g., peptoids), amino acids, amino acid analogs, synthetic polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heterorganic and organometallic compounds) generally having a molecular weight less than about 5,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • Small molecules may include, but are not limited to,
  • the small molecule is a pharmaceutically active agent.
  • the small molecule is an inhibitor of a metabolic activity or component.
  • Useful classes of pharmaceutically active agents include, but are not limited to, antibiotics, antiinflammatory drugs, angiogenic or vasoactive agents, growth factors and chemotherapeutic (anti-neoplastic) agents (e.g., tumor suppressers).
  • antibiotics antibiotics
  • antiinflammatory drugs angiogenic or vasoactive agents
  • growth factors e.g., tumor suppressers
  • chemotherapeutic (anti-neoplastic) agents e.g., tumor suppressers.
  • the invention includes a composition comprising an antibiotic, ant i- inflammatory drug, angiogenic or vasoactive agent, growth factor or chemotherapeutic agent.
  • a heterologous moiety may be a peptide or protein.
  • the peptide moieties may include, but is not limited to, a peptide ligand or antibody fragment that binds a receptor such as an extracellular receptor, neuropeptide, hormone peptide, peptide drug, toxic peptide, viral or microbial peptide, synthetic peptide, and agonist or antagonist peptide.
  • Peptides moieties may be linear or branched.
  • the peptide has a length from about 5 amino acids to about 200 amino acids, about 15 amino acids to about 150 amino acids, about 20 amino acids to about 125 amino acids, about 25 amino acids to about 100 amino acids, or any range therebetween.
  • the peptide has a length from 5 amino acids to 200 amino acids, 15 amino acids to 150 amino acids, 20 amino acids to 125 amino acids, 25 amino acids to 100 amino acids, or any range therebetween.
  • peptides include, but are not limited to, fluorescent tags or markers, antigens, antibodies, antibody fragments such as single domain antibodies, ligands and receptors, proteins from donor-derived cells or tissue, donor-derived MHC, donor- derived antigens, subject-derived MHC, subject-derived antigens, or any combination thereof.
  • Peptides useful as heterologous moiety described herein also include small antigen- binding peptides, e.g., antigen binding antibody or antibody-like fragments, such as single chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076- 113).
  • small antigen binding peptides may bind a cytosolic antigen, a nuclear antigen, an intra-organellar antigen.
  • the composition comprises a polypeptide linked to a ligand that is capable of targeting a specific location, tissue, or cell.
  • Antibody Fusion
  • TCM is linked to an antibody.
  • Antibody targeting strategies include, but are not restricted to, antibodies binding to CD45RO for targeting memory T cells, antibodies binding to CD45RA for naive T cells, antibodies binding to the gamma delta T cell receptor for ⁇ T cells, blocking antibodies against PD-1, CD 160, KLRG-1, or TIM-3 for targeting exhausted or senescent T cells.
  • TCM is linked a bispecific antibody that engages an antigen presenting cell (e.g., by binding to MHC class II without interfering with antigen
  • TCM is linked to an antibody, such as anti-TCM monoclonal antibody.
  • an antibody such as anti-TCM monoclonal antibody.
  • Complexing an agent with its soluble receptor or a blocking antibody prolongs their half-life.
  • TCM is linked to at least one of: anti-IL-7 monoclonal antibody, soluble IL-7Ra (CD127), soluble CD132, and anti-CD132 monoclonal antibody.
  • TCM linked to an immunoglobulin may modulate serum half- life.
  • the TCM is linked to a monoclonal antibody that blocks immune function.
  • monoclonal antibodies include IL-2 receptor-directed antibodies, such as basiliximab (Simulect) and daclizumab (Zenapax), the IgE-inhibiting antibody omalizumab, and the TNF-alpha inhibitors infliximab (Remicade), etanercept (Enbrel), and adalimumab (Humira).
  • TCM may be linked to a monoclonal antibody or antibody fragment that is monospecific, bispecific or multispecific.
  • Bispecific antibodies are useful in a number of biomedical applications. For instance, pre-targeting methods with bispecific antibodies comprising at least one binding site for TCM as well as at least one binding site a tumor-associated antigen (TAA), such as CEACAM 5 and/or CEACAM6, or a tissue specific antigen (see, e.g., U.S. Pat. Nos. 7,300,644; 7,138,103; 7,074,405; 7,052,872; 6,962,702; 6,458,933, the Examples section of each of which is incorporated herein by reference).
  • TAA tumor-associated antigen
  • bispecific antibodies comprising binding moieties targeting two different TAAs, or different epitopes of the same TAA, may be of therapeutic use.
  • Bispecific antibodies comprising the antigen-binding variable region sequences of any known anti-TAA antibody may be utilized, including but not limited to hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,151,164), hA19 (U.S. Pat. No. 7,109,304), MMMU31 (U.S. Pat. No. 7,300,655), hLLl (U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No. 7,387,772), hL243 (U.S. Pat. No.
  • antibodies of use may be commercially obtained from a wide variety of known sources.
  • a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, Va.).
  • a large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published and are available for use in the claimed methods and compositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;
  • antibody sequences or antibody- secreting hybridomas against almost any disease- associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest.
  • the antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art.
  • Bispecific antibodies can be produced by the quadroma method, which involves the fusion of two different hybridomas, each producing a monoclonal antibody recognizing a different antigenic site (Milstein and Cuello, Nature, 1983; 305:537- 540).
  • bispecific antibodies uses heterobifunctional cross- linkers to chemically tether two different monoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez, et al. Nature. 1985; 316:354-356). Bispecific antibodies can also be produced by reduction of each of two parental monoclonal antibodies to the respective half molecules, which are then mixed and allowed to reoxidize to obtain the hybrid structure (Staerz and Bevan. Proc Natl Acad Sci USA. 1986; 83: 1453-1457). Another alternative involves chemically cross-linking two or three separately purified Fab' fragments using appropriate linkers. (See, e.g., European Patent Application 0453082).
  • Other methods include improving the efficiency of generating hybrid hybridomas by gene transfer of distinct selectable markers via retrovirus-derived shuttle vectors into respective parental hybridomas, which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of a hybridoma cell line with expression plasmids containing the heavy and light chain genes of a different antibody.
  • Cognate VH and VL domains can be joined with a peptide linker of appropriate composition and length (usually consisting of more than 12 amino acid residues) to form a single-chain Fv (scFv) with binding activity.
  • a peptide linker of appropriate composition and length usually consisting of more than 12 amino acid residues
  • Methods of manufacturing scFvs are disclosed in U.S. Pat. Nos. 4,946,778 and 5,132,405, the Examples section of each of which is incorporated herein by reference. Reduction of the peptide linker length to less than 12 amino acid residues prevents pairing of VH and VL domains on the same chain and forces pairing of VH and VL domains with complementary domains on other chains, resulting in the formation of functional multimers.
  • Polypeptide chains of VH and VL domains that are joined with linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed triabodies) and tetramers (termed tetrabodies) are favored, but the exact patterns of oligomerization appear to depend on the composition as well as the orientation of V-domains (VH-linker-VL or VL-linker- VH), in addition to the linker length.
  • a heterologous moiety may be an oligonucleotide aptamer.
  • Aptamer moieties are oligonucleotide or peptide aptamers.
  • Oligonucleotide aptamers are single- stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind to pre-selected targets including proteins and peptides with high affinity and specificity.
  • Oligonucleotide aptamers are nucleic acid species that may be engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers provide discriminate molecular recognition, and can be produced by chemical synthesis. In addition, aptamers possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • DNA and RNA aptamers show robust binding affinities for various targets.
  • DNA and RNA aptamers have been selected for lysozyme, thrombin, human immunodeficiency virus trans-acting responsive element (HIV TAR), hemin, interferon ⁇ , vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), dopamine, and the non-classical oncogene, heat shock factor 1 (HSF1).
  • HIV TAR human immunodeficiency virus trans-acting responsive element
  • VEGF vascular endothelial growth factor
  • PSA prostate specific antigen
  • HSF1 heat shock factor 1
  • Diagnostic techniques for aptamer based plasma protein profiling include aptamer plasma proteomics. This technology will enable future multi-biomarker protein
  • a heterologous moiety may be a peptide aptamer.
  • Peptide aptamers have one (or more) short variable peptide domains, including peptides having low molecular weight, 12- 14 kDa.
  • Peptide aptamers may be designed to specifically bind to and interfere with protein- protein interactions inside cells.
  • Peptide aptamers are artificial proteins selected or engineered to bind specific target molecules. These proteins include of one or more peptide loops of variable sequence. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind cellular protein targets and exert biological effects, including interference with the normal protein interactions of their targeted molecules with other proteins. In particular, a variable peptide aptamer loop attached to a transcription factor binding domain is screened against the target protein attached to a transcription factor activating domain. In vivo binding of the peptide aptamer to its target via this selection strategy is detected as expression of a downstream yeast marker gene.
  • Peptide aptamers can also recognize targets in vitro. They have found use in lieu of antibodies in biosensors and used to detect active isoforms of proteins from populations containing both inactive and active protein forms. Derivatives known as tadpoles, in which peptide aptamer "heads" are covalently linked to unique sequence double- stranded DNA "tails”, allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative real-time polymerase chain reaction) of their DNA tails.
  • Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system. Peptide aptamers can also be selected from
  • combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers. All the peptides panned from combinatorial peptide libraries have been stored in a special database with the name MimoDB.
  • compositions described herein may also include a linker.
  • the TCM comprises a linker comprising a Glu and Lys rich sequence to improve solubility.
  • the composition described herein has a linker between the TCM and a heterologous moiety.
  • one or more TCMs described herein are linked with a linker.
  • a linker may be a chemical bond, e.g., one or more covalent bonds or non- covalent bonds.
  • the linker is a peptide linker. Such a linker may be from 2-30 amino acids, or longer.
  • the linker includes flexible, rigid, or cleavable linkers described herein.
  • Flexible linkers may be useful for joining domains that require a certain degree of movement or interaction and may include small, non- polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce unfavorable interactions between the linker and the protein moieties.
  • Cleavable linkers may release free functional domains in vivo.
  • linkers may be cleaved under specific conditions, such as the presence of reducing reagents or proteases.
  • In vivo cleavable linkers may utilize the reversible nature of a disulfide bond.
  • One example includes a thrombin- sensitive sequence (e.g., PRS) between the two Cys residues.
  • PRS thrombin- sensitive sequence
  • In vitro thrombin treatment of CPRSC results in the cleavage of the thrombin- sensitive sequence, while the reversible disulfide linkage remains intact.
