US20210017247A1 - Fusion Molecules Targeting Immune Regulatory Cells and Uses Thereof - Google Patents

Fusion Molecules Targeting Immune Regulatory Cells and Uses Thereof Download PDF

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US20210017247A1
US20210017247A1 US16/628,374 US201816628374A US2021017247A1 US 20210017247 A1 US20210017247 A1 US 20210017247A1 US 201816628374 A US201816628374 A US 201816628374A US 2021017247 A1 US2021017247 A1 US 2021017247A1
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seq
antibody
molecule
linker
amino acid
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Douglas S. Jones
Jesse Lyons
Ulrik B. Nielsen
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Torque Therapeutics Inc
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Torque Therapeutics Inc
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Priority to US16/628,374 priority Critical patent/US20210017247A1/en
Publication of US20210017247A1 publication Critical patent/US20210017247A1/en
Assigned to TORQUE THERAPEUTICS, INC. reassignment TORQUE THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NIELSEN, ULRIK
Assigned to TORQUE THERAPEUTICS, INC. reassignment TORQUE THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LYONS, Jesse
Assigned to TORQUE THERAPEUTICS, INC. reassignment TORQUE THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JONES, DOUGLAS SCOTT
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)

Definitions

  • the present disclosure relates generally to modified immune regulatory cells and engineered fusion molecules. More specifically, the present disclosure relates to modified regulatory T cells and fusion proteins useful for targeted immune therapy and adoptive cell therapy.
  • Adoptive cellular therapies are demonstrating clinical benefits across a range of disorders, with potentially fewer risks and greater efficacy than traditional pharmacological strategies.
  • Regulatory T cells are currently undergoing clinical trials in various immune-mediated pathologies, including transplant rejection and autoimmune conditions.
  • cell therapy relies upon ex vivo expansion of the cell product.
  • In vitro manipulation of cell therapy products prior to administration to patients, offers the opportunity to enhance the efficacy of the final cell therapy product in other ways. For example, cells can be exposed to reagents that enhance their longevity or functional potency after transfer into the patient. Genetic modification strategies can even permit the design of cells with bespoke functionality.
  • in vitro manipulation of therapeutic cells in isolation can exert these influences upon the biology of the therapeutic cells, without systemic exposure of the patient to the reagents being used.
  • Treg manufacturing and ACT requires sorting of Tregs (e.g., CD4+CD25+CD127-by fluorescence-activated cell sorting, FACS) from a patient sample, ex vivo expansion of Tregs, often by expanding the Tregs using interleukin 2 (IL-2) or activating and expanding the T cells by using CD3 and CD28 stimulation and IL-2, and then subsequently administering the Tregs to the patient; see FIG. 1A .
  • Treg cell therapy presents several key challenges: it requires GMP (good manufacturing practice) level sorting and long and costly ex vivo expansion; siginificant deficiencies in Treg viability and activity after freezing and thaw can exist; and cells can exhibit short-term survival following reinfusion into the patient.
  • GMP good manufacturing practice
  • compositions and methods to improve Treg-based cell therapies hold strong potential for delivering meaningful clinical benefits.
  • Treg-targeting fusion molecules can be combined with the backpack technology as disclosed in, e.g., U.S. Publication No. 2017/0080104, U.S. Pat. No. 9,603,944, U.S Publication No. 2014/0081012, and PCT Application No. PCT/US2017/037249, each of which is incorporated herein by reference in its entirety.
  • a fusion molecule comprising:
  • Treg regulatory T cell
  • cytokine or growth factor molecule is operatively linked to the antigen-binding fragment.
  • the antigen is one or more of CD45, CD4, CD25, CD39, Neuropilin 1 (NRP1), or a variant of any of the foregoing.
  • the targeting moiety comprises a bispecific molecule having an antigen-binding fragment specific for CD4 and an antigen-binding fragment specific for CD25.
  • the antigen is one or more of CD4, CD45, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CCR4, CCR5, CD137, CD39, CCR4, CCR5, CCR6 (CD196), CCR8, CCR10, OX40, GITR, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, Neuropilin 1 (NRP1), CD8, CD11a, CD18, or a variant of any of the foregoing.
  • the cytokine or growth factor molecule is one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, or TGF- ⁇ , or a variant of any of the foregoing. In some embodiments, the cytokine or growth factor molecule is one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, TGF- ⁇ , IL-7, or IL-21, or a variant of any of the foregoing.
  • the cytokine or growth factor molecule is one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, TGF- ⁇ , IL-7, IL-21, IL-6, IL-12, or IL-23, or a variant of any of the foregoing.
  • the Treg is a healthy and/or non-malignant Treg.
  • the fusion molecule can further include a linker for operably linking the cytokine or growth factor molecule and the targeting moiety.
  • the linker is selected from one or more of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.
  • the peptide linker is a human serum albumin or a variant thereof.
  • the linker at either end, is fused to the cytokine or growth factor molecule and the targeting moiety via a peptide comprising Gly and Ser.
  • the peptide is a (GGGS) N (SEQ ID NO: 87) or (GGGGS) N (SEQ ID NO: 88) linker, where N indicates the number of repeats of the motif and is an integer selected from 1-10.
  • the antigen-binding fragment is a Fab fragment comprising a light chain and a heavy chain fragment linked by a disulfide bond, and wherein the cytokine or growth factor molecule is operably linked to the Fab fragment at a C-terminus of the light chain, an N-terminus of the light chain, a C-terminus of the heavy chain fragment, or an N-terminus of the heavy chain fragment.
  • the targeting moiety localizes the cytokine or growth factor molecule to the Treg and enhances its concentration, distribution, duration of exposure and/or availability on a cell surface of the Treg.
  • a further aspect relates to a fusion molecule, comprising:
  • a Treg targeting moiety comprising an antibody having an antigen-binding site specific for an antigen on the surface of a Treg, wherein the antibody comprises a light chain having a C-terminus and an N-terminus, and a heavy chain having a C-terminus and an N-terminus,
  • the light chain is linked to the heavy chain by a disulfide bond, wherein the cytokine or growth factor molecule is operably linked to the antibody at the C-terminus of the light chain, the N-terminus of the light chain, or the N-terminus of the heavy chain portion.
  • the antigen is a CD4 or a CD25 receptor expressed on the cell surface of the Treg.
  • the cytokine or growth factor molecule comprises an IL-2, or a variant thereof.
  • the cytokine or growth factor molecule is operably linked to the antibody by a linker.
  • the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.
  • the linker is a peptide linker comprising a Gly and a Ser.
  • the peptide linker is a (GGGS) N (SEQ ID NO: 87) or (GGGGS) N (SEQ ID NO: 88) linker, wherein N indicates the number of repeats of the motif and is an integer selected from 1-10.
  • a fusion molecule comprising: (i) a regulatory T cell (Treg) targeting moiety having an affinity with an immune cell surface receptor on the Treg, wherein the targeting moiety is selected from an antibody or antigen-binding fragment thereof, a non-antibody scaffold, or a ligand that binds to the immune cell surface receptor; and (ii) a cytokine or growth factor molecule; wherein the cytokine or growth factor molecule and the targeting moiety are operably linked together as a fusion molecule.
  • Treg regulatory T cell
  • the targeting moiety is selected from an antibody or antigen-binding fragment thereof, a non-antibody scaffold, or a ligand that binds to the immune cell surface receptor
  • a cytokine or growth factor molecule wherein the cytokine or growth factor molecule and the targeting moiety are operably linked together as a fusion molecule.
  • the immune cell surface receptor is selected from one or more of CD45, CD4, CD25, CD39, Neuropilin 1 (NRP1), or variants thereof.
  • the targeting moiety comprises a bispecific molecule that binds to both CD4 and CD25.
  • the immune cell surface receptor is selected from one or more of CD4, CD45, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CCR4, CCR5, CD137, CD39, CCR4, CCR5, CCR6 (CD196), CCR8, CCR10, OX40, GITR, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, Neuropilin 1 (NRP1), CD8, CD11a, or CD18, or variants thereof.
  • the cytokine or growth factor molecule is selected from one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, or TGF- ⁇ , or variants thereof.
  • the cytokine or growth factor molecule is selected from one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, TGF- ⁇ , IL-7, or IL-21, or variants thereof
  • the cytokine or growth factor molecule is selected from one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, TGF- ⁇ , IL-7, IL-21, IL-6, IL-12, or IL-23, or variants thereof.
  • the Treg targeted by the FMs disclosed herein is a healthy and/or non-malignant Treg.
  • the Treg targeted by the FMs disclosed herein is obtained from patients in need of immunosuppressive therapy that may or may not be marked by Treg disfunction (e.g., an unhealthy or abnormal Treg).
  • the targeting moiety localizes the cytokine or growth factor molecule to the Treg and enhances its concentration, distribution, duration of exposure and/or availability on a cell surface of the Treg.
  • the FM in some embodiments, can further include a linker for operably linking the cytokine or growth factor molecule and the targeting moiety.
  • the linker can be selected from one or more of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.
  • the peptide linker can be human serum albumin or an Fc domain.
  • the linker at either end, can be fused to the cytokine or growth factor molecule and the targeting moiety via a peptide comprising Gly and Ser.
  • the peptide is a (GGGS) N (SEQ ID NO: 87) or (GGGGS) N (SEQ ID NO: 88) linker, where N indicates the number of repeats of the motif and is an integer selected from 1-10.
  • any of the fusion molecules disclosed herein can further include a nanoparticle, a liposome and/or a biodegradable polymer.
  • the nanoparticle comprises a protein nanogel, a nucleotide nanogel, a polymer nanoparticle, or a solid nanoparticle.
  • the nanoparticle comprises a protein nanogel.
  • the nanoparticle optionally comprises at least one polymer, cationic polymer, or cationic block co-polymer on the nanoparticle surface.
  • the nanoparticle comprises a nanogel that is cross linked by a reversible linker that is sensitive to redox (disulfide) or pH (hydrolysable groups) or enzymes (proteases).
  • Another aspect relates to a modified Treg comprising:
  • a further aspect relates to a modified Treg comprising a Treg and any of the fusion molecule disclosed herein bound thereto.
  • each fusion molecule comprising
  • the plurality of fusion molecules are bound to the surface of the Tregs, and wherein the cytokine or growth factor molecule acts in vivo upon the population of Tregs in the human subject to suppress or prevent an immune response in the human subject.
  • composition comprising:
  • Another aspect relates to an isolated nucleic acid molecule engineered to encode the FM disclosed herein.
  • a further aspect relates to a vector comprising the nucleic acid molecule disclosed herein.
  • Also provided herein is method of making the FM disclosed herein, comprising culturing the host cell disclosed herein under suitable conditions.
  • composition comprising the FM disclosed herein and a pharmaceutically acceptable carrier, excipient, or stabilizer is provided.
  • provided herein is a method for providing immunosuppressive therapy, comprising administering to a subject in need thereof a plurality of the fusion molecules disclosed herein, a plurality of the modified Tregs disclosed herein, or the plurality of expanded Tregs disclosed herein.
  • a method for in vivo expansion of Tregs comprising loading a population of sorted Tregs or PBMCs obtained from a subject with CD3/CD28/IL-2 backpacks to provide loaded cells, and administering the loaded cells to the subject without in vitro culturing.
  • a method for providing immunosuppressive therapy comprising administering to a subject in need thereof a plurality of FMs disclosed herein, a plurality of the modified Tregs disclosed herein, the plurality of expanded Tregs disclosed herein, or the loaded cells disclosed herein.
  • the immunosuppressive therapy can be used to treat diseases such as allo-immune diseases, auto-immune diseases, allergy, and inflammatory diseases.
  • the disclosure provides a particle, e.g., a nanoparticle, that comprises an FM as described herein, e.g., nanoparticle that comprises a protein (e.g., a protein nanogel as described herein).
  • the particle comprises the same FM.
  • the particle comprises one or more different types of FM. Nanoparticles and methods of making are disclosed in PCT International Application No. PCT/US2017/037249 filed Jun. 13, 2017, e.g., on pages 57-79, which is incorporated herein by reference in its entirety.
  • compositions e.g., pharmaceutical compositions, comprising the FMs and/or the particles disclosed herein, are also disclosed.
  • the pharmaceutical compositions further include a pharmaceutically acceptable carrier, excipient, or stabilizer.
  • the FM is a bifunctional or bispecific molecule, e.g., it has at least two different kinds of members, e.g., with different functions and/or binding specificities.
  • the FM can comprise, or consist of, a modulatory moiety, e.g., a cytokine molecule, and a targeting moiety, wherein the modulatory moiety and the targeting moiety bind to two different cell surface targets or receptors in the same or different immune regulatory cells.
  • the modulatory moiety and the targeting moiety bind to two different targets on the same regulatory T cell.
  • a bifunctional or bispecific molecule can further comprise additional moieties, e.g., further binding and/or functional moieties.
  • the FM can be a multifunctional or multispecific molecule, e.g., it is a trifunctional or trispecific, or a tetrafunctional or tetraspecific, fusion molecule.
  • the FM can be represented with the following formula in an N to C terminal orientation: R1-(optionally L1)-R2 or R2-(optionally L1)-R1; wherein R1 comprises a targeting moiety, L1 comprises a linker (e.g., a peptide linker described herein), and R2 comprises a modulatory moiety, e.g., a cytokine or growth factor molecule.
  • R1 comprises a targeting moiety
  • L1 comprises a linker (e.g., a peptide linker described herein)
  • R2 comprises a modulatory moiety, e.g., a cytokine or growth factor molecule.
  • the modulatory moiety e.g., the cytokine molecule
  • the modulatory moiety can be covalently coupled indirectly, e.g., via a linker to the targeting moiety.
  • the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.
  • the linker is a peptide linker.
  • the peptide linker can be 1-30, 1-5, 5-20, 8-18, 10-15, or about 8, 9, 10, 11, 12, 13, 14, 15-20, 20-25, or 25-30 amino acids long. In some embodiments, the peptide linker can be 30 amino acids or longer; e.g., 30-35, 35-40, 40-50 50-60 amino acids long.
  • the linker comprises the amino acid sequence of SEQ ID NO: 36, 37, 38, or 39, or an amino acid sequence substantially identical thereto (e.g., having 1, 2, 3, 4, or 5 amino acid substitutions).
  • the linker comprises an amino acid sequence GGGSGGGS (SEQ ID NO: 37).
  • the linker comprises amino acids derived from an antibody hinge region.
  • the linker comprises amino acids derived from the hinge regions of IgG1, IgG2, IgG3, IgG4 , IgGM, or IgGA antibodies.
  • the linker comprises amino acids derived from an IgG hinge region, e.g., an IgG1, IgG2 or IgG4 hinge region.
  • the linker comprises a variant amino acid sequence from an IgG hinge, e.g., a variant having one or more cysteines replaced, e.g., with serines.
  • the linker comprises DKTHT C PPS C APE (SEQ ID NO: 91), having one or both cysteines replaced with another amino acid, e.g., a serine.
  • the linker comprises amino acids DKTHTSPPSPAP (SEQ ID NO: 38), EPKSSDKTHTSPPSPAPE (SEQ ID NO: 92), or a derivative thereof.
  • the linker comprises amino acids derived from an IgG2 hinge region, e.g., amino acids SVESPPSP (SEQ ID NO: 93), ERKSSVESPPSP (SEQ ID NO: 94), or a derivative thereof.
  • the linker comprises amino acids derived from an IgG4 hinge region, e.g., amino acids PPSPSSP (SEQ ID NO: 95), ESKYGPPSPSSP (SEQ ID NO: 96), or a derivative thereof.
  • the linker is a non-peptide, chemical linker.
  • the modulatory moiety is covalently coupled to the targeting moiety by crosslinking.
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • the modulatory moiety is directly covalently coupled to the targeting moiety, without a linker.
  • the modulatory moiety and the targeting moiety of the FM are not covalently linked, e.g., are non-covalently associated.
  • the linker can be a protein or a fragment or derivative thereof, e.g., human albumin or an Fc domain, or a fragment or derivative thereof.
  • the targeting moiety is linked to the N-terminus and the modulatory moiety is linked to the C-terminus.
  • the linker non-covalently associates the targeting moiety to the modulatory moiety.
  • the linker comprises a dimerization domain, e.g., a coiled coil or a leucine zipper.
  • the linker is a reversible linker, in which the association between targeting moiety and the modulatory moiety can be reversed.
  • the linker can be degraded by hydrolysis, reducing environments, or enzymes.
  • the linker can be degraded by hydrolysis in a pH-dependent manner.
  • the linker can be reversed by redox state (e.g. linker containing disulfide bonds).
  • the linker can be reversed by enzymatic cleavae.
  • the linker can be cleaved by a matrix metalloproteinase (MMP; e.g.
  • MMP matrix metalloproteinase
  • MMP-1, MMP-2, MMP-3, or MMP-9 a disintegrin and metalloprotease (ADAM; e.g. ADAM-17), a plasminogen activator (e.g. urokinase-type plasminogen activator, uPA, or tissue plasminogen activator, tPA), or a granzyme (e.g. granzyme A or granzyme B).
  • ADAM disintegrin and metalloprotease
  • a plasminogen activator e.g. urokinase-type plasminogen activator, uPA, or tissue plasminogen activator, tPA
  • a granzyme e.g. granzyme A or granzyme B
  • FIG. 1A depicts the current Treg manufacturing and ACT, and obstacles associated therewith.
  • FIG. 1B depicts exemplary fusion proteins of the present disclosure combining a cytokine and an immunoglobulin moiety for cell-surface targeting and stimulation.
  • FIG. 1C depicts an exemplary process for ex vivo expansion of Tregs.
  • FIG. 1D depicts an exemplary process for in vivo expansion of Tregs, without culturing ex vivo.
  • FIG. 1E depicts exemplary process for IL-2 loading-driven survival, expansion and engraftment of Tregs.
  • FIG. 1F depicts exemplary applications of Treg cell therapy in connection with various cytokines or growth factors.
  • FIG. 1G depicts generation of Treg specific scFvs via phase display and Treg-targeting of exemplary tethered fusions.
  • FIG. 1H depicts exemplary diseases that can benefit from Treg cell therapies disclosed herein.
  • FIGS. 2A-2C depict FMs comprising various antibody formats.
  • FIG. 2A depicts a schematic of IL-15 FMs comprising various antibody formats.
  • FIGS. 2B and 2C depict evaluation of FMs on CD8 T cell expansion in pulse assay using CellTiter Blue ( FIG. 2B ) or flow cytometry counting beads ( FIG. 2C ).
  • FIGS. 3A-3D depict a schematic for various fusion strategies between the IL-15/sushi complex and an anti-CD45 antibody.
  • FIGS. 3A and 3B depict schematics for fusion of IL-15 to the N- or C-terminus of chBC8 IgG ( FIG. 3A ) or chBC8 Fab ( FIG. 3B ) light-chain.
  • Antibody fusions comprise a complex with IL-15R ⁇ -sushi through a high-affinity interaction with IL-15.
  • FIGS. 3C and 3D depict schematics for fusion of IL-15R ⁇ -sushi to the N- or C-terminus of chBC8 IgG ( FIG. 3C ) or chBC8Fab ( FIG. 3D ) light-chain.
  • Antibody fusions comprise a complex with IL-15 N72D through a high-affinity interaction with IL-15R ⁇ -sushi.
  • FIG. 3E depicts a schematic design for a bivalent (bispecific) fusion, comprising 2 anti-CD45 antibodies (h9.4 Fab and BC8 scFv) fused to the IL-15/sushi complex.
  • FIGS. 4A-4B depict the effect of FMs comprising various fusion strategies on CD8 T cell expansion.
  • FMs comprising IL-15 N72D and chBC8 Fab ( FIG. 4A ) or chBC8 IgG ( FIG. 4B ) were evaluated for T cell expansion in pulse bioassay.
  • CD8 T cell proliferation was analyzed using CellTiter Blue; IL15 N72D /sushi L77I -Fc and unstimulated T-cells (neg ctrl) were included for comparison.
  • FIGS. 5A-5D depict biological activity of FMs comprising wild-type or mutated IL-15 and various fusion strategies of IL-15/sushi complex to anti-CD45 antibody.
  • FIGS. 5A and 5B depict FMs comprising wild-type IL-15 or IL-15 N72D and chBC8 Fab ( FIG. 5A ) or chBC8 IgG ( FIG. 5B ) that were evaluated for CD8 T cell expansion in pulse assay using CellTiter Blue three days after pulse incubation with FMs.
  • FIGS. 5C and 5D depict the evaluation of FMs comprising chBC8 Fab ( FIG. 5C ) or chBC8 IgG ( FIG. 5D ) on CD8 T cell expansion in a constant exposure, e.g., static, assay format using CellTiter Blue following incubation with FMs for three days.
  • FIGS. 6A-6B depict FMs comprising C-terminal fusion to chBC8 IgG light- or heavy-chain.
  • FIG. 6A depicts schematics of FMs comprising IL-15 fused to the C-terminus of chBC8 IgG light- or heavy-chain and a noncovalent complex between IL-15 and IL-15R ⁇ -sushi.
  • FIG. 6B depicts effects of FMs on CD8 T cell expansion in a pulse bioassay.
  • FIGS. 7A-7C depict FMs require functional binding of the antibody fragment for improved activity.
  • FIGS. 8A-8C depict cell surface loading and persistence of FMs.
  • FIG. 8A depicts flow cytometry histograms showing MFI after pulse of FMs as detected by staining with fluorescently labeled anti-IL15 or anti-IgG antibodies.
  • FIG. 8B depicts persistence of cell surface staining of IL-15 over time.
  • FIG. 8C depicts cell density over time, measured using flow cytometry counting beads.
  • FIG. 9A-9B depict evaluation of FMs comprising varied linker composition between IL-15 and chBC8.
  • FIG. 9A depicts amino acid sequences of the three different linkers for fusion of IL-15 to the C-terminus of chBC8 Fab light-chain.
  • FIG. 9B depicts evaluation of FMs comprising different linker compositions for CD8 T cell expansion in pulse bioassay using CellTiter Blue.
  • FIGS. 10A-10B depict evaluation of FMs comprising alternative CD45 antibody clones.
  • FIG. 10A depicts binding affinity of various CD45 antibody clones to CD45 expressed on T cells.
  • FIG. 10B depicts evaluation of IL-15 FMs comprising different anti-CD45 antibody clones in pulse bioassay using CellTiter Blue.
  • FIGS. 11A-11B depict FMs comprising antibodies targeting CD8, CD11a, or CD18.
  • FIG. 11A depicts a schematic of IL-15 FMs comprising fusion of IL-15 to the light-chain C-terminus of a Fab antibody fragment targeting alternative cell surface receptors; FMs comprise a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • FIG. 11B depicts evaluation of IL-15 FMs for CD8 T cell expansion in pulse bioassay using CellTiter Blue.
  • FIGS. 12A-12B depict evaluation of FM potency comprising anti-CD8 on CD4 or CD8 T cells.
  • FIG. 12A depicts a schematic of IL-15 FM comprising fusion of IL-15 to the C-terminus of an anti-CD8 Fab light-chain, and a noncovalent association with IL-15R ⁇ -sushi.
  • FIG. 12B depicts evaluation of potency of anti-CD8 FM on purified CD4 or CD8 T cells in pulse bioassay using CellTiter Blue.
  • CD4 or CD8 T cells were purified from total T cells (which naturally comprise mixtures of CD4 and CD8 T cells) using magnetic bead sorting.
  • FIGS. 13A-13C depict graphs showing that nanoparticles comprising IL-15 FMs support T cell expansion.
  • Backpacks comprising IL-15 FM or IL15 N72D /sushiL77I-Fc were evaluated for CD8 T cell expansion in pulse bioassay ( FIG. 13C ).
  • Cell density was analyzed using flow cytometry counting beads; data are presented as fold expansion.
  • FIG. 14 shows example immunotargeted surface receptors and cytokines, illustrating versatility of the platform.
  • FIGS. 15A-15G show tethered fusion platform enables selective cell targeting and improved expansion of CD8 T cells in cultures of activated total CD3 human T cells by pulse incubation with CD8-targeted FMs.
  • FIGS. 16A-16E shows cellular activities of various mutated forms of IL-15 and surface persistence of FMs comprising IL-15 mutants.
  • FIGS. 17A-17C shows selective cell targeting and CD8 T cell expansion by CD8-targeted FMs comprising wild-type or mutated IL-15.
  • FIG. 17D shows surface persistence of CD45-targeted FM comprising wild-type or mutated IL-15.
  • FIG. 18 shows enrichment of Tregs following bead-based magnetic sorting for CD4 + /CD25 + /CD127 dim cells. These Treg enriched CD4s were used to test in vitro and in vivo expansion driven by IL-2 tethered fusions.
  • FIG. 19 depicts a strategy for limiting inflammatory and auto-immune diseases via specific targeting of IL-2 to Treg cells.
  • FIG. 20 shows titration of Fab-HSA-IL2 on Treg-enriched CD4s.
  • Fab-HSA-IL2 shows titratable surface binding immediately post-loading and for more than 3 days.
  • Fab-HSA-IL2 -loaded cells show dose-dependent proliferation, and even very low doses maintains viability.
  • FIG. 21 relates surface staining of pulsed IL-2 tethered fusions (left panel) with proliferation and survival of Treg-enriched cells (right panels). All three tested tethered fusions exhibited strong staining and drove proliferation and survival of the cells. Proliferation and survival were comparable to constant stimulation with recombinant IL-2.
  • FIG. 22 illustrates the effects of pulsed IL-2 tethered fusions over time.
  • Cells pulsed with any of the TF constructs showed equal or better expansion compared to constant stimulation with rhIL2 through 6 days post-pulse, and comparable expansion and survival out to 9 days.
  • IL-2 tethered fusions promote proliferation and long-term survival of Treg-enriched cell populations in vitro.
  • FIG. 23 shows increased expansion of IL-2 tethered fusion pulsed Treg-enriched CD4 cells in the blood of NSG mice. There were increased numbers of CD4 cells in the IL-2 tethered fusion-pulsed group throughout the course of the experiment. This indicates that IL-2 tethered fusions can increase expansion and engraftment in vivo.
  • FIG. 24 shows that the Fab-HSA-IL2 preferentially supports proliferation and survival of loaded cells. Despite having some pro-proliferative effect on non-pulsed cells, there is still stronger effects on the pulsed cells, and the CD4:CD8 ratio is higher in the Fab-HSA-IL2 group than the control group.
  • FIG. 25 provides a schematic describing how dual-targeting, and cytokine affinity modulation will increase on-target effects while reducing off-target effects of IL-2 antibody tethered fusion molecules.
  • the Treg-targeting tethered fusions disclosed herein can be used to improve engraftment, survival and efficacy of adoptive cell therapies, e.g., immunosuppressive cell therapy.
  • the Treg-targeting tethered fusions, or the cytokine molecules alone can be combined with nanoparticle, nanogel and liposome technology as disclosed in, e.g., U.S. Publication No. 2017/0080104, U.S. Pat. No. 9,603,944, U.S Publication No. 2014/0081012. and PCT Application No. PCT/US2017/037249, each of which is incorporated herein by reference in its entirety.
  • Treg-targeting tethered fusions and related immunosuppressive cell therapy disclosed herein are advantageous compared to conventional Treg manufacturing and ACT.
  • Conventional Treg manufacturing requires GMP sorting of pure Tregs.
  • FIG. 1C using Treg-targeting tethered fusions such as Treg-targeted IL-2, selective expansion (ex vivo) of Tregs from a mixed population of peripheral blood mononuclear cells (PBMC) can be achieved, thereby avoiding expensive and time-consuming GMP sorting of Tregs.
  • PBMC peripheral blood mononuclear cells
  • survival and activity of the cells post-thaw can be improved.
  • Treg manufacturing additionally requires long and costly ex vivo expansion.
  • sorted Tregs or PBMC without sorting can be subject to loading (e.g., CD3/CD28/IL-2) followed by direct infusion to patient, whereby the Tregs are expanded in vivo, without in vitro culturing.
  • loaded Tregs e.g., with IL-2 backpacks
  • FIG. 1E loaded Tregs
  • binding of the tethered fusions to Tregs is believed to increase the concentration, e.g., the concentration over time, of the cytokine or growth factor molecule, on the surface of the Treg. This can result in an effect (e.g., stimulatory) on the Treg itself bound by the tethered fusions (autocrine signaling), or on another (e.g., neighboring) immune cell (paracrine signaling).
  • the targeting moiety results in an increase in one or more of: binding, availability, activation and/or signaling of the cytokine or growth factor on the Treg, e.g., over a specified amount of time.
  • CD4/CD25 targeting moieties can result in binding specifically to Tregs.
  • CD45 targeting moieties can be used to target Tregs, although not specifically.
  • the FM does not substantially interfere with the signaling function of the cytokine or growth factor. Such targeting effect results in localized and prolonged stimulation of proliferation and activation of the Tregs, thus inducing controlled Treg expansion and/or activation, and Treg-mediated suppression of an immune response.
  • the targeting moiety can be an antibody molecule or a ligand molecule that binds to a Treg cell surface receptor, e.g., CD45, CD4, CD25, CD39, or Neuropilin 1 (NRP1).
  • the Treg cell surface receptor is selected from one or more of CD4, CD45, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CCR4, CCR5, CD137, CD39, CCR4, CCR5, CCR6 (CD196), CCR8, CCR10, OX40, GITR, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, NRP1, CD8, CD11a, CD18, or variants thereof.
  • the cytokine or growth factor molecule is selected from one or more IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, or TGF- ⁇ , or variants thereof. In some embodiments, the cytokine or growth factor molecule is selected from one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin (Areg), IL-33, TGF- ⁇ , IL-7, or IL-21, or variants thereof.
  • the cytokine or growth factor molecule is selected from one or more of IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, TGF- ⁇ , IL-7, IL-21, IL-6, IL-12, or IL-23, or variants thereof.
  • TGF- ⁇ , IL10 and IL-33 can promote Treg differentiation and prevent conversion to inflammatory cells.
  • IL-35 and IL-10 can promote iTreg in the periphery. Areg drives tissue regeneration during inflammation.
  • the targeting moiety is derived from an anti-CD45 antibody molecule and the cytokine molecule is IL-2 or variants thereof.
  • Allo-immune diseases can include, for example, organ transplant rejection, graft versus host disease or rejection (GVHD) (e.g., post-allogeneic hematopoietic stem cell transplant (HSCT) and other post-allogeneic stem cell transplantation (SCT), and rejection of autologous stem cells and/or genetically modified autologous stem cells (e.g., where CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or the like is used to modify cells ex vivo to fix some deficiency which are then transplanted back into the patient, resulting in rejection due to any new or modified proteins being recognized as antigenic).
  • GVHD organ transplant rejection
  • HSCT post-allogeneic hematopoietic stem cell transplant
  • SCT post-allogeneic stem cell transplantation
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Treg number has been shown to correlate with GVHD control and organ transplant tolerance.
  • expansion of antigen-specific Tregs has been shown to associate with loss of milk allergy, and Treg depletion enhances allergy.
  • Auto-immune diseases include Type 1 diabetes, Multiple Sclerosis and alopecia. These diseases have functional defects in Tregs, phenotypic instability and decreased number of Tregs.
  • Inflammatory diseases include Crohn's Disease, Inflammatory Bowel Disease, Rheumatoid Arthritis and Lupus. These diseases can have increased Treg number, but are marked by decreased Treg function. Tregs have been shown to control/prevent these diseases in mouse models.
  • “about” and “approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.
  • the term “substantially” means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are present in a given embodiment, yet open to the inclusion of unspecified elements.
  • the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced.
  • Antibody or “antibody molecule” as used herein refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence.
  • An antibody molecule encompasses antibodies (e.g., full-length antibodies) and antibody fragments.
  • an antibody molecule comprises an antigen binding or functional fragment of a full length antibody, or a full length immunoglobulin chain.
  • a full-length antibody is an immunoglobulin (Ig) molecule (e.g., IgG) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes).
  • an antibody molecule refers to an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment.
  • An antibody fragment e.g., functional fragment, is a portion of an antibody, e.g., Fab, Fab′, F(ab′) 2 , F(ab) 2 , variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv).
  • a functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody.
  • antibody fragment or “functional fragment” also include isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”).
  • an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues.
  • Exemplary antibody molecules include full length antibodies and antibody fragments, e.g., dAb (domain antibody), single chain, Fab, Fab′, and F(ab′) 2 fragments, and single chain variable fragments (scFvs).
  • Fab and “Fab fragment” are used interchangeably and refer to a region that includes one constant and one variable domain from each heavy and light chain of the antibody, i.e., V L , C L , V H , and C H 1.
  • an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain.
  • the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain.
  • the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.
  • an antibody molecule is monospecific, e.g., it comprises binding specificity for a single epitope.
  • an antibody molecule is multispecific, e.g., it comprises more than one immunoglobulin variable domain sequence, where a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope.
  • an antibody molecule is a bispecific antibody molecule. “Bispecific antibody molecule” as used herein refers to an antibody molecule that has specificity for at least two (e.g., two, three, four, or more) epitopes and/or antigens.
  • Antigen refers to a macromolecule, including all proteins or peptides.
  • an antigen is a molecule that can provoke activation of certain immune cells (including immune regulatory cells) and/or antibody generation. Any macromolecule, including almost all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinant or DNA.
  • any DNA comprising a nucleotide sequence or a partial nucleotide sequence that encodes a protein capable of eliciting an immune response encodes an “antigen.”
  • an antigen does not need to be encoded solely by a full-length nucleotide sequence of a gene, nor does an antigen need to be encoded by a gene at all.
  • an antigen can be synthesized or can be derived from a biological sample, e.g., a tissue sample, a tumor sample, a cell, or a fluid with other biological components.
  • the “antigen-binding site” or “antigen-binding fragment” or “antigen-binding portion” (used interchangeably herein) of an antibody molecule refers to the part of an antibody molecule, e.g., an immunoglobulin (Ig) molecule such as IgG, that participates in antigen binding.
  • the antigen-binding site is formed by amino acid residues of the variable (V) regions of the heavy (H) and light (L) chains.
  • hypervariable regions Three highly divergent stretches within the variable regions of the heavy and light chains, referred to as hypervariable regions, are disposed between more conserved flanking stretches called “framework regions” (FRs).
  • FRs are amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface, which is complementary to the three-dimensional surface of a bound antigen.
  • the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.”
  • CDRs complementarity-determining regions
  • variable chain e.g., variable heavy chain and variable light chain
  • Each variable chain is typically made up of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • VL CDRs are generally defined to include residues at positions 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3).
  • VH CDRs are generally defined to include residues at positions 27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).
  • CDR1 residues at positions 27-33
  • CDR2 52-56
  • CDR3 95-102
  • the loops can be of different length across antibodies and the numbering systems such as the Kabat or Chotia control so that the frameworks have consistent numbering across antibodies.
  • the antigen-binding fragment of an antibody can lack or be free of a full Fc domain.
  • an antibody-binding fragment does not include a full IgG or a full Fc but may include one or more constant regions (or fragments thereof) from the light and/or heavy chains.
  • the antigen-binding fragment can be completely free of any Fc domain.
  • the antigen-binding fragment can be substantially free of a full Fc domain.
  • the antigen-binding fragment can include a portion of a full Fc domain (e.g., CH2 or CH3 domain or a portion thereof).
  • the antigen-binding fragment can include a full Fc domain.
  • the Fc domain is an IgG domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain.
  • the Fc domain comprises a CH2 domain and a CH3 domain.
  • backpacks refer to protein clusters that can be prepared by reacting, e.g., cross-linking various therapeutic protein monomers.
  • a surface modification such as polycation can be introduced on the protein cluster, which can be loaded onto cell surface as cell membranes are negatively charged.
  • Exemplary backpacking technology is disclosed in, e.g., U.S. Publication No. 2017/0080104, U.S. Pat. No. 9,603,944, U.S Publication No. 2014/0081012, U.S. Provisional App. No. 62/554,058 filed Sep. 5, 2017 and PCT Application No. PCT/US2017/037249, each of which is incorporated herein by reference in its entirety.
  • a “cytokine molecule” refers to a full length, a fragment, or a variant of a naturally-occurring, wild type cytokine (including fragments and functional variants thereof having at least 10% of the activity of the naturally-occurring cytokine molecule).
  • the cytokine molecule has at least 30, 50, or 80% of the activity, e.g., the immunomodulatory activity, of the naturally-occurring molecule.
  • the cytokine molecule further comprises a receptor domain, e.g., a cytokine receptor domain, optionally, coupled to an immunoglobulin Fc region. In other embodiments, the cytokine molecule is coupled to an immunoglobulin Fc region.