  • Such linkers are known and described, e.g., in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality.
  • In vivo cleavage of linkers in fusions may also be carried out by proteases that are expressed in vivo under pathological conditions (e.g., cancer or inflammation), in specific cells or tissues, or constrained within certain cellular compartments.
  • pathological conditions e.g., cancer or inflammation
  • the specificity of many proteases offers slower cleavage of the linker in constrained compartments.
  • linking molecules include a hydrophobic linker, such as a negatively chared sulfonate group; lipids, such as a poly (-CH 2 -) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof, noncarbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecule capable of covalently linking two or more polypeptides.
  • lipids such as a poly (-CH 2 -) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof, noncarbon linkers
  • PEG polyethylene glycol
  • Non-covalent linkers are also included, such as hydrophobic lipid globules to which the TCM is linked, for example through a hydrophobic region of the TCM or a hydrophobic extension of the TCM, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine or other hydrophobic residue.
  • the TCM may be linked using charge-based chemistry, such that a positively charged moiety of the TCM is linked to a negative charge of another polypeptide or nucleic acid.
  • two or more TCM are linked with a linker.
  • the linker can be designed to allow each of the TCM in the multimer to bind IL-7Ra.
  • the TCM comprises a linker (e.g., GS linker or another amino acid sequence), wherein the TCM binds to IL-7Ra and CD132 and induces phosphorylation of STAT5.
  • a linker e.g., GS linker or another amino acid sequence
  • Dimers, oligomers, or multimers of TCM can bind to and crosslink/ aggregate several receptor complexes at the same time, clustering thereby also the intracellular signaling machinery which could enhance downstream signaling and thus the biological effect
  • Dimerization, oligomerization or multimerization can be achieved by direct fusion, disulfide bridges, and fusions including one or more linker regions (e.g., GS linker), Fc fusions, and Leucine zippers.
  • linker regions e.g., GS linker
  • Fc fusions Fc fusions
  • Leucine zippers Leucine zippers
  • Heterotypic dimers, oligomers, or multimers to TSLP or gamma chain cytokines would act similar to homotypic fusions, aggregating different receptors and intracellular signaling molecules.
  • cytokines i.e., TCM-IL-2, TCM-IL-4, TCM-IL-15, TCM-IL-9, and TCM-IL-21
  • All the listed cytokines signal at least partially through STAT5, it is expected to have a significantly increased phosphorylation and thus activation of STAT5 signaling that is key to IL-7 mediated survival of T cells.
  • the other signaling pathways may also be enhanced.
  • Dimerization, oligomerization, or multimerization can be achieved by direct fusion, disulfide bridges, fusions including one or several linker regions (e.g., GS linker, Fc fusions, and Leucin zippers).
  • linker regions e.g., GS linker, Fc fusions, and Leucin zippers.
  • a composition described herein includes encapsulated in naturally derived vesicles, e.g., membrane vesicles prepared from cells or tissues, which vesicles carry the composition.
  • naturally derived vesicles e.g., membrane vesicles prepared from cells or tissues, which vesicles carry the composition.
  • compositions or TCM described herein can be encapsulated in engineered substrates such as described in, e.g., in Orive. et al. 2015. Cell encapsulation: technical and clinical advances. Trends in Pharmacology Sciences; 36 (8):537-46; and m Mishra. 2016. Handbook of Encapsulation and Controlled Release. CRC Press.
  • a composition described herein includes the TCM encapsulated in synthetic vesicles, e.g., liposomes or exosomes.
  • a composition described herein is encapsulated in synthetic vesicles, e.g., liposomes or exosomes.
  • Liposomes and exosomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011.
  • BBB blood brain barrier
  • Exosomes are small membrane vesicles that are secreted by a multitude of cell types as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane. Depending on their origin, exosomes can play roles in different physiological processes. Maturing reticulocytes externalize obsolete membrane proteins such as the transferrin receptor by means of exosomes, whereas activated platelets release exosomes whose function is not yet known. Exosomes are also secreted by cytotoxic T cells, and these might ensure specific and efficient targeting of cytolytic substances to target cells. Antigen presenting cells, such as B lymphocytes and dendritic cells, secrete MHC class-I- and class- Il-carrying exosomes that stimulate T cell proliferation in vitro.
  • Antigen presenting cells such as B lymphocytes and dendritic cells, secrete MHC class-I- and class- Il-carrying exosomes that stimulate T cell proliferation in vitro.
  • Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Vesicles may comprise without limitation DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference).
  • vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155/2011/469679 for review).
  • Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.
  • additives may be added to vesicles to modify their structure and/or properties.
  • either cholesterol or sphingomyelin may be added to the mixture in order to help stabilize the structure and to prevent the leakage of the inner cargo.
  • vesicles can be prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and diacetyl phosphate, (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011.
  • vesicles may be surface modified during or after synthesis to include reactive groups complementary to the reactive groups on the carrier cells.
  • reactive groups include without limitation maleimide groups.
  • vesicles may be synthesized to include maleimide conjugated phospholipids such as without limitation DSPE-MaL-PEG2000.
  • a vesicle formulation may be mainly comprised of natural phospholipids and lipids such as l,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.
  • DSPC l,2-distearoryl-sn-glycero-3-phosphatidyl choline
  • sphingomyelin sphingomyelin
  • egg phosphatidylcholines monosialoganglioside.
  • Formulations made up of phospholipids only are less stable in plasma. However, manipulation of the lipid membrane with cholesterol reduces rapid release of the encapsulated bioactive compound into the plasma or 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID
  • lipids may be used to form lipid microparticles.
  • Lipids include, but are not limited to, DLin-KC2-DMA4, C 12-200 and colipids
  • disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4; doi: 10.1038/mtna.2011.3) using a spontaneous vesicle formation procedure.
  • the component molar ratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidyl choline/cholesterol/PEG- DMG).
  • Tekmira has a portfolio of approximately 95 patent families, in the U.S. and abroad, that are directed to various aspects of lipid microparticles and lipid microparticles
  • Some vesicles and lipid-coated polymer particles are able to spontaneously adsorb to cell surfaces.
  • composition described herein includes the TCM
  • a composition described herein is encapsulated in microparticles or microgels.
  • Microparticles are comprised of one or more solidified polymer(s) that is arranged in a random manner.
  • the microparticles may be biodegradable.
  • Biodegradable microparticles may be synthesized using methods known in the art including without limitation solvent evaporation, hot melt microencapsulation, solvent removal, and spray drying. Exemplary methods for synthesizing microparticles are described by Bershteyn et al., Soft Matter 4: 1787-1787, 2008 and in US 2008/0014144 Al, the specific teachings of which relating to microparticle synthesis are incorporated herein by reference.
  • microparticles are biodegradable in nature and thus they gradually degrade in an aqueous environment such as occurs in vivo.
  • TCMs may be released from the microparticles as the microparticle degrades or components of the composition may be released through pores within the microparticles. Release kinetic studies have been performed and they demonstrate that protein and small-molecule drugs can be released from such microparticles over time-courses ranging from 1 day to at least 2 weeks.
  • Exemplary synthetic polymers which can be used to form the biodegradable microparticles include without limitation aliphatic polyesters, poly (lactic acid) (PLA), poly (glycolic acid) (PGA), co-polymers of lactic acid and glycolic acid (PLGA),
  • polycarprolactone PCL
  • PCL polycarprolactone
  • polyanhydrides poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone)
  • natural polymers such as albumin, alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof, including substitutions, additions of chemical groups such as for example alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof.
  • these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • the microparticles' diameter ranges from 0.1-1000 micrometers ( ⁇ ). In some embodiments, their diameter ranges in size from 1-750 ⁇ , or from 50-500 ⁇ , or from 100- 250 ⁇ . In some embodiments, their diameter ranges in size from 50-1000 ⁇ , from 50-750 ⁇ 3 ⁇ 4 from 50-500 ⁇ , or from 50-250 ⁇ . In some embodiments, their diameter ranges in size from .05-1000 ⁇ , from 10-1000 ⁇ , from 100-1000 ⁇ , or from 500-1000 ⁇ .
  • their diameter is about 0.5 ⁇ , about 10 ⁇ , about 50 ⁇ , about 100 ⁇ 3 ⁇ 4 about 200 ⁇ , about 300 ⁇ , about 350 ⁇ , about 400 ⁇ , about 450 ⁇ , about 500 ⁇ 3 ⁇ 4 about 550 ⁇ , about 600 ⁇ , about 650 ⁇ , about 700 ⁇ , about 750 ⁇ , about 800 ⁇ 3 ⁇ 4 about 850 ⁇ , about 900 ⁇ , about 950 ⁇ , or about 1000 ⁇ .
  • the term "about” means+/-5% of the absolute value stated.
  • a ligand is conjugated to the surface of the microparticle via a functional chemical group (carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls) present on the surface of the particle and present on the ligand to be attached.
  • a functional chemical group carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls
  • Functionality may be introduced into the microparticles by, for example, during the emulsion preparation of microparticles, incorporation of stabilizers with functional chemical groups.
  • microparticle preparation Another example of introducing functional groups to the microparticle is during post- particle preparation, by direct crosslinking particles and ligands with homo- or
  • heterobifunctional crosslinkers This procedure may use a suitable chemistry and a class of crosslinkers (CDI, ED AC, glutaraldehydes, etc. as discussed in more detail below) or any other crosslinker that couples ligands to the particle surface via chemical modification of the particle surface after preparation.
  • This also includes a process whereby amphiphilic molecules such as fatty acids, lipids or functional stabilizers may be passively adsorbed and adhered to the particle surface, thereby introducing functional end groups for tethering to ligands.
  • the microparticles may be synthesized to comprise one or more targeting groups on their exterior surface to target a specific cell or tissue type (e.g., cardio myocytes). These targeting groups include without limitation receptors, ligands, antibodies, and the like. These targeting groups bind their partner on the cells' surface. In some embodiments, the microparticles will integrate into a lipid bilayer that comprises the cell surface and the composition is delivered to the cell.
  • a targeting group include without limitation receptors, ligands, antibodies, and the like. These targeting groups bind their partner on the cells' surface.
  • the microparticles will integrate into a lipid bilayer that comprises the cell surface and the composition is delivered to the cell.
  • the microparticles may also comprise a lipid bilayer on their outermost surface.
  • This bilayer may be comprised of one or more lipids of the same or different type. Examples include without limitation phospholipids such as phosphocholines and phosphoinositols. Specific examples include without limitation DMPC, DOPC, DSPC, and various other lipids such as those described herein for liposomes.
  • the vesicles or microparticles described herein are
  • diagnostic agents include, but are not limited to, commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.