  • the cytokine molecule is coupled to an antibody molecule (e.g., an immunoglobulin Fab or scFv fragment, a Fab fragment, a FAB 2 fragment, or an affibody fragment or derivative, e.g. a sdAb (nanobody) fragment, a heavy chain antibody fragment, single-domain antibody, a bispecific or multispecific antibody), or non-antibody scaffolds and antibody mimetics (e.g., lipocalins (e.g. anticalins), affibodies, fibronectin (e.g., monobodies or Adnectins), knottins, ankyrin repeats (e.g,. DARPins), and A domains (e.g. avimers)).
  • an antibody molecule e.g., an immunoglobulin Fab or scFv fragment, a Fab fragment, a FAB 2 fragment, or an affibody fragment or derivative, e.g. a sdAb
  • a “cytokine agonist,” as used herein can include an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor that elicits at least one activity of a naturally-occurring cytokine.
  • an antibody molecule e.g., an agonistic antibody
  • an “immune cell” refers to any of various cells that function in the immune system, e.g., to protect against agents of infection and foreign matter.
  • this term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes.
  • the term “immune cell” includes immune regulatory cells (e.g., Tregs) and immune effector cells described herein.
  • Immunune cell also refers to modified versions of cells involved in an immune response, e.g. modified NK cells, including NK cell line NK-92 (ATCC cat. No.
  • haNK an NK-92 variant that expresses the high-affinity Fc receptor Fc ⁇ RIIIa (158V)
  • taNK targeted NK-92 cells transfected with a gene that expresses a CAR for a given tumor antigen
  • Immuno effector cell refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • immune effector cells include, but are not limited to, T cells, e.g., CD4+ T cells, CD8+ T cells, alpha T cells, beta T cells, gamma T cells, and delta T cells; B cells; natural killer (NK) cells; natural killer T (NKT) cells; dendritic cells; and mast cells.
  • the immune cell is an immune cell (e.g., T cell or NK cell) that comprises, e.g., expresses, a Chimeric Antigen Receptor (CAR), e.g., a CAR that binds to a cancer antigen.
  • the immune cell expresses an exogenous high affinity Fc receptor.
  • the immune cell comprises, e.g., expresses, an engineered T-cell receptor.
  • the immune cell is a tumor infiltrating lymphocyte.
  • the immune cells comprise a population of immune cells and comprise T cells that have been enriched for specificity for a tumor-associated antigen (TAA), e.g.
  • TAA tumor-associated antigen
  • immune cells comprise a population of immune cells and comprise T cells that have been “trained” to possess specificity against a TAA by an antigen presenting cell (APC), e.g. a dendritic cell, displaying TAA peptides of interest.
  • APC antigen presenting cell
  • the T cells are trained against a TAA chosen from one or more of MART-1, MAGE-A4, NY-ESO-1, SSX2, Survivin, or others.
  • the immune cells comprise a population of T cells that have been “trained” to possess specificity against a multiple TAAs by an APC, e.g.
  • the immune cell is a cytotoxic T cell (e.g., a CD8+ T cell).
  • the immune cell is a helper T cell, e.g., a CD4+ T cell.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • Immuno regulatory cell refers to an immune cell that participates in immunomodulation or suppression of immune responses, e.g., a regulatory T cell or regulatory B cell.
  • Tregs or “Treg cells” refer to Regulatory T cells. Regulatory T cells are a class of T cells that suppress the activity of other immune cells, and are defined using flow cytometry by the cell marker phenotype CD4+CD25+FOXP3+. Because FOXP3 is an intracellular protein and requires cell fixation and permeablization for staining, the cell surface phenotype CD4+CD25+CD127—can be used for defining live Tregs. Tregs also include various Treg subclasses, such as tTregs (thymus-derived) and pTregs (peripherally-derived, differentiated from na ⁇ ve T cells in the periphery). All Tregs express the IL2R ⁇ receptor, do not produce their own IL-2 and are dependent on IL-2 for growth, and someone skilled in the art will recognize that both classes will be selectively activated by an IL2R ⁇ selective agonist.
  • CTLs Cytotoxic T lymphocytes
  • CTL activation can occur when two steps occur: 1) an interaction between an antigen-bound MHC molecule on the target cell and a T cell receptor on the CTL is made; and 2) a costimulatory signal is made by engagement of costimulatory molecules on the T cell and the target cell.
  • CTLs then recognize specific antigens on target cells and induce the destruction of these target cells, e.g., by cell lysis.
  • the CTL expresses a CAR.
  • the CTL expresses an engineered T-cell receptor.
  • Modulation refers to any alteration of an existing or potential immune responses against an autoimmune or allergy provoking epitope, including, e.g., nucleic acids, lipids, phospholipids, carbohydrates, self-polypeptides, protein complexes, or ribonucleoprotein complexes, that occurs as a result of administration of the compositions disclosed herein. Such modulation includes any alteration in presence, capacity, or function of an immune regulatory cell, sych as Treg, involved in, or capable of being involved in, an immune response. “Modulation” includes any change imparted on an existing immune response, a developing immune response, a potential immune response, or the capacity to induce, regulate, influence, or respond to an immune response. Modulation includes any alteration in the expression and/or function of genes, proteins and/or other molecules in immune cells as an immune response.
  • Modulation of an immune response includes, for example, the following: elimination, deletion, or sequestration of immune cells; induction or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of an unresponsive state in immune cells (i.e., anergy); increasing, decreasing, or changing the activity or function of immune cells or the capacity to do so, including, but not limited to, altering the pattern of proteins expressed by these cells. Examples include altered production and/or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors; or any combination of these modulatory events.
  • modulation includes suppression or immunosuppression of certain immune responses, e.g., in conditions or diseases where the immune system displays an excessive or overactive response such as autoimmune diseases, allergies, bone marrow or organ transplant, and other inflammatory diseases.
  • Suppression or immunosuppression as used herein includes reduction, inhibition and lessening of an immune response.
  • sample refers to a biological sample obtained from a tissue or bodily fluid of a subject or patient.
  • the source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents (e.g., serum, plasma); bone marrow or any bone marrow constituents; bodily fluids such as urine, cerebral spinal fluid, whole blood, plasma and serum.
  • the sample can include a non-cellular fraction (e.g., urine, plasma, serum, or other non-cellular body fluid).
  • the body fluid from which the sample is obtained from an individual comprises blood (e.g., whole blood).
  • subject includes living organisms in which an immune response can be elicited (e.g., mammals, human).
  • the subject is a patient, e.g., a patient in need of immune cell therapy.
  • the subject is a donor, e.g. an allogenic donor of immune cells, e.g., intended for allogenic transplantation.
  • compositions and methods of the present disclosure encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, 95% identical or higher to the sequence specified.
  • substantially identical is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 85%, 90%.
  • nucleotide sequence 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
  • a reference sequence e.g., a sequence provided herein.
  • substantially identical is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.
  • nucleotide sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence e.g., a sequence provided herein. Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST the default parameters of the respective programs. See http://www.ncbi.nlm.nih.gov. It is understood that the molecules of the present disclosure may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • amino acid includes both the D- or L- optical isomers and peptidomimetics.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • the term “functional variant” or “variant” or “variant form” in the context of a polypeptide refers to a polypeptide that is capable of having at least 10% of one or more activities of the naturally-occurring sequence.
  • the functional variant has substantial amino acid sequence identity to the naturally-occurring sequence, or is encoded by a substantially identical nucleotide sequence, such that the functional variant has one or more activities of the naturally-occurring sequence.
  • molecule can refer to a polypeptide or a nucleic acid encoding a polypeptide, as indicated by the context. This term includes full length, a fragment or a variant of a naturally-occurring, wild type polypeptide or nucleic acid encoding the same, e.g., a functional variant, thereof. In some embodiments, the variant is a derivative, e.g., a mutant, of a wild type polypeptide or nucleic acid encoding the same.
  • polypeptide “peptide” and “protein” (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • the polypeptide can be isolated from natural sources, can be a produced by recombinant techniques from a eukaryotic or prokaryotic host, or can be a product of synthetic procedures.
  • nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • the polynucleotide may be either single-stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (antisense) strand.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • the nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.
  • parent polypeptide refers to a wild-type polypeptide and the amino acid sequence or nucleotide sequence of the wild-type polypeptide is part of a publicly accessible protein database (e.g., EMBL Nucleotide Sequence Database, NCBI Entrez, ExPasy, Protein Data Bank and the like).
  • EMBL Nucleotide Sequence Database NCBI Entrez, ExPasy, Protein Data Bank and the like.
  • mutant polypeptide or “polypeptide variant” or “mutein” refers to a form of a polypeptide, wherein its amino acid sequence differs from the amino acid sequence of its corresponding wild-type (parent) form, naturally existing form or any other parent form.
  • a mutant polypeptide can contain one or more mutations, e.g., replacement, insertion, deletion, etc. which result in the mutant polypeptide.
  • corresponding to a parent polypeptide is used to describe a polypeptide of the present disclosure, wherein the amino acid sequence of the polypeptide differs from the amino acid sequence of the corresponding parent polypeptide only by the presence of at least amino acid variation. Typically, the amino acid sequences of the variant polypeptide and the parent polypeptide exhibit a high percentage of identity. In one example, “corresponding to a parent polypetide” means that the amino acid sequence of the variant polypeptide has at least about 50% identity, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 98% identity to the amino acid sequence of the parent polypeptide.
  • the nucleic acid sequence that encodes the variant polypeptide has at least about 50% identity, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 98% identity to the nucleic acid sequence encoding the parent polypeptide.
  • introducing (or adding etc.) a variation into a parent polypeptide” or “modifying a parent polypeptide” to include a variation (or grammatical variations thereof) do not necessarily mean that the parent polypeptide is a physical starting material for such conversion, but rather that the parent polypeptide provides the guiding amino acid sequence for the making of a variant polypeptide.
  • “introducing a variant into a parent polypeptide” means that the gene for the parent polypeptide is modified through appropriate mutations to create a nucleotide sequence that encodes a variant polypeptide.
  • introducing a variant into a parent polypeptide means that the resulting polypeptide is theoretically designed using the parent polypeptide sequence as a guide. The designed polypeptide may then be generated by chemical or other means.
  • isolated refers to material that is removed from its original or native environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the co-existing materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature.
  • An modulatory moiety can be any agent or molecule capable of modulating, e.g., stimulating or suppressing, a function or activity of another agent, molecule or cell.
  • the modulartory moiety reduces, inhibits or suppresses a function or activity.
  • the modulatory moiety enhances, activates or stimulates a function or activity.
  • the modulatory moiety activates or enhances proliferation of immune regulatory cells.
  • the modulatory moiety activates or enhances proliferation of Tregs, which in turn, regulates, reduces or suppresses certain immune response, thereby providing an immunosuppressive activity.
  • modulatory moieties can be used.
  • the modulatory moiety can be a cytokine or growth factor.
  • Cytokines are proteinaceous signaling compounds that are mediators of the immune response. They control many different cellular functions including proliferation, differentiation and cell survival/apoptosis; cytokines are also involved in several pathophysiological processes including viral infections and autoimmune diseases. Cytokines are synthesized under various stimuli by a variety of cells, including those of both the innate (monocytes, macrophages, dendritic cells) and adaptive (T- and B-cells) immune systems. Cytokines can be classified into two groups: pro- and anti-inflammatory. Pro-inflammatory cytokines, including IFN ⁇ , IL-1 (IL-1 ⁇ , IL-1 ⁇ , IL-6 and TNF- ⁇ , are predominantly derived from the innate immune cells and Th1 cells. Anti-inflammatory cytokines, including IL-10, IL-4, IL-13 and IL-5, are synthesized from Th2 immune cells.
  • Growth factors are capable of stimulating cellular growth, proliferation, healing, and cellular differentiation. Usually it is a protein or a steroid hormone. Growth factor is sometimes used interchangeably with the term cytokine.
  • FMs that include or are engineered to contain one or more cytokine/growth factor molecules, e.g., cytokines, growth factors and/or functional variants thereof.
  • the cytokine molecule is an interleukin (IL) or a variant, e.g., a functional variant thereof.
  • the cytokine is a proinflammatory interleukin.
  • the cytokine is anti-inflammatory.
  • the cytokine molecule is full length, a fragment or a variant of a cytokine, e.g., a cytokine comprising one or more mutations.
  • the cytokine molecule of the FM includes an immunomodulatory cytokine, e.g., a pro-inflammatory cytokine or an anti-inflammatory cytokine.
  • the cytokine is a member of the common y-chain ⁇ (y ⁇ c) family of cytokines.
  • the cytokine molecule comprises a cytokine chosen from one or more of IL-15, IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-13), IL-18, IL-21, IL-27, IL-35), IFN ⁇ , TNF ⁇ , IFN ⁇ , IFN ⁇ , Areg, GM-CSF, or GCSF, including variant forms thereof (e.g., a cytokine derivative, a complex comprising the cytokine molecule with a polypeptide, e.g., a cytokine receptor complex, and other agonist forms thereof).
  • a cytokine derivative e.g., a complex comprising the cytokine molecule with a polypeptide, e.g., a cytokine receptor complex, and other agonist forms thereof.
  • the cytokine molecule is a pro-inflammatory cytokine molecule chosen from an IL-15, IL-2, IL-18, IL-27, IL-10, IL-35, Amphiregulin, IL-33, or TGF- ⁇ molecule.
  • the cytokine is IL-7, IL-21, IL-6, IL-12, or IL-23.
  • the cytokine molecule is an anti-inflammatory cytokine molecule chosen from an IL-4, IL-10, IL-13, IL-35 cytokine molecule.
  • the cytokine molecule is a superagonist (SA).
  • the superagonist can have increased cytokine activity, e.g., by at least 10%, 20%, or 30%, compared to the naturally-occurring cytokine.
  • the cytokine molecule is a monomer or a dimer.
  • the cytokine molecule further comprises a receptor or a fragment thereof, e.g., a cytokine receptor domain.
  • the cytokine molecule comprises an amino acid sequence that is identical or substantially identical to the wild-type cytokine sequence, e.g., a human cytokine sequence. In some embodiments, the cytokine molecule comprises an amino acid sequence at least 95% to 100% identical, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to a wild-type cytokine sequence, e.g., a human cytokine sequence.
  • the cytokine molecule comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the wild-type cytokine sequence, e.g., the human cytokine sequence.
  • the cytokine molecule comprises a complex of a cytokine and an anti-cytokine antibody molecule.
  • the cytokine can be IL-2 and the anti-cytokine antibody can be a non-neutralizing anti-IL-2 antibody molecule (see e.g., Spangler, J.B., et al. (2015) dx.doi.org/10.1016/j.immuni.2015.04.015; Letoumeau, S. et al. (2010) PNAS Vol 107(5): 2171-2176; Boyman, 0. et al. (2006) Science 311, 1924); and Spangler, J.B. (2015) Annu Rev Immunol 33:139-167, the contents of which are entirely incorporated by reference).
  • the cytokine molecule is an IL-15 molecule, e.g., a full length, a fragment or a variant of IL-15, e.g., human IL-15.
  • the IL-15 molecule is a wild-type, human IL-5, e.g., having the amino acid sequence of SEQ ID NO: 10.
  • the IL-15 molecule is a variant of human IL-5, e.g., having one or more amino acid modifications.
  • the IL-15 variant comprises, or consists of, a mutation at position 45, 51, 52, or 72, e.g., as described in US 2016/0184399.
  • the IL-15 variant comprises, or consists of, an N, S or L to one of D, E, A Y or P substitution.
  • the mutation is chosen from L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y, or N72P (in reference to the sequence of human IL-15, SEQ ID NO: 11). As those of skill will realize, any combination of the positions can be mutated.
  • the IL-15 variant comprises two or more mutations.
  • the IL-15 variant comprises three or more mutations.
  • the IL-15 variant comprises four, five, or six or more mutations.
  • the IL-15 molecule comprises a mutation, e.g., an N72D point mutation as shown in SEQ ID NO: 11 herein.
  • the IL-15 molecule comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11, wherein the sequence comprises an N72D mutation relative to wild-type human IL-15, and having IL-15R ⁇ binding activity.
  • the IL-15 variant comprises, or consists of, one or more mutations at amino acid position 8, 10, 61, 64, 65, 72, 101, or 108 (in reference to the sequence of human IL-15, SEQ ID NO: 11).
  • the IL-15 variant possesses increased activity as compared with wild-type IL-15.
  • the IL-15 variant possesses decreased activity as compared with wild-type IL-15.
  • the IL-15 variant possesses approximately two-fold, four-fold, ten-fold, 20-fold, 40-fold, 60-fold, 100-fold, or more than 100-fold decreased activity as compared with wild-type IL-15.
  • the mutation is chosen from D8N, K10Q, D61N, D61H, E64H, N65H, N72A, N72H, Q101N, Q108N, or Q108H (in reference to the sequence of human IL-15, SEQ ID NO: 11).
  • the IL-15 variant comprises two or more mutations.
  • the IL-15 variant comprises three or more mutations.
  • the IL-15 variant comprises four, five, or six or more mutations.
  • the IL-15 variant comprises mutations at positions 61 and 64.
  • the mutations at positions 61 and 64 are D61N or D61H and E64Q or E64H.
  • the IL-15 variants comprises mutations at positions 61 and 108.
  • the mutations at positions 61 and 108 are D61N or D61H and Q108N or Q108H.
  • the cytokine molecule further comprises a receptor domain, e.g., a cytokine receptor domain.
  • the cytokine molecule comprises an IL-15 receptor, or a fragment thereof (e.g., an IL-15 binding domain of an IL-15 receptor alpha) as described herein.
  • the cytokine molecule is an IL-15 molecule, e.g., IL-15 or an IL-15 superagonist as described herein.
  • a “superagonist” form of a cytokine molecule shows increased activity, e.g., by at least 10%, 20%, 30%, compared to the naturally-occurring cytokine.
  • An exemplary superagonist is an IL-15 SA.
  • the IL-15 SA comprises a complex of IL-15 and an IL-15 binding fragment of an IL-15 receptor, e.g., IL-15 receptor alpha or an IL-15 binding fragment thereof, e.g., as described herein.
  • the cytokine molecule further comprises a receptor domain, e.g., an extracellular domain of an IL-15R alpha, optionally, coupled to an immunoglobulin Fc or an antibody molecule.
  • the cytokine molecule is an IL-15 superagonist (IL-15SA) as described in WO 2010/059253.
  • the cytokine molecule comprises IL-15 and a soluble IL-15 receptor alpha domain fused to an Fc (e.g., a sIL-15Ra-Fc fusion protein), e.g., as described in Rubinstein et al PNAS 103:24 p. 9166-9171 (2006).
  • Fc e.g., a sIL-15Ra-Fc fusion protein
  • the IL-15 molecule can further comprise a polypeptide, e.g., a cytokine receptor, e.g., a cytokine receptor domain, and a second, heterologous domain.
  • the heterologous domain is an immunoglobulin Fc region.
  • the heterologous domain is an antibody molecule, e.g., a Fab fragment, a FAB 2 fragment, a scFv fragment, or an affibody fragment or derivative, e.g. a sdAb (nanobody) fragment, a heavy chain antibody fragment.
  • the polypeptide also comprises a third heterologous domain.
  • the cytokine receptor domain is N-terminal of the second domain, and in other embodiments, the cytokine receptor domain is C-terminal of the second domain.
  • Wild-type IL-15 Receptor alpha sequence (Genbank Acc. No. AAI21141.1): SEQ ID NO: 41.
  • Wild-type IL-15 Receptor alpha extracellular domain (portion of accession number Q13261): SEQ ID NO: 63.
  • Isoform CRA_d IL-15 Receptor alpha extracellular domain portion of accession number EAW86418,: SEQ ID NO: 64.
  • IL-15 receptor alpha contains an extracellular domain, a 23 amino acid transmembrane segment, and a 39 amino acid cytoplasmic tail.
  • the extracellular domain of IL-15 Receptor alpha is provided as SEQ ID NO: 63.
  • an IL-15 agonist can be used.
  • an agonist of an IL-15 receptor e.g., an antibody molecule (e.g., an agonistic antibody) to an IL-15 receptor, that elicits at least one activity of a naturally-occurring cytokine.
  • the IL-15 receptor or fragment thereof is from human or a non-human animal, e.g., mammal, e.g., non-human primate.
  • IL-15 receptor alpha contains an extracellular domain, a 23 amino acid transmembrane segment, and a 39 amino acid cytoplasmic tail.
  • the sushi domain has been described in the literature including, e.g., Bergamaschi et al. (2008), JBC VOL. 283, NO. 7, pp. 4189-4199; Wei et al. (2001), Journal of Immunology 167:277-282; Schluns et al. (2004) PNAS Vol 110 (15) 5616-5621; US 2016/0184399 (the contents of each of which is incorporated by reference herein).
  • the extracellular domain of IL-15 Receptor alpha is provided as SEQ ID NO: 63.
  • the extracellular domain of IL-15 Receptor alpha comprises a domain referred to as the sushi domain, which binds IL-15.
  • the general sushi domain also referred to as complement control protein (CCP) modules or short consensus repeats (SCR), is a protein domain found in several proteins, including multiple members of the complement system.
  • the sushi domain adopts a beta-sandwich fold, which is bounded by the first and fourth cysteine of four highly conserved cysteine residues, comprising to a sequence stretch of approximately 60 amino acids (Norman, Barlow, et al. J Mol Biol. 1991 Jun 20;219(4):717-25).
  • amino acid residues bounded by the first and fourth cysteines of the sushi domain in IL-15Ralpha comprise a 62 amino acid polypeptide that we refer to as the minimal domain (SEQ ID NO: 52).
  • a sushi domain as described herein may comprise one or more mutations relative to a wild-type sushi domain.
  • residue 77 of IL-15Ra is leucine in the wild-type gene (and is underlined in SEQ ID NO: 41), but can be mutated to isoleucine (L77I).
  • L77I isoleucine
  • SEQ ID NO: 65 a minimal sushi domain comprising L77I (with the numbering referring to the wild-type IL-15Ra of SEQ ID NO: 41) is provided as SEQ ID NO: 65.
  • An extended sushi domain comprising L77I is provided as SEQ ID NO: 66.
  • Minimal sushi domain wild-type: (SEQ ID NO: 52) CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN VAHWTTPSLKC Extended sushi domain, wild-type: (SEQ ID NO: 9) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR Minimal sushi domain, L77I: (SEQ ID NO: 65) CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVINKATN VAHWTTPSLKCI Extended sushi domain, L77I: (SEQ ID NO: 66) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVINKA TNVAHWTTPSLKCIR
  • a sushi domain consists of 62-171 amino acids of SEQ ID NO: 63 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and having IL-15 binding activity.
  • a sushi domain consists of 65-171 amino acids of SEQ ID NO: 63 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and having IL-15 binding activity.
  • a sushi domain consists of up to 171 amino acids of SEQ ID NO: 63 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and having IL-15 binding activity.
  • a sushi domain consists of 62-171, 62-160, 62-150, 62-140, 62-130, 62-120, 62-110, 62-100, 62-90, 62-80, 62-70, 65-171, 65-160, 65-150, 65-140, 65-130, 65-120, 65-110, 65-100, 65-90, 65-80, or 65-70 amino acids of SEQ ID NO: 63 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and having IL-15 binding activity.
  • a sushi domain consists of 62-171, 62-160, 62-150, 62-140, 62-130, 62-120, 62-110, 62-100, 62-90, 62-80, 62-70, 65-171, 65-160, 65-150, 65-140, 65-130, 65-120, 65-110, 65-100, 65-90, 65-80, or 65-70 amino acids of SEQ ID NO: 63.
  • the sushi domain comprises, or consists of, an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 52.
  • a sushi domain consists of 62-171 amino acids of SEQ ID NO: 63 or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 modifications (e.g., substitutions) relative thereto, and having IL-15 binding activity.
  • a sushi domain consists of up to 171 amino acids of SEQ ID NO: 5 or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 modifications (e.g., substitutions) relative thereto, and having IL-15 binding activity.
  • a sushi domain consists of 62-171, 62-160, 62-150, 62-140, 62-130, 62-120, 62-110, 62-100, 62-90, 62-80, 62-70, 65-171, 65-160, 65-150, 65-140, 65-130, 65-120, 65-110, 65-100, 65-90, 65-80, or 65-70 amino acids of SEQ ID NO: 63 or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 modifications (e.g., substitutions) relative thereto, and having IL-15 binding activity.
  • a sushi domain comprises at least 62 amino acids of SEQ ID NO: 63 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein the sequence comprises an L77I mutation relative to wild-type IL-15Ra, and having IL-15 binding activity.
  • a sushi domain comprises at least 65 amino acids of SEQ ID NO: 63 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein the sequence comprises an L77I mutation relative to wild-type IL-15Ra, and having IL-15 binding activity.
  • a sushi domain comprises a portion of SEQ ID NO: 63 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein the sequence comprises an L77I mutation relative to wild-type IL-15Ra, and having IL-15 binding activity.
  • the sushi domain comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 52.
  • a sushi domain comprises at least 62 amino acids of SEQ ID NO: 63 or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 modifications (e.g., substitutions) relative thereto, wherein the sequence comprises an L77I mutation relative to wild-type IL-15Ra, and having IL-15 binding activity.
  • a sushi domain comprises a portion of SEQ ID NO: 66 or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 modifications (e.g., substitutions) relative thereto, wherein the sequence comprises an L77I mutation relative to wild-type IL-15Ra, and having IL-15 binding activity.
  • the sushi domain comprises at least 10, 20, 30, 40, 50, 60, 62, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 consecutive amino acids of SEQ ID NO: 63, or a sequence having an L77I mutation relative thereto.
  • the sushi domain consists of 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, or 160-170 consecutive amino acids of SEQ ID NO: 63, or a sequence having an L77I mutation relative thereto.
  • the sushi domain is a sushi domain from human or a non-human animal, e.g., mammal, e.g., non-human primate.
  • the polypeptide can have a second, heterologous domain, e.g., an Fc domain or a Fab domain.
  • the polypeptide comprising the IL-15 receptor or fragment thereof comprises an Fc domain.
  • the Fc domain is an effector-attenuated Fc domain, e.g., a human IgG2 Fc domain, e.g., a human IgG2 Fc domain of SEQ ID NO: 54 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the effector-attenuated Fc domain has reduced effector activity, e.g., compared to a wild-type IgG1 Fc domain, e.g., compared to a wild-type IgG1 Fc domain of SEQ ID NO: 67.
  • effector activity comprises antibody-dependent cellular toxicity (ADCC).
  • ADCC antibody-dependent cellular toxicity
  • the effector activity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in an ADCC assay, e.g., compared to a wild-type IgG1 Fc domain of SEQ ID NO: 67.
  • effector activity comprises complement dependent cytotoxicity (CDC).
  • the effector activity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in a CDC assay such as a CDC assay described in Armour et al., “Recombinant human IgG molecules lacking Fc gamma receptor I binding and monocyte triggering activities.” Eur J Immunol (1999) 29:2613-24′′ e.g., compared to a wild-type IgG1 Fc domain of SEQ ID NO: 67.
  • the Fc domain comprises an IgG1 Fc domain of SEQ ID NO: 67 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the Fc domain comprises an IgG2 constant region of SEQ ID NO: 68 or fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the Fc domain comprises an IgG2Da Fc domain of SEQ ID NO: 55 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the Fc domain comprises one or both of A330S and P331S mutations using Kabat numbering system.
  • the Fc domain is one described in Armour et al. “Recombinant human IgG molecules lacking Fc gamma receptor I binding and monocyte triggering activities.” Eur J Immunol (1999) 29:2613-24.
  • the Fc domain has dimerization activity.
  • the Fc domain is an IgG domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain comprises a CH2 domain and a CH3 domain.
  • the nanoparticle comprises a protein having a sequence of SEQ ID NO: 56 (sushi-IgG2Da-Fc)or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • an IgG2 Fc domain e.g., SEQ ID NO: 54 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the FM comprises a sushi domain of SEQ ID NO: 9 and an Fc domain described herein, e.g., an IgG1 Fc domain, e.g., an Fc domain of SEQ ID NO: 67 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the FM comprises a sushi domain of SEQ ID NO: 9 and an IgG2 Fc domain, e.g., an Fc domain of SEQ ID NO: 54 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the FM comprises a sushi domain of SEQ ID NO: 9 and an IgG1 Fc domain, e.g., an Fc domain of SEQ ID NO: 67 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the FM comprises a sushi domain of SEQ ID NO: 9 and an IgG2Da Fc domain of SEQ ID NO: 56 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the FM comprises a sushi domain of SEQ ID NO: 9 and an IgG2Da Fc domain of SEQ ID NO: 56 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the FM comprises a sushi-IgG2Da-Fc protein having a sequence of SEQ ID NO: 56 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the IL-15 molecule is a molecule described in PCT International Application Publication No. WO2017/027843, which is herein incorporated by reference in its entirety.
  • the cytokine molecule is interleukin 2 (IL-2).
  • IL-2 is chiefly secreted by T cells in response to antigenic stimuli, and is the main cytokine for T cell proliferation.
  • IL-2 receptor is expressed by T lymphocytes, NK cells, B cells, macrophages, and monocytes; however, only T lymphocytes are capable of producing this cytokine.
  • the high affinity IL-2 receptor is a heterotrimeric cell surface receptor composed of ⁇ , ⁇ , ⁇ c -polypeptide chains (K D 10 ⁇ 11 M).
  • the 55 kDa ⁇ -chain also known as IL-2R ⁇ , CD25, p55, and Tac (T cell activation) antigen, is unique to the IL-2R.
  • the ⁇ (CD122; 75) and ⁇ c (CD132) chains are part of a cytokine receptor superfamily (hematopoietin receptors) and are functional components of other cytokine receptors, such as IL-15R.
  • the intermediate affinity receptor is a dimer composed of a ⁇ - and a ⁇ c -chain (K D 10 ⁇ 9 M) while the low affinity receptor consists of a monomeric a-subunit that has no signal transduction capacity (K D 10 ⁇ 8 M).
  • Treg cells constitutively express high levels of IL-2 ⁇ -chain, having thus a higher affinity to IL-2, and compete for this growth factor with proliferating cells.
  • Treg cells do not only prevent them from continuing the proliferative process but also leave them without a vital cytokine, causing metabolic interruption and cell death.
  • the IL-2 molecule (e.g., IL-2 polypeptide molecule) comprises a wild-type IL-2 amino acid sequence, e.g., a human IL-2 amino acid sequence, e.g., including the amino acid sequence of SEQ ID NO: 80.
  • the cytokine molecule is an IL-2 molecule, e.g., a full length, a fragment or a variant of IL-2, e.g., human IL-2 that retains at least some IL-2 activity such as binding to IL-2R.
  • the IL-2 molecule is a variant of human IL-2, e.g., having one or more amino acid alterations, e.g., substitutions, to the human IL-2 amino acid sequence.
  • the IL-2 variant comprises, or consists of, one or more mutations while maintaining the IL-2R binding activity.
  • the IL-2 of the present disclosure can have the following sequence (or a functional variant thereof):
  • variant forms of IL-2 can be made by introducing one or more mutations into the wild-type form, so as to fine tune its binding affinity to IL-2R.
  • a library of IL-2 mutants or variants can be made to test their affinity to IL-2R.
  • These variants can also be conjugated or coupled to various targeting moieties, and the resulting FMs can be tested for targeting.
  • the IL-2 variant comprises, or consists of, one or more mutations.
  • the IL-2 variants can be IL-2 ⁇ selective agonists. Functionally they selectively activate the IL2 ⁇ receptor complex relative to the IL2R ⁇ receptor complex. It is derived from a wild type IL-2 protein structurally defined as having at least 95% sequence identity to the wild type IL-2 (e.g., SEQ ID NO: 80) and functionally defined by the ability to preferentially activate Treg cells.
  • the variants can also be functionally defined by its ability to selectively activate IL-2 receptor signaling in Tregs, as measured by the levels of phosphorylated STATS protein in Treg cells compared to CD4+- CD25 ⁇ /low T cells or NK cells, or by the selective activation of Phytohemagglutinin-stimulated T cells versus NK cells.
  • modifications to the cytokine or growth factor amino acid sequence can be made to improve persistence and/or selectivity for an immune regulatory cell such as a regulatory T cell.
  • an immune regulatory cell such as a regulatory T cell.
  • One of the obstacles for prolonged surface persistence and signaling of cytokine fusions is cytokine/receptor internalization and degradation through the endosomal/lysosomal pathway. Extending the persistence of cytokine fusions can be achieved, in some embodiments, by altering the affinity of cytokines in the altered-pH environment of the endosome. By decreasing the affinity of cytokine for its receptor in the endosome, there is decreased lysosomal degradation and increased recycling to the cell surface.
  • histidine mutants can be engineered in receptor-binding motifs of IL-2. Histidine changes protonation state in the altered pH of the endosome, and this reduces the affinity of IL-2/receptor interactions. A number of different substitutions can be tested to find ones that increase recycling and persistence without decreasing surface affinity for the receptor. Alternatively, reduced surface affinity for the receptor can be desirable for enhancing Treg-selective IL-2 signaling. In embodiments, histidine substitutions can be focused in motifs that are involved in IL2R ⁇ interaction in order to enhance IL2R ⁇ binding and Treg selectivity.
  • Antibodies can be used that disrupt IL-2 binding to IL2R ⁇ or IL2 ⁇ bias IL-2 signaling and expansion towards T-effector cells (Teffs) or Tregs respectively.
  • disruption of IL-2 binding to IL2R ⁇ can be achieved via amino acid substitution. This can be tested using the histidine substitution approach detailed above, and through standard binding interface modulation substitutions (sterics, charge, etc.).
  • the cytokine is IL-15 and its variants such as those disclosed in PCT International Application No. PCT/US2017/037249 filed Jun. 13, 2017, e.g., on pages 46-57, which is incorporated herein by reference in its entirety.
  • Amphiregulin is a polypeptide growth factor that belongs to the epidermal growth factor (EGF) family.
  • Areg is synthesized as a type 1 transmembrane protein precursor (proAR) and expressed on the cell surface.
  • EGFR may be activated by Areg in several ways: autocrine or paracrine activation by the soluble form of Areg, a juxtacrine mode enabling the un-cleaved transmembrane form to activate EGFR, or by a newly described mode of signaling entailing Areg containing exosomes, that better enhance invasion of recipient cells in comparison to exosomes containing high affinity ligands.
  • TGF- ⁇ is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes four different isoforms (TGF- ⁇ 1 to 4) and many other signaling proteins produced by all white blood cell lineages.
  • TGF- ⁇ 1 to 4 transforming growth factor superfamily
  • the type 2 receptor kinase phosphorylates and activates the type 1 receptor kinase that activates a signaling cascade. This leads to the activation of different downstream substrates and regulatory proteins, inducing transcription of different target genes that function in differentiation, chemotaxis, proliferation, and activation of many immune cells.
  • Fusion molecules include a targeting moiety to target a reguatory immune cell, such as a regulatory T cell or regulatory B cell.
  • the targeting moiety may be an antibody molecule (e.g., an antigen binding domain as described herein), a receptor or a receptor fragment, or a ligand or a ligand fragment, or a combination thereof.
  • the targeting moiety associates with or binds to a molecule, e.g., surface marker or receptor, present on the surface of a cell (a “targeting moiety”).
  • the FM may be selectively targeted to a regulatory T cell by the binding of the targeting moiety to the cell surface.
  • the FM can be targeted to general Tregs by binding to general Treg surface markers such as CD4 and/or CD25 ( FIG. 1G ).
  • the FM can also be specifically targeted to subsets of functional Tregs by binding to subset-specific surface markers such as CD29 and/or NRP1 ( FIG. 1G ).
  • combinatorial targeting of two or more FMs can be achieved
  • the targeting moiety is chosen from an antibody molecule (e.g., a full antibody (e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fv, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody)), non-antibody scaffold, or ligand that binds to the CD45 receptor.
  • an antibody molecule e.g., a full antibody (e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fv, a single domain antibody,
  • the targeting moiety targets the FM to markers, cell surface receptors, or other molecules present on the surface of a regulatory T cell, including, for example, CD45 (via, e.g., BC8 (ACCT: HB-10507), 9.4 (ATTC: HB-10508)), CD4, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CD8, CCR4, CCR5, CD137, CCR6 (CD196), CCR8, CCR10, OX40, GITR, NRP1, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, CD11a (via, for example, MHM24 monoclonal antibody), CD18 (via, for example, chimeric1B4 monoclonal antibody).
  • CD45 via, e.g., BC8 (ACCT: HB-10507), 9.4 (ATTC: HB-10508)
  • the targeting moiety targets CD19 on a regulatory B cell.
  • a targeting moiety is chosen from an antibody molecule, e.g., an antigen binding domain, non-antibody scaffold, or ligand that binds to CD45, CD4, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CD8, CCR4, CCR5, CD137, CCR6 (CD196), CCR8, CCR10, OX40, GITR, NRP1, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, CD11a, or CD18.