  • PET positron emissions tomography
  • CAT computer assisted tomography
  • single photon emission computerized tomography x-ray, fluoroscopy
  • MRI magnetic resonance imaging
  • contrast agents include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium.
  • Vesicles can be engineered to release TCM (alone or together with other
  • the vesicles can be decorated by proteins that allow organ specific or cell specific targeting. These vesicles can be coated with antibodies or other proteins or peptides that allow organ or cell specific targeting.
  • the TCM is incorporated on the surface of or embedded in a matrix of nanoparticles or microparticles.
  • nanoparticles and microparticles are described elsewhere in and also include iron-based beads, agarose-based hydrogels.
  • the TCM is incorporated on the surface of or inside a liposome. Liposomes are described elsewhere herein.
  • the TCM is incorporated on the surface of or inside exosomes. Exosomes are described elsewhere herein.
  • the TCM is incorporated on the surface of or inside an antibody-conjugated liposome (immunoliposome) to deliver the TCM to a tissue or cell as directed by the specificity of the bound antibody.
  • immunosorbome antibody-conjugated liposome
  • beads are coated with TCM.
  • TCM described herein may be administered alone.
  • TCM may be administered before, concurrently with, or after at least one other therapeutic agent.
  • an antibody, fragment or fusion protein may be covalently or non-covalently attached to at least one therapeutic and/or diagnostic agent to form an immunoconjugate.
  • Therapeutic agents are preferably selected from the group consisting of a
  • radionuclide an immunomodulator, an anti-angiogenic agent, a cytokine, a chemokine, a growth factor, a hormone, a drug, a prodrug, an enzyme, an oligonucleotide, a pro-apoptotic agent, a photoactive therapeutic agent, a cytotoxic agent, which may be a chemotherapeutic agent or a toxin, and a combination thereof.
  • the drugs of use may possess a pharmaceutical property selected from the group consisting of antimitotic, antikinase, alkylating,
  • Exemplary drugs of use include, but are not limited to, 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyc lines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin (CDDP), COX-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin
  • Diagnostic agents are preferably selected from the group consisting of a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a
  • diagnostic agents are well known and any such known diagnostic agent may be used.
  • diagnostic agents may include a radionuclide such as HOIn, l l lln, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 90Y, 89Zr, 94mTc, 94Tc, 99mTc, 1201, 1231, 1241, 1251, 1311, 154-158Gd, 32P, 11C, 13N, 150, 186Re, 188Re, 51Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb, 83Sr, or other gamma-, beta-, or positron-emitters.
  • a radionuclide such as HOIn, l l lln, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga
  • Paramagnetic ions of use may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), or erbium (III).
  • Metal contrast agents may include lanthanum (III), gold (III), lead (II), or bismuth (III).
  • Ultrasound contrast agents may comprise liposomes, such as gas filled liposomes.
  • Radiopaque diagnostic agents may be selected from barium compounds, gallium compounds, and thallium compounds.
  • fluorescent labels are known in the art, including but not limited to fluorescein isothiocyanate (FITC), rhodamine, phycoerytherin (PE), allophycocyanin (APC), o-phthaldehyde and fluorescamine.
  • Chemiluminescent labels of use may include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt,and an oxalate ester.
  • Toxins of use may include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
  • RNase ribonuclease
  • Immunomodulators of use may be selected from a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof. Specifically useful are
  • lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (IL), colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF), interferon, such as
  • interferons-a, - ⁇ or - ⁇ , and stem cell growth factor include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-a and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin;
  • growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone
  • parathyroid hormone such as thyroxine
  • insulin proinsulin
  • relaxin prorelaxin
  • glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (
  • vascular endothelial growth factor vascular endothelial growth factor
  • integrin thrombopoietin
  • TPO nerve growth factors
  • nerve growth factors such as NGF-B ; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, and - ⁇ ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL- 25, LIF, kit-ligand or FL
  • Chemokines of use include CCL7, Fractalkine, I-TAC, MIG, RANTES, MCAF, MlPl-alpha, MIPl-Beta, and IP- 10.
  • Radioactive isotopes useful for treating diseased tissue include, but are not limited to: l l lln, 177Lu, 212Bi, 213Bi, 211 At, 62Cu, 67Cu, 90Y, 1251, 1311, 32P, 33P, 47Sc, l l lAg, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au, and 21 lPb.
  • the therapeutic radionuclide preferably has a decay energy in the range of 20 keV to 6,000 keV, preferably in the ranges 60 keV to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.
  • Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100- 4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles.
  • beta-particle-emitting nuclides are preferably ⁇ 1,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV. Also preferred are radionuclides that substantially decay with generation of alpha-particles.
  • Such radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, and Fm- 255. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000- 10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
  • radioisotopes of use include 11C, 13N, 150, 75Br, 198Au, 224Ac, 1261, 1331, 77Br, 113mln, 95Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 105Rb, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201T1, 225 Ac, 76Br, 169Yb, and the like.
  • Some useful diagnostic nuclides may include 1241, 1231, 1311, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 89Zr, 94Tc, 94mTc, 99mTc, or l l lln.
  • Therapeutic agents may include a photoactive agent or dye.
  • Fluorescent compositions such as fluorochrome, and other chromogens, or dyes, such as porphyrins sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain (1986), 22:430. Moreover, monoclonal antibodies have been coupled with photo activated dyes for achieving
  • TCM can be administered in combination with a corticosteroid hormone to increase the effectiveness of other chemotherapy agents.
  • a corticosteroid hormone for example, prednisone and dexamethasone are corticosteroid hormones.
  • TCM can be administered in combination with a bispecific antibody that has specificity to endogenous IL-7 and a target cell type, organ, tissue or disease state of interest as described herein.
  • TCM can be administered in combination with a molecule for one or more of the following: increases an anti-tumor response, enhance an antigen specific T cell response, enhance cytotoxicity, enhance clearance of tumor, enhance virus clearance, enhance anti- virus response, for treatment of a chronic infection, for treatment of HIV in combination with HAART, for treatment of Hepatitis C in combination with anti- viral agents, increase survival of T cells (naive and memory), for expansion of T cells (naive and memory), treat lymphopenia (induced by chemotherapy, other drugs, radiation, infection or idiopathic), for treatment of sepsis in combination with antibiotics, enhancement of vaccinations, boost thymic T cell development, counteract immune exhaustion, enhance immune function, enhance T cell helpers, ex vivo expansion of CAR-T cells, in vivo expansion of CAR-T cells, ex vivo expansion of patient T cells for autologous T cell therapy, and differentiation of pre-pro B cells.
  • anti-angiogenic agents such as angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGF peptides and antibodies, anti- vascular growth factor antibodies, anti-Flk-1 antibodies, anti- Fit- 1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrophage migration-inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, IP- 10, Gro- ⁇ , thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan poly sulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K
  • oligonucleotides especially antisense oligonucleotides that preferably are directed against oncogenes and oncogene products of B- cell malignancies, such as bcl-2.
  • Preferred antisense oligonucleotides include those known as siRNA or RNAi.
  • TCM can be administered in combination with one or more gamma chain cytokines.
  • a combination of TCM and IL-15 for anti-tumor therapy enhances memory T cell generation, T cell survival and function, and NK cell function.
  • TCM can be administered in combination with one or more compounds that inhibit upregulation of PD-1.
  • type 1 interferon may inhibit TCM induced upregulation of PD- 1.
  • TCM can be administered in combination with one or more checkpoint inhibitors for additive or synergistic effects on the treatment of cancer or chronic infections (e.g., anti-PD-1, anti-PDLl, anti-CTLA4 antibodies, soluble OX40L, soluble 4- 1BB ligand).
  • checkpoint inhibitors for additive or synergistic effects on the treatment of cancer or chronic infections (e.g., anti-PD-1, anti-PDLl, anti-CTLA4 antibodies, soluble OX40L, soluble 4- 1BB ligand).
  • TCM can be administered in combination with one or more cancer therapeutics, such as irradiation and chemotherapy.
  • cancer therapeutics such as irradiation and chemotherapy.
  • Chemotherapy and irradiation lead to death of tumor cells and release of tumor antigens.
  • the liberation of cancer cell antigens in the presence of TCM will enhance tumor antigen specific T cell responses.
  • TCM can be administered in combination with one or more anti- viral drugs.
  • TCM enhances T cell responses and increase efficacy of anti- viral drugs for virus elimination.
  • TCM can be administered in combination with or as a fusion with decorin to amplify the immuno stimulatory effects of TCM by blocking TGF- ⁇ .
  • TCM can be administered in combination with one or more vaccines.
  • TCM boosts memory T cell generation and would thereby increase efficacy of the vaccines.
  • TCM can be administered in combination with one or more antibiotics for the treatment of sepsis.
  • TCM can be administered in combination with IL-12.
  • the combination with TCM may yield additive or synergistic effects of activating antigen presenting cells.
  • patient-derived cells e.g., dendritic cells, stromal cells, induced pluripotent stem cells, and chimeric antigen receptor T cells
  • TCM-encoding DNA e.g., TGF-encoding DNA
  • TCM is linked to a transmembrane domain using a linker that is resistant to metalloprotease cleavage retain accessibility of IL-7R interaction domains.
  • a vector comprising a suicide gene is cloned to also encode this TCM fusion and transfected into patient-derived cells. These transgenic cells are administered to increase the patient's T cell repertoire.
  • a protein may be expressed in genetically engineered cells using recombinant methods known in the art.
  • a nucleic acid sequence coding for a protein of interest can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the protein, by deriving the gene from a vector known to include the same, or by isolating the gene that encodes the protein, transcript of the protein or the protein directly from cells or tissues containing the same, using standard techniques.
  • the protein of interest can be produced synthetically, rather than
  • Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding the gene of interest to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration in eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence.
  • Additional promoter elements e.g., enhancers, regulate the frequency of
  • transcriptional initiation typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor- la (EF- la).
  • EF- la Elongation Growth Factor- la
  • other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus
  • MMTV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV avian leukemia virus promoter
  • Epstein-Barr virus immediate early promoter a Rous sarcoma virus promoter
  • human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • the invention should not be limited to the use of constitutive promoters.
  • Inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced into the source can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes may be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient source and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity.
  • reporter gene Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • TCM or heterologous moiety are routine in the art. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar &
  • the TCM of the composition can be biochemically synthesized by employing standard solid phase techniques. Such methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods can be used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • recombinant methods may be used. Methods of making a recombinant therapeutic polypeptide are routine in the art. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).
  • Exemplary methods for producing a therapeutic pharmaceutical protein or polypeptide involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under the control of appropriate promoters.
  • Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking
  • nontranscribed sequences and 5' or 3' nontranslated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).
  • compositions described herein may include a vector, such as a viral vector, e.g., a lentiviral vector, encoding the recombinant protein.
  • a viral vector e.g., a lentiviral vector
  • the vector e.g., a viral vector, that comprises the nucleic acid encoding the recombinant protein.
  • the invention includes a method of at least one of modulating phosphorylation of STAT5; modulating survival of naive T cells; modulating survival of memory CD4+ T cells; modulating survival of memory CD8+ T cells; modulating proliferation of T cells; modulating activation of PI-3K; modulating gene expression of Bcl- 2; modulating differentiation of pre-pro B cells; and/or modulating proliferation of 2E8 cells (ATCC: TIB -239), comprising contacting the composition described herein to a cell comprising IL-7R.
  • the invention includes a method of at least one of increasing
  • the invention includes a method of increasing thymus cellularity in a subject, comprising administering to the subject an effective amount of the composition described herein.
  • the administration alters at least one of immune cell localization, activation of immune cells, survival of immune cells, differentiation of immune cells, and proliferation of immune cells.
  • the invention includes a method of increasing or enhancing an immune response in a subject comprising administering to the subject the composition described herein in an amount effective to increase or enhance the immune response in the subject.
  • the administration comprises administering subject-derived cells (e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells) modified to express the TCM.
  • subject-derived cells e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells
  • the immune response is an antiviral, anti-bacterial, or anti-parasitic response.
  • the invention includes a method of increasing or enhancing an antitumor response in a subject comprising administering to the subject the composition described herein in an amount effective to increase or enhance the anti-tumor response in the subject.
  • the invention includes a method of screening for a T cell modulator (TCM) that binds to IL-7Ra and at least one of the following: increases phosphorylation of STAT5; increases survival of naive T cells; increases survival of memory CD4+ T cells; increases survival of memory CD8+ T cells; stimulates proliferation of T cells; increases activation of PI-3K; increases gene expression of Bcl-2; stimulates differentiation of pre-pro B cells; and stimulates proliferation of 2E8 cells (ATCC: TIB-239).
  • TCM T cell modulator
  • the invention includes a method of at least one of decreasing phosphorylation of STAT5; decreasing survival of naive T cells; decreasing survival of memory CD4+ T cells; decreasing survival of memory CD8+ T cells; inhibiting proliferation of T cells; decreasing activation of PI-3K; increasing gene expression of Bcl-2; decreasing differentiation of pre-pro B cells; and/or inhibiting proliferation of 2E8 cells (ATCC: TIB- 239), comprising contacting the composition described herein to a cell comprising IL-7R.
  • the invention includes a method of decreasing thymus cellularity in a subject, comprising administering to the subject an effective amount of the composition described herein.
  • the administration alters at least one of immune cell localization, activation of immune cells, survival of immune cells, differentiation of immune cells, and proliferation of immune cells.
  • the invention includes a method of decreasing or inhibiting an immune response in a subject comprising administering to the subject the composition described herein in an amount effective to decrease or inhibit the immune response in the subject.
  • the administration comprises administering subject-derived cells (e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells) modified to express the TCM.
  • subject-derived cells e.g., dendritic cells, stromal cells, induced pluripotent stem cells, chimeric antigen receptor T cells
  • the immune response is an autoimmune, allergic or inflammatory response, e.g., multiple sclerosis, type 1 diabetes, rheumatoid arthritis, lupus, sarcoidosis and inflammatory bowel disease.
  • the invention includes a method of decreasing or inhibiting an autoimmune, allergic or inflammatory response in a subject comprising administering to the subject the composition described herein in an amount effective to decrease or inhibit the auto-immune, allergic or inflammatory response in the subject.
  • the invention includes a method of screening for a T cell modulator (TCM) that binds to IL-7Ra or CD132 and at least one of the following: decreases phosphorylation of STAT5; decreases survival of naive T cells; decreases survival of memory CD4+ T cells; decreases survival of memory CD8+ T cells; inhibits proliferation of T cells; decreases activation of PI-3K; decreases gene expression of Bcl-2; inhibits differentiation of pre-pro B cells; and inhibits proliferation of 2E8 cells (ATCC: TIB-239).
  • TCM T cell modulator
  • compositions comprising TCM, to be delivered to a subject can comprise one or more pharmaceutically acceptable vehicles, one or more additional ingredients, or some combination of these.
  • TCM can be formulated according to known methods to prepare pharmaceutically useful compositions.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable vehicle.
  • the admin stration of a pharmaceutical composition comprising the TCM described herein may be by way of oral, inhaled, transdermal or parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, and subcutaneous) administration.
  • the active compound may be administered alone or preferably formulated as a
  • a unit dose will normally contain 0.01 mg to 500 nig, for example 0.01 mg to 50 mg, or 0.01 mg to 10 mg, or 0.05 mg to 2 mg of compound or a pharmaceutically acceptable salt thereof.
  • Unit doses will normally be administered once or more than once a day, for example 2, 3, or 4 times a day, more usually 1 to 3 times a day, such that the total daily dose is normally in the range of 0.0001 mg/kg to 10 mg kg; thus a suitable total daily dose for a 70 kg adult is 0.01 mg to 700 mg, for example 0.01 nig to 100 mg, or 0.01 mg to 10 mg or more usually 0.05 mg to 10 mg.
  • the compound or a pharmaceutically acceptable salt thereof is administered in the form of a unit-dose composition, such as a unit dose oral, parenteral, transdermal or inhaled composition.
  • a unit-dose composition such as a unit dose oral, parenteral, transdermal or inhaled composition.
  • Such compositions are prepared by admixture and are suitably adapted for oral, inhaled, transdermal or parenteral administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and infusable solutions or suspensions or suppositories or aerosols.
  • Tablets and capsules for oral administration are usually presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tableting agents, lubricants, disintegrants, colourants, flavorings, and wetting agents.
  • the tablets may be coated according to well-known methods in the art.
  • Suitable fillers for use include cellulose, mannitol, lactose and other similar agents.
  • Suitable disintegrants include starch,
  • polyvinylpyrrolidone and starch derivatives such as sodium starch glycollate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylceilulose, carboxymethyi cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
  • suspending agents for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylceilulose, carboxymethyi cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example le
  • Oral formulations also include conventional sustained release formulations, such as tablets or granules having an enteric coating.
  • compositions for inhalation are presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose,
  • the particles of active compound suitably ha ve diameters of less than 50 microns, preferably- less than 10 microns, for example from 1 microns and 5 microns, such as from 2 microns and 5 microns.
  • coated nanoparticles can be used, with a particle size from 30 nm and 500 nm.
  • a favored inhaled dose will be in the range of 0.05 mg to 2 mg, for example 0.05 mg to 0.5 mg, 0.1 mg to 1 mg or 0.5 mg to 2 mg.
  • fluid unit dose forms are prepared containing a compound of the present invention and a sterile vehicle.
  • the active compound depending on the vehicle and the concentration, can be either suspended or dissolved.
  • Parenteral solutions are normally prepared by dissolving the compound in a vehicle and filter sterilising before filling into a suitable vial or ampoule and sealing.
  • adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle.
  • the composition can be frozen after filling into the vial and the water removed under vacuum.
  • Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilised by exposure to ethylene oxide before suspending in the sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound.
  • small amounts of bronchodilators for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamoi, phenylephrine, and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included.
  • TCM binds to the IL-7Ra (CD 127) and to the common gamma chain (CD 132) to induce signaling and to mediate its immunomodulatory effect.
  • TCM is a 4 helix bundle protein.
  • TCM is a 4 helix bundle based on IL-7 in which helix B is replaced by a linker that takes the structure of an alpha helix (AEAAAKEAAAKEAAAKA (SEQ ID NO: 25)) followed by the flexible linker (GGGGS) 4 (SEQ ID NO: 26).
  • AEAAAKEAAAKEAAAKA SEQ ID NO: 25
  • GGGGS flexible linker
  • a C-terminal His 6 tag facilitates purification in this example.
  • Helix A EGKDGKQYESLLMLSIDELL (SEQ ID NO: 28)
  • Helix B AEAAAKEAAAKEAAAKA (SEQ ID NO: 25)
  • Helix D LCFLKRLLQEIKTCWNKILM (SEQ ID NO: 3)
  • TCM is a polypeptide consisting of a 4 helix bundle fused to a Fc domain that prolongs TCM's serum half- life. Dimerization of TCM increases avidity and thus receptor engagement by TCM.
  • the Fc domain of this example has the following sequence:
  • EPKS CD KTHTCPPCP APELLGGPS VFLFPPK KDTLMIS RTPE VTC V VD VS HE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 31)
  • the TCM is fused to the N-terminus of the Fc domain, the C- terminus of the Fc domain or to a side group in the Fc domain.
  • the C-terminus of the TCM is fused to the N-terminus of the Fc domain via the flexible linker GGGGSGGGGSGGGGS (SEQ ID NO: 32).
  • the TCM is based on IL-7, and the following amino acid substitutions are included to stabilize H2-bonds to IL-7Ra. Note that the amino acid number designations refer to the final amino acid sequence below.
  • Helix A EGKDGKRYEEVLMVEIDELL (SEQ ID NO: 33)
  • Helix B AEAAAKEAAAKEAAAKA (SEQ ID NO: 25)
  • Helix D LCFLKRLLQEIKTCWNKILM (SEQ ID NO: 3)
  • TCM is composed of 3 alpha helices from IL-7 and one alpha helix derived from human IL-21 that are connected by the linker GGGGS GGGGS (SEQ ID NO: 35).
  • TCM is a fusion protein linked to an anti-CEA antibody (cited in Patent US 20150125386 Al) for treatment of CEA+ frontline ovarian cancer.
  • anti-CEA antibody heavy chain sequence is as follows:
  • VHTFP A VLQS S GLYS LS S V VT VPS S S LGTQT YICN VNHKPS NTKVD KRVEPKS CD K
  • anti-CEA antibody light chain sequence is as follows:
  • the heavy chain and light chain are connected via disulfide bonds.
  • the TCM is linked to the C-terminus of the heavy chain via the flexible linker KESGSVSSEQLAQFRSLD (SEQ ID NO: 17).
  • TCM sequence is as follows:
  • TCM-NGR fusion protein is described for the treatment of cancer.
  • TCM-NGR fusion protein improves accumulation of TCM at tumor sites.
  • amino acid substitutions in the TCM which is based on IL-7, are included to stabilize hydrogen-bond interactions with IL-7Ra. Note that the amino acid number designations refer to the final amino acid sequence below.
  • a flexible (GGGGS) 2 linker (SEQ ID NO: 35) is inserted C-terminal to the second alpha helix.
  • the TCM is connected to the NGR motive GCNGRC (SEQ ID NO: 39) via another flexible (GGGGS) 2 linker (SEQ ID NO: 35).
  • polyethylene glycol (PEG) coated nanoparticles carrying TCM and anti-PD-1 antibody for treatment of chronic infections or cancer is described.
  • Nanoparticles are coated with hydrophilic polymers to avoid wash out and remain in the bloodstream for a longer period of time that can sufficiently target cancerous cells.
  • Hydrophilic polymer coating on the nanoparticle surface repels plasma proteins and escapes from being opsonized and cleared. This is described as a "cloud” effect.
  • Commonly used hydrophilic polymers include PEG, poloxamines, poloxamers, polysaccharides, and so forth.
  • Cancerous cells have some unique properties that differentiate them from the healthy cells at molecular level. Some receptors are over expressed on the surface of them that make the distinguishing feature. Attachment of the complementary ligands on the surface of nanoparticles makes them able to target only the cancerous cells (reviewed for example by Sutradhar and Amin. ISRN Nanotechnology. Volume 2014 (2014)). When these
  • TCM in this example is useful for the treatment of cancer, and its function assessed in a mouse model of cancer, for example the melanoma xenograft model B 16F10, implanted in C57B1/6 mice.
  • the melanoma-B 16F10 cell line is obtained from American Type Culture Collection (ATCC; Rockville MD) and B 16F10 cells are maintained in culture as adherent monolayers in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1-glutamine and penicillin-streptomycin (Gibco BRL; Rockville MD). Cells in the exponential growth phase are harvested at 80% confluence, using brief exposure to 0.1% trypsin solution. Only single-cell suspensions with 95% viability are used for inoculation.
  • DMEM Dulbecco's modified Eagle's minimal essential medium
  • mice For tumorigenesis experiments, syngeneic C57BL/6J male mice (6 animals per group, 10-12 weeks of age), are subcutaneously inoculated with 5 x 10 5 cells of B 16F10 cells suspended in 150 ⁇ of PBS into each flank. Tumor growth is monitored daily from day 7. Mice are treated with TCM (nanoparticles coated with anti-PD-1 monoclonal antibody and TCM) or empty nanoparticles as negative control, on days 7, 9 and 11. The tumor volume is measured daily until the end point and is calculated using the formula (0-4 ab2), with 'a' as the larger diameter and 'b' as the smaller diameter.
  • TCM nanoparticles coated with anti-PD-1 monoclonal antibody and TCM
  • TCM is a fusion protein with 3 helices from TSLP and 1 helix from IL-7 linked via a (GGGGS) 4 (SEQ ID NO: 26) linker.
  • the TCM also contains a C-terminal His6 tag (SEQ ID NO: 27).
  • the first 3 helices are derived from human TSLP.
  • a Cys to Ser mutation (underlined below) is introduced to avoid formation of unwanted disulfide bonds:
  • TCM is linked to an anti-TCM antibody by a linker sensitive to
  • MMP-2 and MMP-9 for treating MMP-2 / MMP-9 positive tumors.
  • TCM is covalently bound to its targeted receptor CD 127 via the
  • MMP-2 and MMP-9 sensitive linker IPVSLRSG SEQ ID NO: 47.
  • the short linker GGGG SEQ ID NO: 48 is included both N-terminal and C-terminal to the IPVSLRSG linker (SEQ ID NO: 47).
  • This TCM-CD127 complex keeps the TCM inactive until the MMP-2/MMP-9 sensitive linker is cleaved by MMP-2 or MMP-9. Once the linker is cleaved, the presence of soluble CD 127 (sCD127) enhances the biological function of TCM. It is known that many cytokines are more potent when complexed by a cognate receptor or antibody. It is also known that MMP-2 and MMP-9 are overexpressed in a variety of tumors. Thus, TCM in this example is specifically activated at the site of MMP-2 and/or MMP-9 positive tumors.
  • MMP-2 and MMP-9 have preferential cleavage sites such as PLG x MWS (SEQ ID NO: 44); PVG x LIG (SEQ ID NO: 45); IPVS x LRSG (SEQ ID NO: 47); IPVS x LYSG (SEQ ID NO: 46) (wherein "x" defines the cleavage site).
  • the linker construct is shown here: GGGGIPVSLRSGGGGG (SEQ ID NO: 49)
  • the TCM is a dimer with the linker GGGGSGGGGSGGGGS (SEQ ID NO: 32) to connect them. This increases TCM stability and half-life.
  • the TCM has the following sequence:
  • TCM is a heterodimer with IL-21 to boost T cell function and induce memory T cell formation. Their combined actions increase downstream signaling events by bringing the receptors with their respective adapter proteins into proximity.
  • the linker GGGGSGGGGSGGGGS (SEQ ID NO: 32) is used to connect the TCM and IL-21.
  • the TCM-IL-21 heterodimer in this example has the following sequence:
  • TCM-HGF-beta is a heterodimer to increase the biological activity of TCM, in part by upregulating expression of the receptor that TCM uses for signaling.
  • TCM is fused to human recombinant HGF-beta via a flexible (GGGGS) 3 linker (SEQ ID NO: 32).
  • TCM-HGF-beta fusion protein The full sequence of the TCM-HGF-beta fusion protein is:
  • RMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS SEQ ID NO: 53.
  • the TCMs block IL-7 mediated activation of the IL-
  • TCM is a truncated version of human IL-7, missing exon 2, and dimerized via a fusion to an Fc domain.
  • TCM is a shorter version of human IL-7, missing exon 4, thereby lacking the domain that is known to engage IL-7Ra.
  • the TCM in this example has the following point mutations in exon 2 (Helix A):
  • the TCM is a homodimer with human IL-7 is which exons 5 and 6 are deleted and the following amino acid substitutions are included to enhance binding affinity to IL-7Ra. Note that the amino acid substitutions indicate the substitutions in the N- terminal TCM domain. The C-terminal TCM domain also contains the equivalent substitutions.
  • any one of the TCMs described above is cloned into a pET plasmid vector (pET17b) to express TCM with six histidine residues (SEQ ID NO: 27) at the amino- terminal portion using a T7 RNA polymerase expression system (Novagen) in E. coli BL21(DE3) or pLysE (Novagen).
  • the His-tagged TCM is cloned into the pFUSE-hFcl vector
  • HEK293 cells ATCC
  • DMEM Dulbecco's Modified Eagle Medium
  • GlutaMAX GlutaMAX
  • GIBCO GlutaMAX
  • GIBCO fetal calf serum
  • zeocin zeocin
  • TCM-IL-15 fusion protein Mammalian expression of a TCM-IL-15 fusion protein using CHO cells (alternative expression in BHK or HEK293 cells is possible):
  • the cells are seeded at an appropriate density in Corning Erlenmeyer Flasks.
  • DNA and transfection reagent are mixed at an optimal ratio and then added into the flask with cells ready for transfection.
  • the recombinant plasmids encoding target protein is transiently transfected into suspension CHO- 3E7 cell cultures. The cell culture supernatant is collected on day 6 and is used for purification.
  • codon-optimized TCM DNA is fused to mouse Fc (TCM-mFc) or human Fc (TCM-hFc) and cloned into pcDNA3.1 vector.
  • the encoding plasmids are stably transfected into Chinese hamster ovary (CHO) cell lines. Cells are cultured in Ex-Cell CHO DHFR animal-component-free medium (SAFC, USA). Production of TCM in bioreactor culture conditions
  • the most stable positive clone expressing TCM is adapted to serum-free suspension culture by several media and components screenings in order to produce a clone optimized for productivity and growth in high cell density culture.
  • pre-cultures are performed in the "wave bag" system.
  • Cell culture is then performed in a 100 L to 2000 L bioreactor with a perfusion system or a fed-batch system for 10 to 15 days.
  • Cells are amplified to a concentration of 10 millions cells/ml in a low- glutamine content medium supplemented with plant peptones.
  • the culture temperature is regulated at 37°C to increase cell density.
  • TCM expression is boosted by addition of 0.5- lOmM sodium butyrate in the medium.
  • TCM expression is monitored both inside the cells and in the culture medium. Presence of glucose and glutamine as well as good oxygenation support production of high molecular weight glycoforms. Amino acids consumption is monitored and amino acids are added to the culture as required. Cells are harvested as soon as cell viability decreases below 90%.
  • Supernatants are filtered through a 0.22 ⁇ filter. Sterile supernatant is then fractionated utilizing an Amersham HisTrap FF 5 ml Nickel affinity column. Protein is eluted over a 20- 500 mM imidazole gradient with the bulk of the purified PA2934-His eluting at -100 mM. Fractions containing the protein are concentrated using an Amicon Ultra 15 centrifugation filter with a cutoff of 10 kDa as per manufacturer's instructions followed by dialysis against 20 mM HEPES buffer pH 7.5 containing mM NaCl. Protein concentration is determined utilizing the Biorad protein assay kit. Other filtration systems with similar porosity such as Centrasette Cassette apparatus, membrane cut off 10 kDa (Pall Life Sciences) can be used to reduce the volume of supernatant.
  • TCM is purified from cell culture medium by in depth filtration on clarification capsules or modules such as Mustang XT capsule (Pall), Sartoclear P. (Sartorius), Millistak+ Opticap (Millipore) or hollow fiber cartridges (AXH cross flow 10 (GE)) or equivalent.
  • clarification capsules or modules such as Mustang XT capsule (Pall), Sartoclear P. (Sartorius), Millistak+ Opticap (Millipore) or hollow fiber cartridges (AXH cross flow 10 (GE)) or equivalent.
  • recombinant (His-tagged) protein produced by E. coli (strain BL21) is purified from the soluble supernatant or the insoluble inclusion body of isopropyl-b-D- thiogalactopyranoside (IPTG)-induced batch cultures by affinity chromatography with the one-step QIAexpress Ni-nitrilotriacetic acid (NT A) agarose matrix (Qiagen) in the presence of 8 M urea. Briefly, an overnight saturated culture of BL21 containing the pET construct is added to yeast extract-tryptone medium containing ampicillin and chloramphenicol and grown at 37°C with shaking.
  • the bacterial cultures are induced with IPTG at an optical density (OD) at 560 nm and grown for an additional 3 h.
  • Cells are harvested from batch cultures by centrifugation and resuspended in binding buffer (0.1 M sodium phosphate, pH 8; 10 mM Tris-HCl, pH 8) containing PMSF and leupeptin.