  • an antibody molecule e.g., an antigen binding domain, non-antibody scaffold, or ligand that binds to CD45, CD4, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CD8, CCR4, CCR5, CD137, CCR6 (CD196),
  • the targeting moiety selectively targets the FM to regulatory T cells over other immune cells or other cell types.
  • the targeting moiety directs the FM to regulatory T cells by targeting cell surface receptors or other molecules that are expressed on the surface of a regulatory cell buy may also be present on other immune cells or other cell types.
  • the targeting moiety targets the FM to cell surface receptors or other molecules that are present in higher abundance on the surface of regulatory T cells as compared with other immune cells or other cell types that may be co-located with the regulatory T cells of interest.
  • the targeting moiety can be a bispecific moiety that binds to more than one target.
  • Exemplary bispecific Treg targeting moieties include CD4:CD45, CD8:CD45, CD4:CD39, CD4:NRP1, and CD4:CD25.
  • CD127 is a negative surface marker for Tregs
  • the pairing of CD127 with a positive Treg surface marker can be used to negatively enrich and/or sort Tregs.
  • CD45 also known as leukocyte common antigen, refers to human CD45 protein and species, isoforms, and other sequence variants thereof.
  • CD45 can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein.
  • CD45 is a receptor-linked protein tyrosine phosphatase that is expressed on leukocytes, and which plays an important role in the function of these cells (reviewed in Altin, JG (1997) Immunol Cell Biol. 75(5):430-45, incorporated herein by reference).
  • the extracellular domain of CD45 is expressed in several different isoforms on T cells, and the particular isoform(s) expressed depends on the particular subpopulation of cell, their state of maturation, and antigen exposure. Expression of CD45 is important for the activation of T cells via the TCR, and that different CD45 isoforms display a different ability to support T cell activation.
  • CD4 is a co-receptor for MHC Class II (with TCR, T-cell receptor); found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.
  • CD4+ T cells are crucial in achieving a regulated effective immune response to pathogens.
  • Naive CD4+ T cells are activated after interaction with antigen-MHC complex and differentiate into specific subtypes depending mainly on the cytokine milieu of the microenvironment.
  • T-helper 1 and T-helper 2 other subsets have been identified, including T-helper 17, regulatory T cell, follicular helper T cell, and T-helper 9, each with a characteristic cytokine profile.
  • CD4+ T cells carry out multiple functions, ranging from activation of the cells of the innate immune system, B-lymphocytes, cytotoxic T cells, as well as nonimmune cells, and also play critical role in the suppression of immune reaction. See e.g., Rishi Vishal et al. “CD4+ T Cells: Differentiation and Functions,” Clinical and Developmental Immunology, vol. 2012, Article ID 925135, 12 pages, 2012. doi:10.1155/2012/925135.
  • CD25 also called interleukin-2 receptor alpha chain
  • Tregs are type I transmembrane protein present on Tregs as well as activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes.
  • CD127 is part of the heterodimeric IL-7 receptor that is composed of CD127 and the common g chain, which is shared by other cytokine receptors (IL-2R, IL-4R, IL-9R, IL-15R, and IL-21R). CD127 is expressed on thymocytes, T- and B-cell progenitors, mature T cells, monocytes, and some other lymphoid and myeloid cells. CD127 expression is down-modulated on the Treg cells, inversely correlating with the expression of Treg marker FoxP3. Studies have shown that IL-7R plays an important role in the proliferation and differentiation of mature T cells, and in vitro experiments show that the expression of CD127 is down-regulated following T cell activation. It is believed that FoxP3 interacts with the CD127 promoter and might contribute to reduced expression of CD127 in Tregs.
  • CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule, but is specific for the class I MHC protein. There are two isoforms of the protein, alpha and beta, each encoded by a different gene. In humans, both genes are located on chromosome 2 in position 2p12. The CD8 co-receptor is predominantly expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. It is expressed in T cell lymphoblastic lymphoma and hypo-pigmented mycosis fungoides.
  • MHC major histocompatibility complex
  • CD8 forms a dimer, consisting of a pair of CD8 chains.
  • the most common form of CD8 is composed of a CD8- ⁇ and CD8- ⁇ chain, both members of the immunoglobulin superfamily with an immunoglobulin variable (IgV)-like extracellular domain connected to the membrane by a thin stalk, and an intracellular tail. Less-common homodimers of the CD8- ⁇ chain are also expressed on some cells.
  • the extracellular IgV-like domain of CD8- ⁇ interacts with the ⁇ 3 portion of the Class I MHC molecule. This affinity keeps the T cell receptor of the cytotoxic T cell and the target cell bound closely together during antigen-specific activation. Cytotoxic T cells with CD8 surface protein are called CD8+ T cells.
  • CD39 is an integral membrane protein with two transmembrane domains and a large extracellular region with nucleoside triphosphate diphosphohydrolase activity. In humans, CD39 is mainly expressed by regulatory T cells but also other leukocytes. It is also named as ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1). CD39 is an ectoenzyrne that hydrolases ATP/UTP and ADP/UDP to the respective nucleosides such as AMP.
  • CD39 is a cell surface marker of Foxp3 Treg cells which may regulate immune T cell suppression by the downstream production of adenosine and thus, might represent a target for the development of novel therapeutic methods useful for treating diseases associated with Treg activity (Deaglio et al.,. J Exp Med. 2007 Jun. 11; 204(6):1257-65).
  • NRP1 is a multi-functional receptor that contributes to the development of the nervous and vascular systems. NRP1 was described as a receptor that binds the sernaphorin 3A ligand, acting with plexin co-receptors to regulate axon guidance (He and Tessier-Lavigne, Cell (1997) 90:739-51). It was later shown that NRP1 also binds members of the vascular endothelial growth factor (VEGF) ligand family to mediate vascular development (Soker et al, Cell (1998) 92:735-45; Kawasaki et al, Development (1999) 126:4895-902).
  • VEGF vascular endothelial growth factor
  • NRP1 is expressed at high levels on nTreg cells and can be used to separate nTreg versus iTreg cells in certain physiological settings (Yadav et al., J Exp Med. 2012 Sep. 24;209(10):1713-22, S1-19).
  • iTreg cells generated through antigen delivery or converted under homeostatic conditions lack NRP1 expression.
  • NRP1(lo) iTreg cells show similar suppressive activity to nTreg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings.
  • NRP1 provides a marker to distinguish distinct Treg subsets and can be useful in studying the role of nTreg versus iTreg cells in different disease settings.
  • the fusion proteins described herein may comprise one or more antibody molecule.
  • the targeting moiety may comprise an antibody molecule.
  • the antibody molecule binds to a cell surface protein on a regulatory T cell, e.g., a human Treg antigen.
  • the antibody molecule binds specifically to an epitope, e.g., linear or conformational epitope, on the Treg antigen.
  • an antibody molecule is a monospecific antibody molecule and binds a single epitope, e.g., a monospecific antibody molecule having a plurality of immunoglobulin variable domain sequences, each of which binds the same epitope.
  • an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap. In an embodiment, the first and second epitopes do not overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain.
  • a multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope.
  • a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope.
  • a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody molecule comprises a scFv or a Fab, or fragment thereof, have binding specificity for a first epitope and a scFv or a Fab, or fragment thereof, have binding specificity for a second epitope.
  • an antibody molecule comprises a diabody, and a single-chain molecule, as well as an antigen-binding fragment of an antibody (e.g., Fab, F(ab′) 2 , and Fv).
  • an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL).
  • VH heavy chain variable domain sequence
  • VL light chain variable domain sequence
  • an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody.
  • an antibody molecule in another example, includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab′, F(ab′) 2 , Fc, Fd, Fd′, Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor.
  • Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies.
  • a preparation of antibody molecules can be monoclonal or polyclonal.
  • An antibody molecule can also be a human, humanized, CDR-grafted, or in vitro generated antibody.
  • the antibody can have a heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, or IgG4.
  • the antibody can also have a light chain chosen from, e.g., kappa or lambda.
  • immunoglobulin (Ig) is used interchangeably with the term “antibody” herein.
  • antigen-binding fragments of an antibody molecule include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al.
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • a F(ab′)2 fragment a bivalent fragment comprising two Fab fragment
  • Antibody molecules can also be single domain antibodies.
  • Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be any of the art, or any future single domain antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine.
  • a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example.
  • variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
  • VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the present disclosure.
  • Antibody molecules can include non-antibody scaffolds and antibody mimetics.
  • Exemplary non-antibody scaffolds include: lipocalins (e.g. anticalins), affibodies, fibronectin (e.g. monobodies or Adnectins), knottins, ankyrin repeats (e.g. DARPins), and A domains (e.g. avimers).
  • VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR or FW).
  • CDR complementarity determining regions
  • FR framework regions
  • CDR complementarity determining region
  • the precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme). As used herein, the CDRs defined according the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the antibody molecule can be a polyclonal or a monoclonal antibody.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • a monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).
  • the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.
  • Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No.
  • antibody repertoires can be displayed on the surface of filamentous bacteriophage, and the constructed library may be screened for phages that bind to the immunogen (e.g., CD4, CD25, CD39, NRP1).
  • the immunogen e.g., CD4, CD25, CD39, NRP1.
  • Antibody phage is based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection and phage propagation. This technique allows in vitro selection of monoclonal antibodies.
  • the phage display process begins with antibody-library preparation followed by ligation of the variable heavy (VH) and variable light (VL) PCR products into a phage display vector, culminating in analysis of clones of monoclonal antibodies.
  • VH variable heavy
  • VL variable light
  • the VH and VL PCR products, representing the antibody repertoire, are ligated into a phage display vector (e.g., the phagemid pComb3X) that is engineered to express the VH and VL as an scFv fused to the pIII minor capsid protein of a filamentous bacteriophage of Escherichia coli that was originally derived from the M13 bacteriophage.
  • a phage display vector e.g., the phagemid pComb3X
  • the phage display vector pComb3X does not have all the other genes necessary to encode a full bacteriophage in E. coli .
  • a helper phage is added to the E. coli that are transformed with the phage display vector library.
  • the result is a library of phages, each expressing on its surface a monoclonal antibody and harboring the vector with the respective nucleotide sequence within.
  • the phage display can also be used to produce the monoclonal antibody itself (not attached to phage capsid proteins) in certain strains of E. Coli .
  • Additional cDNA is engineered, in the phage display vector, after the VL and VH sequences to allow characterization and purification of the mAb produced.
  • the recombinant antibody may have a hemagglutinin (HA) epitope tag and a polyhistidine to allow easy purification from solution.
  • HA hemagglutinin
  • the antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody.
  • a rodent mouse or rat
  • the non-human antibody is a rodent (mouse or rat antibody).
  • Methods of producing rodent antibodies are known in the art.
  • Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al.
  • An antibody molecule can be one in which the variable region, or a portion thereof, e.g., the CDRs, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the present disclosure. Antibody molecules generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the present disclosure.
  • Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al.
  • a humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immuoglobulin chains) replaced with a donor CDR.
  • the antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen.
  • the donor will be a rodent antibody, e.g., a rat or mouse antibody
  • the recipient will be a human framework or a human consensus framework.
  • the immunoglobulin providing the CDRs is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.”
  • the donor immunoglobulin is a non-human (e.g., rodent).
  • the acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.
  • the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.
  • a “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.
  • An antibody molecule can be humanized by methods known in the art (see e.g., Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are hereby incorporated by reference).
  • Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced.
  • CDR-grafting or CDR substitution wherein one, two, or all CDRs of an immunoglobulin chain can be replaced.
  • humanized antibody molecules in which specific amino acids have been substituted, deleted or added. Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.
  • the antibody molecule can be a single chain antibody.
  • a single-chain antibody (scFv) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52).
  • the single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein.
  • the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4.
  • the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda.
  • the constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, cell function, and/or complement function).
  • the antibody e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, cell function, and/or complement function.
  • it is an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.
  • Antibodies with altered function e.g. altered affinity for an effector ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 A1, U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of all of which are hereby incorporated by reference). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.
  • an antibody molecule can be derivatized or linked to another functional molecule (e.g., a cytokine molecule as described herein or other chemical or proteinaceous groups).
  • a “derivatized” antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules of the present disclosure are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules.
  • an antibody molecule can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as a cytokine molecule, another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • a cytokine molecule e.g., another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody molecule is produced by crosslinking an antibody molecule to one or more proteins, e.g., a cytokine molecule, another antibody molecule (of the same type or of different types, e.g., to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • Such linkers are available from Pierce Chemical Company, Rockford, Ill.
  • the targeting moiety of the FM includes an antibody molecule or a ligand that selectively binds to an immune regulatory cell surface target, e.g., an immune cell surface receptor.
  • the immune regulatory cell surface target or receptor can have one, two, three or more of the following properties: (i) is abundantly present on the surface of an immune cell (e.g., outnumbers the number of receptors for the cytokine molecule present on the immune cell surface); (ii) shows a slow downregulation, internalization, and/or cell surface turnover, e.g., relative to the receptors activated by the cytokine of the FM; (iii) is present on the surface of the immune regulatory cell for a prolonged period of time, e.g., relative to the receptors activated by the cytokine of the FM; or (iv) once internalized is substantially recycled back to the cell surface, e.g., at least 25%, 50%, 60%, 70%, 80%, 90% or more of the immune regulatory cell surface
  • the targeting moiety of the FM binds to a recycling cell surface receptor. In some embodiments, the targeting moiety of the FM binds to a receptor expressed on a cell (e.g., an immune regulatory cell), e.g. the surface membrane of the cell, and further the cell also expresses a cytokine receptor (e.g., a receptor to the cytokine molecule of the FM).
  • a cell e.g., an immune regulatory cell
  • a cytokine receptor e.g., a receptor to the cytokine molecule of the FM.
  • the targeting moiety of the FM can be chosen from an antibody molecule or a ligand molecule that binds to an immune regulatory cell surface target, e.g., a target chosen from CD45, CD4, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CD8, CCR4, CCR5, CD137, CCR6 (CD196), CCR8, CCR10, OX40, GITR, NRP1, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, CD11a, or CD18.
  • the immune regulatory cell surface target is chosen from CD4, CD25, CD39, CD45, and/or NRP1.
  • the targeting moiety comprises an antibody molecule or a ligand molecule that binds to CD4. In one embodiment, the targeting moiety comprises an antibody molecule or a ligand molecule that binds to CD25. In one embodiment, the targeting moiety comprises an antibody molecule or a ligand molecule that binds to CD127. In one embodiment, the targeting moiety comprises a bispecific antibody molecule or a ligand molecule that binds to CD4 and CD25. In one embodiment, the targeting moiety comprises a bispecific antibody molecule or a ligand molecule that binds to CD127 and CD25 (e.g., for negative enrichment of Tregs).
  • the targeting moiety comprises a bispecific antibody molecule or a ligand molecule that binds to CD127 and CD4 (e.g., for negative enrichment of Tregs).
  • the antibodies can be selected to fine tune the selective binding to the immune cell such as Treg.
  • the targeting moiety comprises an antibody molecule or a ligand molecule that binds to CD45 (also interchangeably referred to herein as “CD45 receptor” or “CD45R”).
  • CD45R also interchangeably referred to herein as “CD45 receptor” or “CD45R”).
  • the target is CD45 (e.g., a CD45 isoform chosen from CD45RA, CD45RB, CD45RC or CD45RO).
  • CD45 is primarily expressed on T cells.
  • CD45RA is primarily expressed on na ⁇ ve T cells
  • CD45RO is primarily expressed on activated and memory T cells.
  • the targeting moiety comprises an antibody molecule or a ligand molecule that binds to CD8.
  • the targeting moiety comprises a bispecific antibody molecule or a ligand molecule that binds to CD8 and CD45.
  • the targeting moiety of the FM comprises an antibody molecule (e.g., an antigen binding domain), a receptor molecule (e.g., a receptor, a receptor fragment or functional variant thereof), or a ligand molecule (e.g., a ligand, a ligand fragment or functional variant thereof), or a combination thereof, that binds to the Treg target or receptor.
  • an antibody molecule e.g., an antigen binding domain
  • a receptor molecule e.g., a receptor, a receptor fragment or functional variant thereof
  • a ligand molecule e.g., a ligand, a ligand fragment or functional variant thereof
  • the antibody molecule of the targeting moiety of the FM comprises a full antibody (e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fv, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody)) that binds to the Treg target or receptor.
  • a full antibody e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains
  • an antigen-binding fragment e.g., a Fab, F(ab′)2, Fv, a single chain Fv, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof,
  • the heavy chain constant region of the antibody molecule can be chosen from IgG1, IgG2, IgG3, or IgG4, or a fragment thereof, and more typically, IgG1, IgG2 or IgG4.
  • the Fc region of the heavy chain can include one or more alterations, e.g., substitutions, to increase or decrease one or more of: Fc receptor binding, neonatal-Fc receptor binding, antibody glycosylation, the number of cysteine residues, cell function, complement function, or stabilize antibody formation (e.g., stabilize IgG4).
  • the heavy chain constant region for an IgG4, e.g., a human IgG4, can include a substitution at position 228 (e.g., a Ser to Pro substitution), as shown in, e.g., SEQ ID NO: 67 (see e.g., Angal, S, King, DJ, et al. (1993) Mol Immunol 30:105-108 (initially described as S241P using a different numbering system); Owens, R, Ball, E, et al. (1997) Immunotechnology 3:107-116).
  • a substitution at position 228 e.g., a Ser to Pro substitution
  • SEQ ID NO: 67 see e.g., Angal, S, King, DJ, et al. (1993) Mol Immunol 30:105-108 (initially described as S241P using a different numbering system); Owens, R, Ball, E, et al. (1997) Immunotechnology 3:107-116).
  • the light chain constant region of the antibody molecule can be chosen from the light chain constant regions of kappa or lambda, or a fragment thereof.
  • the antibody molecule of the targeting moiety of the FM can bind to the target antigen with a dissociation constant of less than about 100 nM, 50 nM, 25 nM, 10 nM, e.g., less than 1 nM (e.g., about 10-100 pM). In embodiments, the antibody molecule binds to a conformational or a linear epitope on the antigen. In certain embodiments, the antigen bound by the antibody molecule of the targeting moiety is stably expressed on the surface of the immune cell. In embodiments, the antigen is a cell surface receptor that is more abundant on the cell surface relative to a receptor for the cytokine molecule of the FM on the cell surface.
  • the targeting moiety is chosen from an antibody molecule (e.g., a full antibody (e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fv, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody)), or a non-antibody scaffold, or a ligand that binds to an immune cell surface target or ligand.