  • binding buffer 0.1 M sodium phosphate, pH 8; 10 mM Tris-HCl, pH 8) containing PMSF and leupeptin.
  • E. coli is lysed by adding lysozyme and incubated at 4°C following sonication, then spun to pellet the inclusion bodies.
  • the inclusion bodies are washed three times in 1% 3-[(3-cholamido-proply)dimethyl ammonio]- 1-propanesulfonic acid in 10 mM Tris-HCl (pH 8).
  • the inclusion body is solubilized in binding buffer containing 8 M urea.
  • Recombinant Ags with His-Tag residues are batch bound to Ni-NTA.
  • the concentrated supernatant is centrifuged, adjusted to pH 7.5, and applied to a Q Sepharose Fast Flow (General Electric Healthcare) column equilibrated with 0.05M sodium phosphate pH 7.5. The protein is recovered in the flow through.
  • Mustang Q membrane cassettes are used for better yield and/or slightly faster process.
  • Another alternative is to capture the protein on a strong Anion exchanger resin such as Q Ceramic Hyper D (Biosepra), Capto Q (GE), or membrane (Sartobind Q, Sartorius). After this prepurification step, a capture step is performed on a strong cation exchanger resin. The flow from the previous step is loaded onto a Fractogel EMD S03' (Merck) column
  • HIC Hydrophobic Interaction Chromatography
  • Tosoh Hydrophobic Interaction Chromatography
  • TCM is eluted with 25 column volumes of a salt gradient ranging from 1.5 M to 2.0 M ammonium sulphate in 0.05 M sodium phosphate pH 7.
  • HIC resin such as hexyl Toyopearl 650-M (Tosoh), Butyl/Octyl SepharoseTM 4 Fast Flow (General Electric Healthcare), is utilized for this step.
  • HIC resin such as hexyl Toyopearl 650-M (Tosoh), Butyl/Octyl SepharoseTM 4 Fast Flow (General Electric Healthcare)
  • Another alternative to HIC for scaling up purposes is to use another matrix such as MEP HyperCel (Pall Biosepra) for similar results.
  • the protein fractions obtained from G25 step is loaded onto a Source 15S (General Electric Healthcare) column equilibrated with the loading buffer (20 mM sodium acetate pH 6). This polishing step results in protein concentration and elimination of the residual contaminants.
  • the column is washed with sodium acetate loading buffer and the TCM protein is eluted with 15 column volumes of a salt gradient ranging from 0 to 1 M NaCI in 20 mM sodium acetate pH 6. Eluted fractions are separated by SDS-PAGE and stained with either Coomassie blue or silver nitrate. Only the fractions containing TCM are pooled.
  • purification process may also include an additional combination of two filtrations to guaranty optimal viral clearance.
  • Viral removal can be achieved by filtration using a prefiltration device (Planova 75, Asahi Kasei Medical) followed by a nanoporous cellulose membranes (Planova 20N, Asahi Kasei Medical) or by other viral removal membranes (Virosart, Sartorius; DV20, Millipore).
  • the separated proteins are transferred onto 0.45 um pore- size nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio- Rad), and the membranes are blocked by a 2 hour incubation with phosphate buffered saline (PBS) containing 0.2% Tween-20 and 5% non-fat dry milk (Sigma).
  • PBS phosphate buffered saline
  • the membranes are then probed overnight at 4°C with mouse or anti-his tag antibodies and washed in PBS containing 0.2% Tween 20.
  • the membranes are subsequently incubated for 1 hour at room temperature with europium-conjugated goat-anti-mouse IgG antibody (AbCam), washed thoroughly and imaged using the ScanLater Western Blot Detection Cartridge in the SpectramAx i3x multimode detection platform (Molecular Devices).
  • AbCam europium-conjugated goat-anti-mouse IgG antibody
  • ScanLater Western Blot Detection Cartridge in the SpectramAx i3x multimode detection platform (Molecular Devices).
  • the above protocol is identically conducted except the TCM solutions are separated on a 12% acrylamide SDS-PAGE gel under reducing conditions. Purified TCM is used as standard protein.
  • CHO cell-based expression systems are currently the most validated and most commonly used expression systems for the production of recombinant human therapeutic glycoprotein. It has been shown that CHO cells, including genetically modified CHO cell lines expressing sialyl-a-1-6 transferase, are able to glycosylate recombinant proteins in a manner qualitatively similar to that observed in human cells. This particular feature is of major importance to reduce potential immunogenicity of recombinant glycoprotein administered to patients.
  • Purified TCM or fractions enriched for particular glycoforms obtained from transfected CHO cells are analysed by western blot to confirm glycosylation status in comparison to E. co/z-derived TCM.
  • the different glycoforms of the CHO-produced TCM are differentially characterized using PolyAcrilamide Gel Electrophoresis. Enzymatic deglycosylation of TCM is used to confirm the obtained results.
  • Glycoforms of the CHO- produced and purified TCM are differentially characterized using mass spectrometry. The amount of glycosylations increases the molecular weight of TCM.
  • oligosaccharides Purified glycosylated TCM samples are enzymatically digested with an endoglycosidase such as peptide-N-glycosidase F (PNGaseF, Roche). Released N-linked oligosaccharides are separated from the peptide structure and sorted using a graphite
  • Glycosylation complexity is also assessed via determination of molar ratio of the different monosaccharides found on all the glycans (N- and O-glycan if applicable) of the purified CHO-derived TCM. All glycans of purified glycosylated TCM samples are chemically treated by methanolysis reaction to hydrolyze all the glycosidic links between sugars. Released monosaccharides are separated from the peptide structure and sorted using a coupled Gas Chromatography-Mass Spectrometry Automass apparatus (Finnigan). Molar ratios are determined as compared to known internal standards.
  • Sites- specific N-glycan pattern heterogeneity of the CHO-derived hIL-7 is assayed by endoprotease digestion, followed by fractionation and Mass Spectrometry analyses of the generated peptides. Purified samples are digested with Trypsin or other endo-proteases for generation of glycopeptides corresponding to each N-glycosylation site of the expressed TCM. Each glycopeptide is identified by N-terminal micro sequencing and by its specific retention time when analyzed by reverse phase HPLC. Each glycopeptide is therefore separated. The heterogeneity of N-glycans is analyzed by MALDI-TOF MS (Q Star, Applied Biosystems). The m/z values corresponding to each peak of the MS spectrum allow identification of the N-Glycan pattern for each designated site of TCM.
  • O-glycosylation is assayed via the use of O-glycan specific lectins (Lectin blot).
  • Purified CHO-derived TCM samples are separated by SDS-PAGE and blotted to PVDF membranes. Immobilized proteins are probed with (but not limited to) peroxidase-labelled PNA (peanut agglutinin) and/or MAA (Maackia amurensis agglutinin) and stained for visualization.
  • Glycan heterogeneity and composition are also determined via the use of Lectin affinity to the purified CHO-derived TCM.
  • An array of lectins having affinity for N- and O- glycan structures is used to coat 96 well microplates. Identical amounts of recombinant purified TCM preparations are added to the microplate wells and incubated. The amount of bound TCM is proportional to the affinity of a given lectin to the glycan decoration of TCM. Binding is assessed using a TCM specific antibody coupled to biotin followed by a streptavidin-peroxidase conjugate.
  • the used lectins are: LEA (lectin from Lycopersicon esculentum), WGA (from Triticum vulgare), UEA.I (from Ulex europeus), MAA (from Maackia amurensis), ACA (from Amaranthus caudatus), AIA (from Artocarpus
  • ACA from Agaricus bisporus
  • PHA.L from Phaseolus vulgaris
  • ACA from Agaricus bisporus
  • AIA have affinity for Gal and GalNAc.
  • ABA reveals the presence of O-glycans.
  • LEA has affinity for GalNAc indicating the presence of N-Glycan structures.
  • WGA binds to core structures of N-linked glycans but highly complex N-Glycans mask the core structure and render lectin affinity difficult to operate.
  • UEA.I has affinity to branched fucose.
  • MAA has affinity to terminal sialic acids that can be present on both N and O-glycans.
  • PHA.L has affinity to complex branched structures of N-Glycan.
  • the SPR experiments are performed with a BIAcore3000 (GE).
  • Recombinant human CD 127 is immobilized on research-grade CM5 chips (GE).
  • the binding response at each concentration is calculated by subtracting the equilibrium response, measured in the control (BSA) flow cell, from the response in each sample flow cell.
  • Kinetic constants are derived by using the curve-fitting function of the Biaevaluation 4.1.1 software (GE), to fit the rate equations derived from the simple 1: 1 Langmuir binding model (A + B M AB) or the bivalent model.
  • a coating of IL-7Ra-Fc (RnD Systems) is placed in wells of a 96 well microtiter plate in Buffer A (Buf A; 100 mM KCl, 3 mM MgC12, and 10 mM PIPES, pH 7.0) by incubation at 4°C sealed overnight. After washing with Buffer T (Buf T; buffer A + 0.1% Tween-20), the plate is blocked with 1% BSA in Buf T by incubation at room temperature for 1 hr.
  • Buffer A Buffer A
  • Buf T buffer A + 0.1% Tween-20
  • the plate is washed three times with Buf T to remove free protein and serial dilutions of TCM in Buffer B (Buf B; buffer T + 0.1% BSA) are then added to the wells and incubated with the immobilized IL-7Ra-Fc at room temperature for 2 hrs sealed. After three washes with Buf T biotinylated anti-His tag monoclonal antibody in Buf B is added to all the wells and incubated sealed at room temperature for 1 hr. The plate is then washed 3 times. Streptavidin- HRP (RnD Systems) in Buf B is added to all the wells (according to manufacturer's suggestions) and incubated at room temperature for 45 min. The plate is then washed 3 times prior to adding ABTS-H202 substrate solution.
  • Buf B buffer T + 0.1% BSA
  • ABTS/H202 horseradish peroxidase-conjugated first or second antibody
  • 0.04% ABTS diammonium is dissolved in 65.7 mM citric acid monohydrate, 34 mM sodium citrate dihydrate, pH 4.0, adjusted with sodium citrate dihydrate.
  • the substrate solution is brought to room temperature and immediately prior to its application, 0.03% H202 is added.
  • the substrate reaction is incubated at room temperature with periodic mixing (e.g., 2-3 seconds shaking in the plate reader) and the color development monitored by absorbance reading at 415 nm or 405 nm using a microplate reader at several time intervals. Chemicals are acquired from Sigma- Aldrich).