  • an antibody molecule e.g., a full antibody (e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain
  • the targeting moiety is an antibody molecule or a ligand that binds to CD45, CD4, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CD8, CCR4, CCR5, CD137, CCR6 (CD196), CCR8, CCR10, OX40, GITR, NRP1, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, CD11a, or CD18, e.g., a bispecific antibody that binds to two or more of the aforesaid targets.
  • CD45 CD4, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CD8, CCR4, CCR5, CD137, CCR6 (CD196), CCR8, CCR10, OX40, GITR, NRP1, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR
  • the antibody molecule binds to one or more of CD45, CD4, CD25, CD39, CD137, or NRP1, e.g., it is an IgG, e.g., human IgG4, or an antigen binding domain, e.g., a Fab, a F(ab′)2, Fv, a single chain Fv, that binds to CD45, CD4, CD25, CD39, CD137, or NRP1.
  • the antibody molecule is a human, a humanized or a chimeric antibody. In embodiments, the antibody molecule is a recombinant antibody.
  • antibody molecules having the amino acid sequences disclosed herein, or an amino acid sequence substantially identical thereof), nucleic acid molecules encoding the same, host cells and vectors comprising the nucleic acid molecules.
  • the targeting moiety can be a binding agent for CD45, such as an antibody or antigen-binding fragment thereof.
  • the anti-CD45 antibody is a human anti-CD45 antibody, a humanized anti-CD45 antibody, or a chimeric anti-CD45 antibody.
  • the anti-CD45 antibody is an anti-CD45 monoclonal antibody.
  • Exemplary anti-CD45 antibodies include antibodies BC8, 4B2, GAP8.3 or 9.4.
  • Antibodies against other immune cell surface targets are also disclosed, e.g., anti-CD8 antibodies, such as OKT8 monoclonal antibodies, anti-CD18 antibodies, such as 1B4 monoclonal antibodies, and anti-CD1la antibodies, such as MHM24 antibodies.
  • the antibody molecule that binds to CD45 is specific to one CD45 isoform or binds to more than on CD45 isoforms, e.g., is a pan-CD45 antibody.
  • the anti-CD45 antibody molecule binds to CD45RA and CD45RO.
  • the anti-CD45 antibody molecule is a BC8 antibody.
  • the BC8 antibody binds to CD45RA and CD45RO.
  • the anti-CD45 antibody molecule is CD45RO-specific or is a pan-CD45 antibody molecule, e.g., it binds to activated and memory T cells. Additional examples of anti-CD45 antibody molecules include, but is not limited to, GAP8.3, 4B2, and 9.4.
  • Exemplary formats for the FM include a cytokine molecule (e.g., one or more cytokine molecules) coupled to an antibody molecule that binds to an immune cell surface target (e.g., immunoglobulin moiety (Ig), for example an antibody (e.g., a full antibody, IgG) or antibody fragment (Fab, scFv, a half antibody, or a single domain antibody and the like).
  • an immune cell surface target e.g., immunoglobulin moiety (Ig), for example an antibody (e.g., a full antibody, IgG) or antibody fragment (Fab, scFv, a half antibody, or a single domain antibody and the like).
  • the FM includes a fusion to the amino-terminus (N-terminus) or carboxy-terminus (C-terminus) of the antibody molecule, typically, the C-terminus.
  • the cytokine molecule can be coupled to the antibody molecule, optionally, via a linker.
  • the cytokine or cytokine receptor e.g., receptor fragment (e.g., sushi domain) is coupled to the N-terminus or the C-terminus of the antibody molecule.
  • the cytokine or cytokine receptor e.g., receptor fragment (e.g., sushi domain) is coupled to the N-terminus or the C-terminus of the light chain of the antibody molecule (e.g., a full antibody or fragment thereof, e.g., a Fab, a scFv, or a half antibody).
  • the cytokine or cytokine receptor e.g., receptor fragment (e.g., sushi domain) is coupled to the N-terminus or the C-terminus of the heavy chain of the antibody molecule (e.g., a full antibody or fragment thereof, e.g., a Fab, a scFv, a half antibody, or a single domain antibody (VH)).
  • a full antibody or fragment thereof e.g., a Fab, a scFv, a half antibody, or a single domain antibody (VH)
  • VH single domain antibody
  • the FM includes an antibody molecule or fragment thereof, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD4, anti-CD25, anti-CD8, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-15, IL-2, IL-7, IL-21, IL-27, or a cytokine receptor, e.g., IL-15Ralpha sushi domain, at either the N-terminal region or the C-terminal region of the light chain or heavy chain (as depicted in, e.g., FIGS. 1B, 1G, 2A, 3A-3D , 6 A, 11 A and 12 A). Any combination of orientation of light chain- or heavy chain-cytokine molecule can be present in the FM.
  • anti-CD45 e.g., BC8 antibody
  • the FM can include a full antibody or a tetramer of two identical half-antibodies, e.g., a first and second antibody, each having a light chain and a heavy chain (e.g., a scFv) of the antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-2 or a variant thereof, IL-15 or IL-15 sushi domain, at the N-terminal region of the light chain (e.g., a depicted in FIGS. 3A and 3C ).
  • a cytokine molecule e.g., IL-2 or a variant thereof, IL-15 or IL-15 sushi domain
  • the FM can include a full antibody or a tetramer of two identical half-antibodies, e.g., a first and second antibodies, each having a light chain and a heavy chain of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-2 or its variant, IL-15 or an IL-15 sushi domain, at the C-terminal region of the light chain (e.g., a depicted in FIGS. 2A, 3A, 3C and 6A ).
  • a cytokine molecule e.g., IL-2 or its variant, IL-15 or an IL-15 sushi domain
  • the FM can include a full antibody or a tetramer of two identical half-antibodies, e.g., a first and second antibodies, each having a light chain and a heavy chain of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-2 or its variant, IL-15 or an IL-15 sushi domain, at the C-terminal region of the heavy chain (e.g., a depicted in FIG. 6A ).
  • a cytokine molecule e.g., IL-2 or its variant, IL-15 or an IL-15 sushi domain
  • the FM can include a full antibody or a tetramer of two identical half-antibodies, e.g., a first and second antibodies, each having a light chain and a heavy chain of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-2 or its variant, IL-15 or an IL-15 sushi domain, at the N-terminal region of the heavy chain.
  • a cytokine molecule e.g., IL-2 or its variant, IL-15 or an IL-15 sushi domain
  • the FM can include an antibody fragment (e.g., a scFv, a Fab) having a light chain variable domain and a heavy chain variable domain of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-2 or its variants, IL-15 or an IL-15 sushi domain, at the N- or C-terminal region of the heavy chain variable domain (e.g., a depicted in FIGS. 2A, 3B, 3D, 11A , and 12 A).
  • an antibody fragment e.g., a scFv, a Fab having a light chain variable domain and a heavy chain variable domain of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody),
  • the FM can include an antibody fragment (e.g., a scFv, a Fab) having a light chain variable domain and a heavy chain variable domain of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-2 or its variants, IL-15 or an IL-15 sushi domain, at the N- or C-terminal region of the light chain variable domain.
  • an antibody fragment e.g., a scFv, a Fab having a light chain variable domain and a heavy chain variable domain of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e
  • the FM includes a tetramer of two different half-antibodies, e.g., a first and second antibodies, wherein the first antibody has a light chain and a heavy chain (or an fragment thereof, e.g., scFv or Fab) of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4, anti-CD25, anti-CD39, or anti-NRP1, that is coupled to, e.g., covalently linked, to a cytokine molecule, e.g., IL-2 or its variants, IL-15 or an IL-15 sushi domain, at the N-terminal region of the light chain; and the second antibody has a light chain and a heavy chain (or an fragment thereof, e.g., scFv or Fab) of the targeting antibody molecule, e.g., anti-CD45 (e.g., BC8 antibody), anti-CD8, anti-CD4,
  • any pairing of antibodies to the same or different targets can be used, such as CD4:CD45, CD8:CD45, CD4:CD39, CD4:NRP1, and CD4:CD25.
  • CD127 is a negative surface marker for Tregs
  • the pairing of CD127 with a positive Treg surface marker can be used to negatively enrich and/or sort Tregs.
  • a blocking antibody can be targeted to non-Treg cells (for example activated Tcons having CD25).
  • the CD127 can target the antibody to the Tcons and the CD25 can then block binding of the IL-2 tethered fusion.
  • an anti-CD127 antibody can be attached to an inhibitor of CD25, such that even if an IL-2 tethered fusion bound the Tcon, it would not activate it. This improves enrichment of IL-2 on Tregs in mixed-cell populations.
  • the FM can be represented with the following formula in an N to C terminal orientation: R1-(optionally L1)-R2 or R2-(optionally L1)-R1; wherein R1 comprises a targeting moiety, L1 comprises a linker (e.g., a peptide linker described herein), and R2 comprises a modulatory moiety, e.g., a cytokine molecule.
  • R1 comprises a targeting moiety
  • L1 comprises a linker (e.g., a peptide linker described herein)
  • R2 comprises a modulatory moiety, e.g., a cytokine molecule.
  • the modulatory moiety e.g., the cytokine molecule
  • the modulatory moiety is connected to, e.g., covalently linked to, the targeting moiety.
  • the modulatory moiety e.g., the cytokine molecule
  • is functionally linked e.g., covalently linked (e.g., by chemical coupling, fusion, noncovalent association or otherwise) to the targeting moiety.
  • the modulatory moiety can be covalently coupled indirectly, e.g., via a linker to the targeting moiety.
  • the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.
  • the linker is a peptide linker.
  • the peptide linker can be 5-20, 8-18, 10-15, or about 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long.
  • the linker comprises the amino acid sequence of SEQ ID NO: 36, 37, 38, or 39, or an amino acid sequence substantially identical thereto (e.g., having 1, 2, 3, 4, or 5 amino acid substitutions).
  • the linker comprises an amino acid sequence GGGSGGGS (SEQ ID NO: 37).
  • the linker comprises amino acids from an IgG4 hinge region, e.g., amino acids DKTHTSPPSPAP (SEQ ID NO: 38).
  • the linker can be a human serum albumin (HSA) linker. It has been surprisingly discovered that the HSA linker can improve IL-2 expression efficiency and increase overall stability of the FM.
  • the FM can have the formula R1-L1-R2 and can be constructed as a single genetic molecule for expression in cells.
  • the linker is a non-peptide, chemical linker.
  • the modulatory moiety is covalently coupled to the targeting moiety by crosslinking.
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • the modulatory moiety is directly covalently coupled to the targeting moiety, without a linker.
  • the linker can be a biodegradable or cleavable linker.
  • a cleavable linker allows for cleavage of the FM such that the immune stimulating moiety, e.g., the cytokine molecule, can be released from the immune targeting moiety.
  • the cleavage of the linker may be caused by biological activation within the relevant tissue or, alternatively, by external stimuli such as, e.g., electromagnetic radiation e.g., UV-radiation.
  • the cleavable linker is configured for cleavage exterior to a cell, e.g., to be cleaved in conditions associated with cell or tissue damage or disease.
  • conditions include, for example, acidosis; the presence of intracellular enzymes (that are normally confined within cells), including necrotic conditions (e.g., cleaved by calpains or other proteases that spill out of necrotic cells); hypoxic conditions such as a reducing environment; thrombosis (e.g., a linker may be cleavable by thrombin or by another enzyme associated with the blood clotting cascade); immune system activation (e.g., a linker may be cleavable by action of an activated complement protein); or other condition associated with disease or injury.
  • necrotic conditions e.g., cleaved by calpains or other proteases that spill out of necrotic cells
  • hypoxic conditions such as a reducing environment
  • a cleavable linker may include an S-S linkage (disulfide bond), or may include a transition metal complex that falls apart when the metal is reduced.
  • S-S linker may have the following structure (as disclosed in U.S. Pat. No. 9,603,944, incorporated herein by reference in its entirety:
  • pH sensitive linkers which are cleaved upon a change in pH, e.g., at low pH, which will facilitate hydrolysis of acid (or base) labile moieties, e.g. acid labile ester groups etc.
  • acid (or base) labile moieties e.g. acid labile ester groups etc.
  • Such conditions may be found in the extracellular environment, such as acidic conditions which may be found near cancerous cells and tissues or a reducing environment, as may be found near hypoxic or ischemic cells and tissues; by proteases or other enzymes found on the surface of cells or released near cells having a condition to be treated, such as diseased, apoptotic or necrotic cells and tissues; or by other conditions or factors.
  • An acid-labile linker may be, for example, a cis-aconitic acid linker.
  • pH-sensitive linkages include acetals, ketals, activated amides such as amides of 2,3dimethylmaleamic acid, vinyl ether, other activated ethers and esters such as enol or silyl ethers or esters, imines, iminiums, orthoesters, enamines, carbamates, hydrazones, and other linkages known in the art (see, e.g., PCT Publication No. WO 2012/155920 and Franco et al. AIMS Materials Science, 3(1): 289-323, incorporated herein by reference).
  • pH sensitive refers to the fact that the cleavable linker in question is substantially cleaved at an acidic pH (e.g., a pH below 6.0, such as in the range of 4.0-6.0).
  • the cleavable linker is configured for cleavage by an enzyme, such as a protease (e.g., pepsin, trypsin, thermolysine, matrix metalloproteinase (MMP), a disintegrin and metalloprotease (ADAM; e.g. ADAM-10 or ADAM-17)), a glycosidase (e.g., ⁇ -, ⁇ -, ⁇ -amylase, ⁇ -, ⁇ -glucosidase or lactase) or an esterase (e.g. acetyl cholinesterase, pseudo cholinesterase or acetyl esterase).
  • a protease e.g., pepsin, trypsin, thermolysine, matrix metalloproteinase (MMP), a disintegrin and metalloprotease (ADAM; e.g. ADAM-10 or ADAM-17
  • ADAM disintegrin and metalloprotease
  • cleave the cleavable linker include urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), granzyme A, granzyme B, lysosomal enzymes, cathepsins, prostate-specific antigen, Herpes simplex virus protease, cytomegalovirus protease, thrombin, caspase, and interleukin 1 beta converting enzyme.
  • uPA urokinase plasminogen activator
  • tPA tissue plasminogen activator
  • granzyme A granzyme B
  • lysosomal enzymes cathepsins
  • cathepsins prostate-specific antigen
  • Herpes simplex virus protease Herpes simplex virus protease
  • cytomegalovirus protease cytomegalovirus protease
  • thrombin thrombin
  • caspase and interleukin 1 beta
  • Still another example is over-expression of an enzyme, e.g., proteases (e.g., pepsin, trypsin), in the tissue of interest, whereby a specifically designed peptide linker will be cleaved in upon arrival at the tissue of interest.
  • proteases e.g., pepsin, trypsin
  • suitable linkers in this respect are Gly-Phe-Ser-Gly (SEQ ID NO: 97), Gly-Lys-Val-Ser (SEQ ID NO: 98), Gly-Trp-Ile-Gly (SEQ ID NO: 99), Gly-Lys-Lys-Trp (SEQ ID NO: 100), Gly-Ala-Tyr-Met (SEQ ID NO: 101).
  • over-expression of an enzyme e.g. of glycosidases (e.g. a-amylase)
  • a specifically designed carbohydrate linker to be cleaved upon arrival at the tissue of interest.
  • suitable linkers in this respect are—( ⁇ -1-4-D-Glucose)n—where n ⁇ 4.
  • the cleavable linker is configured for cleavage by electromagnetic radiation, e.g., UV-radiation.
  • UV-exposure of the tissue of interest resulting in cleavage of the linker B can facilitate drug release or facilitate nanoparticles uptake in the desired tissue.
  • the cleavable linker may include a total of from 2 to 60 atoms, such as from 2 to 20 atoms.
  • the cleavable linker may include amino acid residues, and may be a peptide linkage, e.g., of from 1 to 30, or from 2 to 10, amino acid residues.
  • the cleavable linker B consists of from 1 to 30, such as from 2 to 10, or from 2 to 8, or from 3 to 9, or from 4-10, amino acids.
  • the number of atoms is typically from 2 to 50, such as from 2-30.
  • the linker includes an aminocaproic acid (also termed aminohexanoic acid) linkage or a linkage composed of from 1 to 30, or from 2 to 10 carbohydrate residues.
  • aminocaproic acid also termed aminohexanoic acid
  • the linker includes a peptide that can serve as a substrate of a matrix metalloproteinase.
  • a matrix metalloproteinase for example, MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3, MMP-7, MMP-9 (gelatinase B), and the like are known, and a substrate peptide that can serve as a substrate of one or more kinds of matrix metalloproteinases among those mentioned above can be used.
  • matrix metalloproteinases see for example, “Molecular mechanism of cancer metastasis”, Ed. by Tsuruo T., pp. 92-107, Medical View Co., Ltd., published in 1993.
  • the substrate peptide that can serve as a substrate of a matrix metalloproteinase for example, the matrix metalloproteinases of particular types and substrate peptides specifically recognized thereby are explained in Nature Biotechnology, 19, pp. 661-667, 2001. Therefore, by referring to this publication, a substrate peptide specifically cleaved by a particular type of matrix metalloproteinase can be chosen.
  • a substrate peptide specifically cleaved by a particular type of matrix metalloproteinase can be chosen.
  • Val-Pro-Leu-Ser-Leu-Tyr-Ser-Gly SEQ ID NO: 102
  • it is preferable to use the aforementioned octapeptide as a substrate peptide that can serve as a substrate of MMP-9.
  • Illustrative examples of the substrate peptide that can serve as a substrate of a matrix metalloproteinase include Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln (SEQ ID NO: 103), Val-Pro-Met-Ser-Met-Arg-Gly-Gly (SEQ ID NO: 104), Ile-Pro-Val-Ser-Leu-Arg-Ser-Gly (SEQ ID NO: 105), Arg-Pro-Phe-Ser-Met-Ile-Met-Gly (SEQ ID NO: 106), Val-Pro-Leu-Ser-Leu-Thr-Met-Gly (SEQ ID NO: 107), Ile-Pro-Glu-Ser-Leu-Arg-Ala-Gly (SEQ ID NO: 108), Arg-His-Asp, Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys (SEQ ID NO: 109), Arg-
  • the linker includes a peptide that can serve as a substrate of a disintegrase and metalloproteas (ADAM), an MMP, or a granzyme.
  • ADAM disintegrase and metalloproteas
  • the linker could comprise a peptide substrate of ADAM-8, ADAM-10, ADAM-12, ADAM-17, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, granzyme A, or granzyme B.