  • Memory T cells are isolated from human PBMCs by magnetic enrichment (CD45RO beads, Miltenyi Biotech). Memory T cells (100,000 cells/well) are incubated in FACS buffer (HBSS, 2% FBS, 2mM EDTA, 25mM Hepes) together with TCM in a 96 well round bottom microplate for 30 minutes at 4°C and after a wash in FACS buffer stained with CD127-PE antibody (eBioscience) and anti-6-His-Alexa647 ("6-His" disclosed as SEQ ID NO: 27) (ThermoFisher Scientific) in FACS buffer for 30 minutes at 4°C. Cells are washed in FACS buffer, fixed in FACS buffer containing 4% formaldehyde and acquired by a flow cytometer.
  • FACS buffer HBSS, 2% FBS, 2mM EDTA, 25mM Hepes
  • 6-His-Alexa647 disclosed as SEQ ID NO: 27
  • Memory T cells are isolated from human PBMCs by magnetic enrichment (CD45RO beads, Miltenyi Biotech). Memory T cells (100,000 cells/well) are incubated in FACS buffer (HBSS, 2% FBS, 2mM EDTA, 25mM Hepes) together with biotinylated IL-7, serial dilutions of TCM, or IL-7 and TCM in FACS buffer in a 96 well round bottom microplate for 30 minutes at 4°C and after a wash in FACS buffer stained with CD127-PE antibody
  • CD127-Fc (RnD Systems) and TCM are added to wells and incubated for 60 minutes, and sealed at room temperature. Serial dilutions are performed and a negative control is added as recommended by the manufacturer (Cisbio). IL-7 is used as a positive control.
  • Anti- 6His-d2 (“6His” disclosed as SEQ ID NO: 27) and anti-tag-Tb cryptate are added to each well, incubated for 120 minutes, sealed, at room temperature and read on an I3X microplate reader (Molecular Devices).
  • the binding affinity of TCM to IL-7R is optimized by using yeast surface display to select for variations in the TCM helices (e.g., a part of Helix A) that modulate TCM binding affinity to soluble IL-7Ra or CD 132.
  • This example is equally applicable to target selection of TCM. It is also equally applicable to target selection of TCM variants with mutations in CD132 (common gamma chain) interacting strands.
  • the nine amino acids IL-7Ra binding stretches are mutated to random amino acids as shown in the amino acid sequence below (shown as XXXXXXXX):
  • Established yeast surface display protocols (Bin Liu (ed.), Yeast Surface Display: Methods, Protocols, and Applications, Methods in Molecular Biology, vol. 1319; 2015) are used to clone a library of nucleotide sequence coding for the TCM fusion mutants into an appropriate DNA expression vector to enable fusion to Aga2p, and inducible expression, secretion and placement on the yeast cell surface.
  • the DNA encoding a library of these mutated TCM fusion are cloned into a DNA vector and transformed into a yeast strain that enables surface display of the mutated TCM.
  • a molecule containing an Fc fusion to the extracellular region of IL-7Ra (RnD Systems; Cat: 306-IR-050) is mixed with the yeast cell library, and fluorescently labeled antibodies that bind the Fc domain are used to identify yeast cells that contain TCMs with increased binding affinity to IL-7Ra.
  • Differential isolation by flow cytometry or other selection methods enables enrichment of yeast containing TCMs with optimized binding characteristics.
  • the DNA from these TCMs is isolated and sequenced to identify the TCM variants that provide modulated IL-7Ra binding affinity, and these TCMs variants are tested in immune cell assays to identify the TCM variants with the best T cell modulating activity.
  • Naive CD4+ T cells are labeled with proliferation dye (CellTrace Violet) according to the manufacturer's instructions. Labeled cells are cultured (100,000 cells/ 200 ⁇ ) in 96-well microtiter plates with medium only, coated with anti-CD3 antibody and soluble anti-CD28 antibody alone or in combination with TCM or rhuIL-7 as a positive control. Proliferation and percentage of live CD4+ T cells is assessed after 5 days using flow-cytometry. Cells are stained with a fixable viability dye before acquisition on a flow-cytometer.
  • proliferation dye CellTrace Violet
  • proliferation of human T cells are assessed by measuring radiolabeled thymidine ([ H]-thymidine, Amersham) incorporation by dividing cells. After 72 hrs supernatants are removed for analysis of cytokines production and cells are pulsed with ICi/well [ H]-thymidine in medium. Proliferation is assessed using a 1450 Microbeta liquid scintillation counter (Perkin Elmer).
  • proliferation and survival are assessed using a colorimetric assay based on uptake of a dye marker reflecting the general metabolism of the cell, such as MTT dye (3- (4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium, reduced by mitochondrial RedOx activity) or MTS dye (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium).
  • a dye marker reflecting the general metabolism of the cell such as MTT dye (3- (4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium, reduced by mitochondrial RedOx activity) or MTS dye (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium).
  • Intensity of the staining is proportional to the amount of viable cells.
  • ED50 concentration (ng/ml) is measured for each sample. A higher ED50 indicates a lower activity.
  • ED50 corresponds to the TCM dose necessary to induce one half of the possible maximal induction activity in vitro.
  • highly bioactive molecules correspond to low ED50 values whereas higher ED50 concentrations are typically from less bioactive preparations.
  • Intracellular P-STAT5 levels are assessed using intracellular FACS staining.
  • Human CD4+ T cells are enriched using magnetic cell sorting, and are cultured in 96-well microtiter plates (10 5 cells) with medium or IL-7 for 15 min. Cells are fixed by adding IC fixation buffer (eBioscience), permeabilized with 90% methanol for at least 30 minutes and stained with fluorochrome labelled mouse anti-human STAT5 pY694 (Cell Signaling Technologies) using the eBioscience protocol C2 for intracellular FACS staining and acquired on a flow cytometer. The percentage of P-STAT5-positive cells is measured from a cut-off set using an unstained control.
  • intracellular P-STAT5 is assessed using the phospho-STAT5 (Tyr694) kit for HTRF (Cisbio) according to the manufacturer's protocol.
  • Tyr694 phospho-STAT5
  • HTRF HTRF
  • 100,000 cells are plated in 96 half- well plate in HBSS.
  • TCM or IL-7 as positive control are diluted in HBSS and added to the plate for 15 minutes.
  • Supplemented lysis buffer is added directly to the plate and the plate is incubated for at least 30 minutes at room temperature under shaking.
  • cell lysate is transferred from the 96- well cell-culture plate to a 384-well small volume white plate and premixed antibody solutions (vol/vol) are added, the plate is covered with a plate sealer and incubated overnight at room temperature. The following day the fluorescence emission (Eu3+ Cryptate) is read at two different wavelenghts (665nm and 620nm) on a compatible HTRF reader (I3X,
  • Intracellular Bcl-2 expression is assessed after culturing human CD4+ T cells
  • Tumor biopsies and PBMCs are obtained from cancer patients after informed consent.
  • PBMCs Peripheral blood mononuclear cells
  • TILs Tumor infiltrating lymphocytes
  • the tumor cells are stained with CellTrace Far Red (ThermoFisher Scientific), plated as hanging drops using Perfecta3D Hanging Drop Plates in complete cell culture medium (DMEM with 10% fetal bovine serum (FBS) (Fisher Scientific) and 1% penicillin/streptomycin) at 37°C with 10% C02 at 50,000- 250,000 cells/drop.
  • DMEM complete cell culture medium
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • T cells are harvested, stained with CellTrace Violet according to manufacturer's instructions (ThermoFisher Scientific), washed, and resuspended in complete cell culture medium (50,000).
  • TCM, IL-7, anti-PD-1 alone or in combination are added to the tumor spheroids.
  • Spheroid generation in hanging drops is captured using the ImageXpress Micro Confocal High Content Screening System with environmental control (Molecular Devices).
  • spheroids are collected for imaging and FACS staining: To collect spheroids, drops are harvested, transferred to a conical tube (Falcon), washed with PBS, and centrifuged for 5-10 min.
  • spheroids are incubated with trypsin/EDTA at 37°C for 5-30 min, while pipetting every 2-3 min. When no cell aggregates are visible, spheroid derived cells are collected by centrifugation for 5-10 min to be used in described assays. Phenotype, proliferation, and survival of patient derived T cells and tumor cells are assessed after coculture using flow cytometry. Cells are stained for CD4, CD8, TCRa/b, CD56, PD-1, Lag- 3, TIM-3, TIGIT, OX40, 4-1BB, CD25, CD69, CD45RO, PD-L1, CD160, CD127, CD28, KLRG-1, and fixable viability dye (ebio science).
  • cytokines IFN-g, IL-17, IL-10, TGF-b
  • Luminex or HTRF or ATP as a measure of cytotoxicity using HTRF (Cisbio). Cytotoxicity assay in coculture of patient derived T cells with syngeneic tumor cells
  • Tumor biopsies and PBMCs are obtained from cancer patients after informed consent.
  • PBMCs Peripheral blood mononuclear cells
  • Tumor cells are stained with CellTrace Far Red (ThermoFisher Scientific) and cultured in X-VIVO 15 medium on hydrogel for 2 days using 24 well plates and 500,000 cells per well.
  • PBMCs are cultured in T cell medium for 2 days. After 2 days, PBMCs are harvested, stained with stained with CellTrace Violet according to manufacturer's
  • Example 8 In vivo models for assessing activation of immune function
  • mice Using TCM for treatment of irradiation-induced lymphopenia in mice
  • Sublethal irradiation causes a large scale loss of naive T-cells, thereby leading to lymphopenia in irradiated mice.
  • lymphopenia can be observed (Xu et al. Cell & Bioscience. 2016 6:30).
  • Proliferation dye labelled naive splenic T cells that are obtained from spleens of 6-12 weeks old CD45.2 B6 mice by magnetic enrichment and FACS sorting, are injected into irradiated CD45.1 mice 24 hours after irradiation (10 x 10 6 cells per mouse).
  • mice are injected with effective amounts of TCM, rhuIL-7 (positive control), or PBS (negative control) three times per week for two weeks.
  • Tail blood samples are collected post T-cell transfer, stained for CD3, CD4, CD8, CD45.1 and CD45.2.
  • mice are euthanized for testing in vitro proliferation and cytokine production.
  • cytokines staining splenocytes are isolated and stimulated with PMA and Ionomycin in the presence of Brefeldin A for 4 hours. Cells are stained for CD3, CD4, CD8, CD45.1, and CD45.2, and fixed using Fixation/Permeabilization buffer (eBioscience) and processed according to manufacturer's suggestion (see eBioscience protocol B2 for intracellular staining). After intracellular staining for IL-17, IFNy, and IL-2, cells are analyzed using a flow cytometer.