  • Substrates of ADAMs, MMPs, and granzymes have been well-described (e.g. in Miller, MA, et al. Integrative Biology (2011) 3:422-438, Van Damme et al.
  • the linker may, besides the substrate peptide, contain connectors, involved in the bond or bonds with the therapeutic protein.
  • Such connectors may each consist of one amino acid residue or of an oligopeptide containing from 2 to 10, such as from 3 to 9, or from 4 to 8, or from 2 to 8, amino acid residues.
  • the amino acid residue or oligopeptide as the connectors may, if present, bind to both ends of the substrate peptide, or may bind only to one end of the substrate peptide so as to represent one of the structures.
  • Types of one amino acid usable as the connector(s), and amino acid residues constituting an oligopeptide usable as the connector(s) are not particularly limited, and one amino acid residue of an arbitrary type, or an arbitrary oligopeptide containing, e.g., from 2 to 8 of the same or different amino acid residues of arbitrary types can be used.
  • Examples of the oligopeptide usable as the connector(s) include, for example, connectors that are rich in Gly amino acids. Other organic moieties can also be used as connectors.
  • the modulatory moiety and the targeting moiety of the FM are not covalently linked, e.g., are non-covalently associated.
  • Exemplary formats for fusion of a cytokine molecule to an antibody molecule can include a fusion to the amino-terminus (N-terminus) or carboxy-terminus (C-terminus) of the antibody molecule, typically, the C-terminus of the antibody molecule.
  • a cytokine-Ig moiety fusion molecule comprising a cytokine polypeptide, cytokine-receptor complex, or a cytokine -receptor Fc complex joined to an Ig polypeptide, a suitable junction between the cytokine polypeptide chain and an Ig polypeptide chain includes a direct polypeptide bond, a junction having a polypeptide linker between the two chains; and, a chemical linkage between the chains.
  • a typical junction is a flexible linker composed of small Gly4Ser linker (Gly-Gly-Gly-Gly-Ser) N , where N indicates the number of repeats of the motif (Gly4Ser) 2 and (Gly4Ser) 3 are preferred embodiments of linkers for use in the fusion constructs of the present disclosure.
  • Exemplary fusion proteins described herein can comprise the amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:
  • fusion proteins or portion thereof of the present disclosure. It should be noted that in certain scFv the arrangement is VH-linker-VL. However, the VL-linker-VH arrangement can also be used without affecting functionality. In addition, while specific linkers (Linker-1, Linker-2, etc.) were used in various constructs, other linkers disclosed herein can also be used interchangeably.
  • Light-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental BC8 mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15R ⁇ -sushi domain genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a chimeric BC8 Fab is shown fused via a peptide linker to an IL-15-binding sushi domain.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and single underline; and the sushi domain is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the chimeric BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable region of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 1.
  • Light-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental BC8 mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15R ⁇ -sushi domain genetically fused to antibody light-chain N-terminus using a flexible linker.
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable region of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 2.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 2.
  • the FMs described herein can comprise the amino acid sequence selected from SEQ ID NO: 2, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 2, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 2.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 2.
  • LC-chBC8-IL15 Also Referred to as LC-chBC8-L1-IL15
  • Light-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a chimeric BC8 Fab is shown fused via a peptide linker to an IL-15 cytokine.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and single underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the chimeric BC8 antibody comprises the light chain variable amino acid sequence (optionally, further including a kappa light chain sequence) shown in SEQ ID NO:3, or an amino acid sequence substantially identical thereof (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to SEQ ID NO:3).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 3.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 3.
  • the FMs described herein can comprise the amino acid sequence selected from SEQ ID NO: 3, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 3, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 3.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 3.
  • Light-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain N-terminus using a flexible linker.
  • an IL-15 cytokine fused via a peptide linker to a light chain portion of a chimeric BC8 Fab is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 4.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 4.
  • the FMs described herein can comprise the amino acid sequence selected from SEQ ID NO: 4, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 2, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 4.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 4.
  • An exemplary corresponding heavy chain of the chimeric BC8 antibody molecule e.g., a Fab or IgG, is shown below as SEQ ID NO:5 or SEQ ID NO:6, respectively.
  • Fab heavy-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental BC8 mouse monoclonal antibody and CH1 domain from human IgG1.
  • the chimeric BC8 antibody comprises the heavy chain variable amino acid sequence (optionally, further including a CH1 domain sequence from human IgG1) shown in SEQ ID NO:5, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to SEQ ID NO:5).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the CDR sequence of SEQ ID NO: 5.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the CDR sequence of SEQ ID NO: 5.
  • Heavy-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental BC8 mouse monoclonal antibody and constant region from human IgG4 containing S228P mutation (IgG4-S228P).
  • the chimeric BC8 antibody comprises the heavy chain variable amino acid sequence (optionally, further including a constant domain sequence from human IgG4 containing S228P mutation (IgG4-S228P)) shown in SEQ ID NO:6, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to SEQ ID NO:6).
  • Light-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental BC8 mouse monoclonal antibody and human constant kappa domain.
  • the BC8 antibody comprises the light chain variable amino acid sequence (optionally, further including a constant domain sequence from human kappa) shown in SEQ ID NO:7, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to SEQ ID NO:7).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 7.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 7.
  • Heavy-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental BC8 mouse monoclonal antibody and constant region from human IgG4 containing S228P mutation (IgG4-S228P); contains wild-type IL-15 genetically fused to antibody heavy-chain C-terminus using a flexible linker.
  • a heavy chain portion of a chimeric BC8 IgG4 fused to an IL-15 cytokine fused via a peptide linker is shown.
  • the sequence of the heavy chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the BC8 antibody comprises the heavy chain variable amino acid sequence (optionally, further including human IgG4 constant sequence) shown in SEQ ID NO:8, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 95% or higher identical to SEQ ID NO:8).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 8.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 8.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 8, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 8.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 8.
  • Fab heavy-chain of chimeric 9.4 anti-CD45 antibody contains variable domain from parental 9.4 mouse monoclonal antibody and CH1 domain from human IgG1.
  • the chimeric 9.4 antibody comprises the heavy chain variable amino acid sequence (optionally, further including a human IgG1 heavy chain sequence) shown in SEQ ID NO:14, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 95% or higher identical to SEQ ID NO:14).
  • the 9.4 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the 9.4 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO:14.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO:14.
  • Light-chain of chimeric 9.4 anti-CD45 antibody contains variable domain from parental 9.4 mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a chimeric 9.4 IgG1 fused to an IL-15 cytokine fused via a peptide linker is shown.
  • the sequence of the light-chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the chimeric 9.4 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:15).
  • the 9.4 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 15.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 15.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence substantially identical thereto(e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 15, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 15.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 15.
  • Fab heavy-chain of chimeric 4B2 anti-CD45 antibody contains variable domain from parental 4B2 mouse monoclonal antibody and CH1 domain from human IgG1.
  • the chimeric 4B2 antibody comprises the heavy chain variable amino acid sequence (optionally, further including a human IgG1 heavy chain sequence) shown in SEQ ID NO:16, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 95% or higher identical to SEQ ID NO:16).
  • the 4B2 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the 4B2 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO:16.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO:16.
  • Light-chain of chimeric 4B2 anti-CD45 antibody contains variable domain from parental 4B2 mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a chimeric 4B2 IgG1 fused to an IL-15 cytokine via a peptide linker is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the chimeric 4B2 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:17).
  • the 4B2 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the 4B2 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 17.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the any of the CDR sequences of SEQ ID NO: 17.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 17, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 17.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 17.
  • Fab heavy-chain of humanized BC8 anti-CD45 antibody contains humanized variable domain and CH1 domain from human IgG1.
  • the heavy chain variable amino acid sequence of a humanized BC8 antibody (optionally, further including a CH1 domain sequence from human IgG1) has the amino acid sequence shown in SEQ ID NO:18, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:18).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 18.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 18.
  • Light-chain of humanized BC8 anti-CD45 antibody contains humanized variable domain and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a humanized BC8 IgG1 fused to an IL-15 cytokine via a peptide linker is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the humanized BC8 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:19).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any the CDR sequences of SEQ ID NO: 19.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any the CDR sequences of SEQ ID NO: 19.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 19, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 19.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 19.
  • Fab heavy-chain of humanized BC8 anti-CD45 antibody contains humanized variable domain, an SGGGS (SEQ ID NO: 90) substitution in CDR-H3, and CH1 domain from human IgG1.
  • Light-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker with sequence GGGSGGGS (SEQ ID NO: 37).
  • a light chain portion of a chimeric BC8 IgG1 fused to an IL-15 cytokine via a peptide linker (L2) is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the chimeric BC8 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:21).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any the CDR sequences of SEQ ID NO: 21.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any the CDR sequences of SEQ ID NO: 21.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:21, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 21.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 21.
  • Light-chain of chimeric BC8 anti-CD45 antibody contains variable domain from parental mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a linker related to the human IgG1 hinge (SEQ ID NO: 38).
  • a light chain portion of a chimeric BC8 IgG1 fused to an IL-15 cytokine via a peptide linker (L3) is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the chimeric BC8 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:22).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the BC8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 22.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 22.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:22, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 22.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 22.
  • Heavy-chain variable domain of humanized BC8 anti-CD45 antibody contains an SGGGS substitution in CDR-H3.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:25, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 25.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 25.
  • Fab heavy-chain of chimeric MHM24 anti-CD11a antibody contains variable domain from parental mouse monoclonal antibody and CH1 domain from human IgG1.
  • the heavy chain variable amino acid sequence of a chimeric MHM24 antibody (optionally, further including a CH1 domain sequence from human IgG1) has the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:26).
  • the MHM24 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the MHM24 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 26.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 26.
  • Fab heavy-chain of humanized MHM24 anti-CD11a antibody contains variable domain from a humanized MHM24 heavy-chain variable domain and CH1 domain from human IgG1.
  • the heavy chain variable amino acid sequence of a humanized MHM24 antibody (optionally, further including a CH1 domain sequence from human IgG1) has the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO: 27).
  • the MHM24 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the MHM24 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 27.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 27.
  • Light-chain of chimeric MHM24 anti-CD11a antibody contains variable domain from parental mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a chimeric MHM24 IgG1 fused to an IL-15 cytokine via a peptide linker is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the chimeric MHM24 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:27).
  • the chimeric MHM24 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 27.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 27.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:27, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 27.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 27.
  • Fab heavy-chain of chimeric 1B4 anti-CD18 antibody contains variable domain from parental mouse monoclonal antibody and CH1 domain from human IgG1.
  • the heavy chain variable amino acid sequence of a chimeric 1B4 antibody (optionally, further including a CH1 domain sequence from human IgG1) has the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:28).
  • the chimeric 1B4 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the 1B4 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 28.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 28.
  • Light-chain of chimeric 1B4 anti-CD18 antibody contains variable domain from parental mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a chimeric 1B4 IgG1 fused to an IL-15 cytokine via a peptide linker is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the chimeric 1B4 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:29).
  • the chimeric 1B4 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 29.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 29.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:29, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 29.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 29.
  • Fab heavy-chain of chimeric OKT8 anti-CD8 antibody contains variable domain from parental mouse monoclonal antibody and CH1 domain from human IgG1.
  • the heavy chain variable amino acid sequence of a chimeric OKT8 anti-CD8 antibody (optionally, further including a CH1 domain sequence from human IgG1) has the amino acid sequence shown in SEQ ID NO:30, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:30).
  • the chimeric OKT8 anti-CD8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the heavy chain variable domain of the OKT8 anti-CD8 antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from the CDR sequence of SEQ ID NO: 30.
  • Light-chain of chimeric OKT8 anti-CD8 antibody contains variable domain from parental mouse monoclonal antibody and human constant kappa domain; contains wild-type IL-15 genetically fused to antibody light-chain C-terminus using a flexible linker.
  • a light chain portion of a chimeric OKT8 IgG1 fused to an IL-15 cytokine via a peptide linker is shown.
  • the sequence of the light chain is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the kappa constant region is shown as SEQ ID NO: 74.
  • the light chain variable amino acid sequence of the chimeric OKT8 antibody (optionally, further including a kappa light chain sequence) corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:31).
  • the chimeric OKT8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences of SEQ ID NO: 31.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:31, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 31.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 31.
  • IL-15 fused to the C-terminus of BC8-scFv using a short flexible linker and a hexahistidine tag.
  • a light chain portion of a BC8 scFv fused to an IL-15 cytokine via a peptide linker is shown.
  • the scFv sequence is shown in normal font; the location of the peptide linker is shown by italics and underline; and the cytokine molecule is shown by the double underline.
  • the light chain variable amino acid sequence of the BC8 scFv antibody corresponding to the antibody portion of the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95% or higher identical to the antibody portion of SEQ ID NO:32).
  • the BC8 antibody comprises one, two, or all three CDR1, CDR2 or CDR3 of the light chain variable domain of the antibody, e.g., according to the Kabat definition, or a closely related CDR, e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences within SEQ ID NO: 32.
  • a closely related CDR e.g., CDRs which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) from any of the CDR sequences within SEQ ID NO: 32.
  • the FMs described herein can comprise the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:32, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 32.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to SEQ ID NO: 32.
  • the FM comprises a constant lamba or lamda region.
  • exemplary constant lamba or lamda regions include SEQ ID NOS: 74-78.
  • the FMs described herein can comprise one or more of the amino acid sequences of SEQ ID NOS: 1-86, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to any one of SEQ ID NOS: 1-86, or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to any one of SEQ ID NOS: 1-86.
  • the FM comprises no more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to any one of SEQ ID NOS: 1-86.
  • IL-15R ⁇ -sushi also Sushi domain from wild-type IL-15R ⁇ . referred as sushi
  • 10 IL15-WT Wild-type IL-15 corresponds to mature polypeptide from full IL-15 sequence described in SEQ ID NO: 40.
  • 11 IL15-N72D IL-15 containing N72D mutation 12 sushiL77I-Fc Sushi domain from IL-15R ⁇ fused to IgG1-Fc; sushi domain contains L77I mutation 13 sushi-Fc Sushi domain from IL-15R ⁇ fused to IgG1-Fc 23 hBC8(23) heavy-chain Humanized BC8(23) heavy-chain variable domain.
  • variable domain 24 hBC8(23) light-chain Humanized BC8(23) light-chain variable domain.
  • variable domain 33 Leader-1 Leader sequence (e.g. signal sequence, signal peptide) used for antibody light-chain, IL-15, N-terminal IL-15 fusions, and N-terminal fusion of sushi to Fab-light- chain.
  • Leader-2 Leader sequence e.g. signal sequence, signal peptide used for antibody heavy-chain.
  • Linker-1 (L1) (G4S)3 linker 37 Linker-2 (L2) (G3S)2 linker 38 Linker-3 (L3) Linker derived from IgG1 hinge 39 Linker-4 (L4) Linker for fusing IL-15 to C-terminus of BC8-scFy 40 Human IL-15 full Genbank Accession No. CAA62616.1 sequence 41 Human IL-15R ⁇ full Genbank Accession No. AAI21141.1 sequence 42 IL-7 full sequence Genbank Accession No. AAA59156, AAC63047, and NP_000871, and UniProtKB/Swiss-Prot- P13232 43 IL-7 mature sequence 44 IL-21 full sequence Genbank Accession No.
  • AAG29348, AAH66262, AAH69124, and EAX05226 45 IL-21 mature sequence 46 IL-12A full sequence Genbank Accession No. P29459 (also referred to as IL- 12p35) 47 IL-12A mature sequence 48 IL-12B full sequence Genbank Accession No.
  • P29460 (also referred to as IL- 12p40) 49 IL-12B mature sequence 50 scIL-12p70-BA Synthetic sequence; IL-12B and IL-12A joined by flexible linker 51 scIL-12p70-AB Synthetic sequence; IL-12A and IL-12B joined by flexible linker 52
  • Minimal sushi domain 53 IgG1-Fc Fc domain (CH2 and CH3 domains) from human IgG1 54
  • IgG2Da-Fc Fc domain from human IgG2 containing two point mutations 56 sushi-IgG2Da-Fc (also Sushi domain from IL-15Ra fused to IgG2Da-Fc referred to as sushi- Fc2Da)
  • BC8 heavy-chain variable domain 58 BC8 light-chain variable domain 59 9.4 heavy-chain variable domain 60 9.4 light-chain variable domain 61 4B2 heavy-
  • HSA Human Serum Albumin HSA
  • HSA Human Serum Albumin
  • scFv-h9.4-HSA-IL2 An scFv comprising a humanized 9.4 antibody; heavy- chain and light-chain variable domains are genetically fused using a flexible linker (Linker-6; SEQ ID NO: 101); contains an HSA peptide (SEQ ID NO: 82) genetically fused to the c-terminus of the scFv domain using a flexible linker (Linker-1, SEQ ID NO: 36); contains a human IL-2 (SEQ ID NO: 80) genetically fused to the HSA c-terminus with a flexible linker (GGGGS (SEQ ID NO: 88)) 84 FcgR3B 85 scFv-h9.4-FcgR3B-IL2 An scFv comprising a humanized 9.4 antibody; heavy- chain and light-chain variable domains are genetically fused using a flexible linker (Linker-6
  • HC-h9.4Fab-HSA-IL2 Heavy-chain of a humanized anti-CD45 antibody contains variable domain from h9.4 heavy-chain and the CH1 domain from human IgG1; contains an HSA peptide (SEQ ID NO: 82) genetically fused to the antibody heavy-chain C-terminus using a flexible linker (Linker-1, SEQ ID NO: 36); contains a human IL-2 (SEQ ID NO: 80) genetically fused to the c-terminus of the HSA peptide using a flexible linker (GGGGS, SEQ ID NO: 88) Protein variants
  • Full length polypeptides and variants thereof are described below.
  • Full-length IL-15 sequence (SEQ ID NO: 40) is taken from Genbank Accession No. CAA62616.1; mature IL-15 is devoid of the signal sequence and is defined in SEQ ID NO: 10.
  • Full-length IL-15R ⁇ (SEQ ID NO: 40) is taken from Genbank Accession No. CAA62616.1; mature IL-15 is devoid of the signal sequence and is defined in SEQ ID NO: 10.
  • Full-length IL-15R ⁇ SEQ ID NO: 40
  • IL-15R ⁇ (IL-15R ⁇ -sushi) is taken from Genbank Accession No. AAI21141.1.
  • the sushi domain of IL-15R ⁇ (IL-15R ⁇ -sushi) is given by SEQ ID NO: 9.