  • the melanoma-B 16F10 cell line is obtained from American Type Culture Collection (ATCC; Rockville MD) and B 16F10 cells are maintained in culture as adherent monolayers in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1-glutamine and penicillin-streptomycin (Gibco BRL; Rockville MD). Cells in the exponential growth phase are harvested at 80% confluence, using brief exposure to 0.1% trypsin solution. Only single-cell suspensions with 95% viability are used for inoculation.
  • DMEM Dulbecco's modified Eagle's minimal essential medium
  • mice For tumorigenesis experiments, syngeneic C57BL/6J male mice (6 animals per group, 10-12 weeks of age), are subcutaneously inoculated with 5 x 10 5 cells of B 16F10 cells suspended in 150 ⁇ of PBS into each flank. Tumor growth is monitored daily from day 7. Mice are treated with anti-PD-1, TCM, a combination of anti-PD-1 and TCM, or isotype control antibody in PBS as negative control.
  • the tumor volume is calculated using the formula (0-4 ab2), with 'a' as the larger diameter and V as the smaller diameter.
  • B 16F10 melanoma cells are transferred intravenously into syngeneic C57BL/6J male mice 10-12-weeks old. The lungs and liver are removed 14 days after the transfer, and visible metastatic colonies on the lungs and the liver are counted.
  • Splenocytes are prepared 14 days after the intravenous injection B 16 melanoma cells. Then, these cells are incubated with irradiated B 16 melanoma cells for 96 hr with or without IL-2. Assay is performed for 4 hrs. Cytotoxicity of spleen cells is measured using the HTRF based CellTiter-Glo Assay from CysBio using a microplate reader.
  • PDX models are generated by subcutaneous or orthotopical (organ of cancer origin) implantation of sectioned patient tumor fragments into immunodeficient NOD/scid/IL2y- receptor null (NSG) mice.
  • NSG mice have defects in both humoral and innate immunity, and support implantation of human hematopoietic cells and solid tumors.
  • An important advantage of PDX models as compared to in vitro models or tumor models using cell lines is that they retain key characteristics of patients' tumors. The histologic characteristics and genomic signature of the patient tumor, and the heterogeneity of cancer cells, are highly preserved in PDX tumors.
  • PDX models are the most clinically relevant cancer models developed to date, and represent a highly predictive drug response platform that recapitulates the therapeutic outcome in human patients.
  • the procedures for the generation of PDX comprise collecting fresh surgical tumor- containing tissue, dividing the tumor into pieces, and then implanting, either subcutaneously or orthotopically, the cells into an immunodeficient mouse.
  • the tumors are removed from the mouse in 2-4 mm fragments and reimplanted into new hosts for the next passage, as described by Cho et al. (Cho et al. Mol Cells. 2016 Feb 29; 39(2): 77-86).
  • Tumor growth is monitored daily. Mice are treated with anti-PD-1, TCM, a combination of anti-PD-1 and TCM, or isotype control antibody in PBS (negative control).
  • the tumor volume is calculated using the formula (0-4 ab2), with 'a' as the larger diameter and V as the smaller diameter.
  • mice are anesthetized with isoflurane and a midline incision performed. The cecum is ligated, punctured, and the abdomen closed. One ml of normal saline mixed with buprenorphine is administered immediately postoperatively. Imipenem is administered subcutaneously 4 hours postoperatively.
  • TCM is administered subcutaneously after C. albicans injection. Mice in the control group received saline only.
  • Spleens and peripheral lymph nodes are harvested from naive and septic animals after C. albicans injection, cells are isolated, and
  • mice and cell culture supernatants are tested for TNF-a, IL-1, IL-6, IL-12, IFN-y, MIF, IL-10, TGF-b, and IL-4 using ProcartaPlex Multiplex Immunoassays
  • Infection is initiated by injecting LCMV clones into the tail vein of 4 to 5-week-old C57BL/6 mice. Virus is propagated on L929 cells.
  • Infected mice are injected with TCM or PBS after infection, when chronicity has been established.
  • Spleens are harvested for phenotypic analysis.
  • Specific monoclonal antibodies for CD4, CD8, CD19, CD25, CD44, CD69, B220, Foxp3, IFN- ⁇ , IL-2, IL-17, CD107a, PD-1, TIM-3, Lag-3, TIGIT, CD160, KLRGl, CD56, CD127, and CD210 (IL-10 receptor) are used for phenotyping.
  • Antigen specific T cells are detected using the following dextramers H- 2Db/GP33 (KAVYNFATM (SEQ ID NO: 57)), H-2Db/GP276 (SGVENPGGYCL (SEQ ID NO: 58)) and H-2Db/NP396 (FQPQNGQFI (SEQ ID NO: 59)) according to the following dextramers H- 2Db/GP33 (KAVYNFATM (SEQ ID NO: 57)), H-2Db/GP276 (SGVENPGGYCL (SEQ ID NO: 58)) and H-2Db/NP396 (FQPQNGQFI (SEQ ID NO: 59)) according to the following dextramers H- 2Db/GP33 (KAVYNFATM (SEQ ID NO: 57)), H-2Db/GP276 (SGVENPGGYCL (SEQ ID NO: 58)) and H-2Db/NP396 (FQPQNGQFI (SEQ
  • CD4+ antigen- specific cells are quantified by restimulating splenocytes in vitro with the CD4 epitope GP61 peptide (GLNGPDIYKGVQFKSVEFD (SEQ ID NO: 60)) or an irrelevant OVA323 control peptide (IS Q A VH A AH AEINE AGR (SEQ ID NO: 61)) in the presence of intracellular protein transport inhibitor GolgiStop (BD Biosciences).
  • splenocytes are isolated and stimulated with PMA and Ionomycin in the presence of Brefeldin A for 4 hours.
  • Cell are stained for CD3, CD4, and CD8, and fixed using Fixation/Permeabilization buffer (eBioscience) and processed according to manufacturer's suggestion (see eBioscience protocol B2 for intracellular staining). After intracellular staining for IL-17, IFNy, and IL-2, cells are analyzed using a flow cytometer.
  • Fixation/Permeabilization buffer eBioscience
  • Viral titers are quantified by focus-forming assays using MC57 fibroblast cells, as previously described (Battegay et al., J. Virol. Methods 33, 191-198).
  • Example 9 Assays for assessing inhibition of immune function
  • Human PBMCs are cultured in media supplemented with a single dose of
  • IL-7-biotin recombinant human IL-7-biotin
  • sIL-7Ra recombinant human IL-7-biotin
  • TCM recombinant human IL-7-biotin
  • Phospho-STAT5 a measure for response of PBMCs to IL-7, is assessed using intracellular flow cytometry.
  • the concentration of IL-7 in the supernatant is measured by ELIS A using a coating antibody for IL-7 as well as SA-HRP and TMB for detection of bound IL-7-biotin. Testing inhibitory TCM in delaying or decreasing EAE in C57BL/6 mice.
  • EAE Experimental autoimmune encephalomyelitis
  • mice receive pertussis toxin (List Biological Laboratories).
  • PBS control group

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Abstract

L'invention concerne des molécules de modulateur de cellules T et des compositions pharmaceutiques qui ont des caractéristiques bénéfiques appropriées pour une administration à un tissu ou à une cellule cible (par exemple, ex vivo ou in vivo), utiles dans des procédés pour modifier (par exemple, modifier l'activation ou l'inhibition de) une voie de signalisation cible dans un tissu ou une cellule (par exemple, ex vivo ou in vivo), et/ou pour traiter un sujet (par exemple, un mammifère tel qu'un être humain). Les préparations et les compositions de l'invention peuvent également être modifiées, par exemple, des variants, des fusions ou des combinaisons.
PCT/US2018/019044 2017-02-22 2018-02-21 Compositions de molécules de modulateur cellules t (tcm) et utilisation connexe Ceased WO2018156649A1 (fr)

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WO2020009938A1 (fr) * 2018-07-03 2020-01-09 Catalent Pharma Solutions, Llc Molécules protéiques multifonctionnelles comprenant de la décorine et utilisation associée
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WO2020077190A1 (fr) * 2018-10-12 2020-04-16 Jumaa Weinacht Hassan Anticorps monoclonal pour le traitement de la leucémie lymphoblastique aiguë
WO2021209402A3 (fr) * 2020-04-15 2021-12-02 F. Hoffmann-La Roche Ag Immunoconjugués
CN114432441A (zh) * 2022-01-11 2022-05-06 中国人民解放军陆军军医大学第一附属医院 一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用
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Publication number Priority date Publication date Assignee Title
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
WO2020009938A1 (fr) * 2018-07-03 2020-01-09 Catalent Pharma Solutions, Llc Molécules protéiques multifonctionnelles comprenant de la décorine et utilisation associée
CN112638403A (zh) * 2018-07-03 2021-04-09 康泰伦特药物解决方案有限责任公司 包括饰胶蛋白聚糖的多功能蛋白质分子及其用途
US11377488B2 (en) 2018-07-03 2022-07-05 Catalent Pharma Solutions, Llc Multifunctional protein molecules comprising decorin and use thereof
US11879009B2 (en) 2018-07-03 2024-01-23 Catalent Pharma Solutions, Llc Multifunctional protein molecules comprising decorin and use thereof
US12398176B2 (en) 2018-08-27 2025-08-26 Regeneron Pharmaceuticals, Inc. Use of Raman spectroscopy in downstream purification
US12312584B2 (en) 2018-10-02 2025-05-27 Exosome Therapeutics, Inc. cGMP exosome loaded therapeutics for treating sickle cell disease
US20200101016A1 (en) * 2018-10-02 2020-04-02 Exosome Therapeutics, Inc. Compositions and methods for producing exosome loaded therapeutics for treating cardiovascular disease
WO2020077190A1 (fr) * 2018-10-12 2020-04-16 Jumaa Weinacht Hassan Anticorps monoclonal pour le traitement de la leucémie lymphoblastique aiguë
US11629195B2 (en) 2019-02-01 2023-04-18 Regeneron Pharmaceuticals, Inc. Anti-IL2 receptor gamma antigen-binding proteins
WO2021209402A3 (fr) * 2020-04-15 2021-12-02 F. Hoffmann-La Roche Ag Immunoconjugués
CN114432441A (zh) * 2022-01-11 2022-05-06 中国人民解放军陆军军医大学第一附属医院 一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用
CN114432441B (zh) * 2022-01-11 2023-04-25 中国人民解放军陆军军医大学第一附属医院 一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用
WO2025038450A1 (fr) * 2023-08-11 2025-02-20 Cornell University Immunothérapies à particules à base de cellules immunitaires ultra-petites, compositions de cellules immunitaires et leurs utilisations

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