  • a minimal sushi domain encompassing the first and fourth cysteines and the intervening amino acids (SEQ ID NO: 52) have also been described elsewhere and are plausible substitutes.
  • optional N-terminal additions to the minimal sushi domain comprising the native Thr or Ile-Thr and/or optional C-terminal additions to the minimal sushi domain comprising Ile or Ile-Arg residues are also plausible.
  • Protein variants described below specify protein subunit names and SEQ ID NOs corresponding to the mature proteins. Each protein subunit was recombinantly expressed with an N-terminal signal peptide to facilitate secretion from the expressing cell.
  • the native IL-15R+ signal peptide (SEQ ID NO: 35) was used for sushi, sushi-L77I-Fc, and sushi-Fc.
  • the leader sequence in SEQ ID NO: 33 was used to support secretion of antibody light-chains, IL15-WT, N-terminal IL-15 fusions, IL15-N72D, and N-terminal fusion of sushi to Fab-light-chain.
  • the leader sequence in SEQ ID NO: 34 was used to support secretion of antibody heavy chains.
  • variable light-chain domain and constant kappa domain and cytokine fusion comprise the “light-chain” while the variable heavy-chain domain and CH1 domain comprises the “heavy-chain”.
  • chBC8-IL15/sushi Fab (Also Referred to as chBC8-L1-IL15/sushi Fab).
  • This protein was made by coexpression of three subunits: HC-chBC8-Fab (SEQ ID NO: 5), LC-chBC8-IL15 (SEQ ID NO: 3), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the chimeric BC8 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with IL-15R ⁇ -sushi (via interaction with IL-15).
  • the chimeric BC8 Fab is an anti-human CD45R antibody Fab fragment comprising variable-heavy and variable-light chain domains (VH and VL) from the parental mouse monoclonal antibody (mAb) and constant domains from human (human constant kappa domain and human IgG1-CH1 domain).
  • This protein was made by coexpression of three subunits: HC-chBC8-Fab (SEQ ID NO: 5), IL15-LC-chBC8 (SEQ ID NO: 4), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of IL-15 to the N-terminus of the chimeric BC8 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • This protein was made by coexpression of three subunits: HC-chBC8-Fab (SEQ ID NO: 5), LC-chBC8-sushi (SEQ ID NO: 1), and IL15-N72D (SEQ ID NO: 11).
  • the resulting protein comprises a fusion of IL-15R ⁇ -sushi to the C-terminus of the chimeric BC8 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL15-N72D and IL-15R ⁇ -sushi.
  • This protein was made by coexpression of three subunits: HC-chBC8-Fab (SEQ ID NO: 5), sushi-LC-chBC8 (SEQ ID NO: 2), and IL15-N72D (SEQ ID NO: 11).
  • the resulting protein comprises a fusion of IL-15R ⁇ -sushi to the N-terminus of the chimeric BC8 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL15-N72D and IL-15R ⁇ -sushi.
  • This protein was made by coexpression of three subunits: HC-chBC8-IgG4S228P (SEQ ID NO: 6), LC-chBC8-IL15 (SEQ ID NO: 3), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the chimeric BC8 IgG-S228P light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with IL-15R ⁇ -sushi (via interaction with IL-15).
  • the chimeric BC8 IgG4-S228P is an anti-human CD45R antibody comprising variable-heavy and variable-light chain domains (VH and VL) from the parental mouse mAb and constant domains from human (human constant kappa domain and human IgG4 containing an S228P point mutation, which reduces susceptibility of Fab-arm-exchange of IgG4).
  • VH and VL variable-heavy and variable-light chain domains
  • This protein was made by coexpression of three subunits: HC-chBC8-IgG4S228P (SEQ ID NO: 6), IL15-LC-chBC8 (SEQ ID NO: 4), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the N-terminus of the chimeric BC8 IgG4-S228P light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • This protein was made by coexpression of three subunits: HC-chBC8-IgG4S228P (SEQ ID NO: 6), LC-chBC8-sushi (SEQ ID NO: 1), and IL15-N72D (SEQ ID NO: 11).
  • the resulting protein comprises a fusion of IL-15R ⁇ -sushi to the C-terminus of the chimeric BC8 IgG4-S228P light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with between IL-15R ⁇ -sushi and IL-15.
  • This protein was made by coexpression of three subunits: HC-chBC8-IgG4S228P (SEQ ID NO: 6), sushi-LC-chBC8 (SEQ ID NO: 2), and IL15-N72D (SEQ ID NO: 11).
  • the resulting protein comprises a fusion of IL-15R ⁇ -sushi to the N-terminus of the chimeric BC8 IgG4-S228P light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with between IL-15R ⁇ -sushi and IL-15.
  • This protein was made by coexpression of three subunits: HC-chBC8-IgG4S228P-IL15 (SEQ ID NO: 8), LC-chBC8 (SEQ ID NO: 7), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of IL-15 to the C-terminus of the chimeric BC8 IgG4-S228P heavy-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with between IL-15 and IL-15R ⁇ -sushi.
  • BC8scFv-IL15 SEQ ID NO: 32
  • IL-15R ⁇ -sushi SEQ ID NO: 9
  • the resulting protein comprises a fusion of IL-15 to the C-terminus of BC8-scFv using linker-L1 (SEQ ID NO: 36), and a noncovalent association with between IL-15 and IL-15R ⁇ -sushi.
  • BC8-scFv comprises the variable domains from the parental mouse mAb joined by a flexible linker.
  • This protein was made by coexpression of two subunits: IL15-N72D (SEQ ID NO: 11) and sushi-L77I-Fc (SEQ ID NO: 12).
  • the resulting protein comprises IL-15R ⁇ -sushi containing an L77I mutation fused to the N-terminus of human IgG1-Fc region, and a noncovalent association with between IL15-N72D and IL-15R ⁇ -sushi.
  • This protein was made by coexpression of two subunits: IL15-WT (SEQ ID NO: 10) and sushi-Fc (SEQ ID NO: 13).
  • the resulting protein comprises a fusion of IL-15R ⁇ -sushi to the N-terminus of human IgG1-Fc region, and a noncovalent association with between IL15-WT and IL-15R ⁇ -sushi.
  • This protein was made by coexpression of two subunits: IL15-WT (SEQ ID NO: 10) and sushi-IgG2Da-Fc (SEQ ID NO: 56).
  • the resulting protein comprises a fusion of IL-15R ⁇ -sushi to the N-terminus of human IgG2Da-Fc region, and a noncovalent association with IL15-WT (via interaction with IL-15R ⁇ -sushi).
  • This protein was made by coexpression of three subunits: HC-ch9.4-Fab (SEQ ID NO: 14), LC-ch9.4-IL15 (SEQ ID NO: 15), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the chimeric 9.4 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • the chimeric 9.4 Fab is an anti-CD45R antibody Fab fragment comprising variable-heavy and variable-light chain domains (VH and VL) from the parental mouse mAb (corresponding to ATCC hybridoma HB-10508) and constant domains from human (human constant kappa domain and human IgG1-CH1 domain).
  • This protein was made by coexpression of three subunits: HC-ch4B2-IL15 (SEQ ID NO: 16), LC-ch4B2-IL15 (SEQ ID NO: 17), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the chimeric 4B2 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • the chimeric 4B2 Fab is an anti-CD45R antibody Fab fragment comprising variable-heavy and variable-light chain domains (VH and VL) from the parental mouse mAb (corresponding to ATCC hybridoma HB-11186) and constant domains from human (human constant kappa domain and human IgG1-CH1 domain).
  • This protein was made by coexpression of three subunits: HC-hBC8(23)-Fab (SEQ ID NO: 18), LC-hBC8(23)-IL15 (SEQ ID NO: 19), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the humanized BC8 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • the humanized BC8(23) Fab is an anti-CD45R antibody Fab fragment comprising variable-heavy and variable-light chain domains (VH and VL) humanized from the parental mouse mAb and constant domains from human (human constant kappa domain and human IgG1-CH1 domain).
  • VH and VL variable-heavy and variable-light chain domains
  • This protein was made by coexpression of three subunits: HC-chBC8(23)-null-Fab (SEQ ID NO: 20), LC-hBC8(23)-IL15 (SEQ ID NO: 19), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the humanized BC8 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • This protein differs from hBC8(23)-IL15/sushi Fab in that it's heavy chain contains a GGGS substitution within the CDR-H3 loop, which results in ablated binding affinity towards CD45R (hence, the designation as “null” binding variant).
  • This IL15 fusion protein thus serves as a negative control for the effect of CD45R engagement.
  • This protein was made by coexpression of three subunits: HC-chBC8-Fab (SEQ ID NO: 5), LC-chBC8-L2-IL15 (SEQ ID NO: 21), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of IL-15 to the N-terminus of the chimeric BC8 Fab fragment light-chain using linker-L2 (SEQ ID NO: 37), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • Protein Name: chBC8-L3-IL15/sushi Fab This protein was made by coexpression of three subunits: HC-chBC8-Fab (SEQ ID NO: 5), LC-chBC8-L3-IL15 (SEQ ID NO: 22), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of IL-15 to the N-terminus of the chimeric BC8 Fab fragment light-chain using linker-L3 (SEQ ID NO: 38), and a noncovalent association between IL-15 and IL-15R ⁇ -sushi.
  • This protein was made by coexpression of three subunits: HC-chMHM24-Fab (SEQ ID NO: 26), LC-MHM24-IL15 (SEQ ID NO: 27), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the chimeric MHM24 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with IL-15R ⁇ -sushi (via interaction with IL-15).
  • the chimeric MHM24 Fab is an anti-human CD11a antibody Fab fragment comprising variable-heavy and variable-light chain domains (VH and VL) from the parental mouse mAb and constant domains from human (human constant kappa domain and human IgG1-CH1 domain).
  • This protein was made by coexpression of three subunits: HC-ch1B4-Fab (SEQ ID NO: 28), LC-ch1B4-IL15 (SEQ ID NO: 29), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the chimeric 1B4 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with IL-15R ⁇ -sushi (via interaction with IL-15).
  • the chimeric 1B4 Fab is an anti-human CD18 antibody Fab fragment comprising variable-heavy and variable-light chain domains (VH and VL) from the parental mouse mAb and constant domains from human (human constant kappa domain and human IgG1-CH1 domain).
  • This protein was made by coexpression of three subunits: HC-chOKT8-Fab (SEQ ID NO: 30), LC-chOKT8-IL15 (SEQ ID NO: 31), and IL-15R ⁇ -sushi (SEQ ID NO: 9).
  • the resulting protein comprises a fusion of wild-type IL-15 to the C-terminus of the chimeric OKT8 Fab fragment light-chain using linker-L1 (SEQ ID NO: 36), and a noncovalent association with IL-15R ⁇ -sushi (via interaction with IL-15).
  • the chimeric OKT8 Fab is an anti-human CD8 antibody Fab fragment comprising variable-heavy and variable-light chain domains (VH and VL) from the parental mouse mAb and constant domains from human (human constant kappa domain and human IgG1-CH1 domain).
  • This protein was made by expression of a fusion protein comprising three subunits: the targeting antibody (e.g., anti-CD45, anti-CD4 and/or anti-CD25), human serum albumin (SEQ ID NO: 79, no underline), and IL-2 (SEQ ID NO: 80).
  • the targeting antibody e.g., anti-CD45, anti-CD4 and/or anti-CD25
  • human serum albumin SEQ ID NO: 79, no underline
  • IL-2 SEQ ID NO: 80.
  • the three subunits are operably linked together via a peptide linker comprising G and S (SEQ ID NO: 79, single underline).
  • This protein is made by coexpression of two subunits: LC-h9.4Fab (SEQ ID NO: 79), and HC-h9.4Fab-IL2 (SEQ ID NO: 81).
  • the resulting protein comprises a fusion of a human IL-2 to the C-terminus of h9.4 Fab fragment heavy-chain using Linker-1 (SEQ ID NO: 36).
  • This protein is made by coexpression of two subunits: LC-h9.4Fab (SEQ ID NO: 79), and HC-h9.4Fab-HSA-IL2 (SEQ ID NO: 81).
  • the resulting protein comprises a fusion of HSA to the C-terminus of h9.4 Fab fragment heavy-chain using Linker-1 (SEQ ID NO: 36), and IL-2 to the C-terminus of HSA using a flexible linker (GGGGS) (SEQ ID NO: 88).
  • binding of the immune targeting moiety to the immune cell surface target is believed to increase the concentration, e.g., the concentration over time, of the immune modulatory moiety, e.g., cytokine molecule, with its corresponding receptor, e.g., a cytokine receptor, on the surface of the immune cell, e.g., relative to the association of the free cytokine molecule with its cytokine receptor.
  • the immune modulatory moiety e.g., cytokine molecule
  • its corresponding receptor e.g., a cytokine receptor
  • the immune regulatory cell surface target is abundantly present on the surface of an immune regulatory cell (e.g., outnumbers the number of receptors for the cytokine molecule present on the immune cell surface).
  • the targeting moiety can be an antibody molecule or a ligand molecule that binds to an immune cell surface target, e.g., CD45, CD4, CD25, CD39, or Neuropilin 1 (NRP1) on Tregs.
  • the immune cell surface receptor is selected from one or more of CD4, CD45, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CCR4, CCR5, CD137, CD39, CCR4, CCR5, CCR6 (CD196), CCR8, CCR10, OX40, GITR, CTLA4, LAG3, CD73, CD103, CD62L, CCR2, CCR9, Neuropilin 1 (NRP1), CD8, CD11a, or CD18, or variants thereof.
  • the targeting moiety comprises an antibody molecule or a ligand molecule that binds to CD45. In other embodiments, the targeting moiety binds bispecifically to CD4 and CD25. In embodiments, the targeting moiety is believed to specifically deliver and/or increase the concentration of the cytokine molecule (e.g., IL-2) to the surface of an immune cell, thereby resulting in one or more of increased localization, altered distribution and/or enhanced cell surface availability of the cytokine molecule. In embodiments, the FM does not substantially interfere with the signaling function of the cytokine molecule. Such targeting effect results in localized and prolonged stimulation of proliferation and activation of the immune cells, thus inducing the controlled expansion and activation of an immune response.
  • the cytokine molecule e.g., IL-2
  • the immune cell is a regulatory T cells.
  • the immune regulatory cell e.g., a regulatory T cell acquired from a patient, e.g., a patient's blood.
  • the immune regulatory cell is acquired from a healthy donor.
  • Regulatory T cells express the transcription factor Foxp3 and restrain immune responses to self and foreign antigens.
  • Treg cells express abundant amounts of the interleukin 2 receptor a-chain (IL-2R ⁇ ; CD25), but are unable to produce IL-2.
  • IL-2 binds with low affinity to IL-2R ⁇ or the common ⁇ -chain ( ⁇ c)-IL-2R ⁇ heterodimers, but receptor affinity increases ⁇ 1,000 fold when these three subunits together interact with IL-2.
  • IL-2 and STATS a key IL-2R downstream target, are indispensable for Foxp3 induction and differentiation of Treg cells in the thymus.
  • IL-2R ⁇ and ⁇ c are shared with the IL-15 receptor, whose signaling can also contribute to the induction of Foxp3.
  • CD4 + T cells are commonly divided into regulatory T (Treg) cells and conventional T helper (Th) cells. Th cells control adaptive immunity against pathogens and cancer by activating other effector immune cells. Treg cells are defined as CD4 ++ T cells in charge of suppressing potentially deleterious activities of Th cells. This review briefly summarizes the current knowledge in the Treg field and defines some key questions that remain to be answered. Functions for Treg cells include:
  • the most widely used markers for Treg cells are:
  • Human Treg cells in some embodiments, can be identified by the CD4+CD25+CD127-markers.
  • Exemplary Treg includes:
  • the Tregs can be ex vivo induced Tregs.
  • CD4 cells can be isolated and induced to become Tregs by activation and culturing with, e.g., TGF-B, IL-10.
  • the Tregs can be antigen trained (specific) Tregs, which can be used in, e.g., organ or cell transplant rejection.
  • the Tregs can be CAR-Tregs or transgenic TCR Tregs.
  • the FMs can be used for the in vitro expansion of Treg cells.
  • the FMs can be used as a cell culturing agent, e.g., in place of free stocks of cytokines currently used in Treg manufacturing and expansion during ex vivo culturing prior to injection. FMs can provide greater persistence than natural form of cytokine.
  • cell specific targeting is also possible with FMs for ex vivo expansion of sorted Tregs as well as selective expansion of Tregs from a population of mixed PBMCs.
  • the FMs disclosed herein can be directly administered in vivo as an immune therapy such as immunosuppressive therapy.
  • an immune therapy such as immunosuppressive therapy.
  • the FMs can facilitate cell specific targeting, e.g., to Tregs.
  • the FMs can be designed to target to cell surface receptors nonselectively expressed (e.g., CD45, CD2, CD11a, CD18) and to increase the pharmacokinetics and/or alter cytokine biodistribution.
  • the FMs disclosed herein can be pre-loaded to immune cells prior to a cell therapy, e.g., autologous cell therapy.
  • FMs can be pre-loaded onto a Treg or CAR-T cell.
  • the disclosure also features nucleic acids comprising nucleotide sequences that encode the fusion proteins described herein.
  • vectors comprising the nucleotide sequences encoding an FM described herein.
  • the vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).
  • YAC yeast artificial chromosome
  • Numerous vector systems can be employed.
  • one class of vectors utilizes DNA elements which are derived from animal viruses such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus.
  • Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses.
  • cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells.
  • the marker may provide, for example, prototropy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper, or the like.
  • the selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.
  • the expression vectors may be transfected or introduced into an appropriate host cell.
  • Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid based transfection or other conventional techniques.
  • protoplast fusion the cells are grown in media and screened for the appropriate activity.
  • Methods and conditions for culturing the resulting transfected cells and for recovering the antibody molecule produced are known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed, based upon the present description.
  • the application features host cells and vectors containing the nucleic acids described herein.
  • the nucleic acids may be present in a single vector or separate vectors present in the same host cell or separate host cell.
  • the host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli .
  • the mammalian cell can be a cultured cell or a cell line.
  • Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.
  • lymphocytic cell lines e.g., NSO
  • CHO Chinese hamster ovary cells
  • COS cells e.g., COS cells
  • oocyte cells e.g., oocyte cells
  • cells from a transgenic animal e.g., mammary epithelial cell.
  • the disclosure also provides host cells comprising a nucleic acid encoding an antibody molecule as described herein.
  • the host cells are genetically engineered to comprise nucleic acids encoding the antibody molecule.
  • the host cells are genetically engineered by using an expression cassette.
  • expression cassette refers to nucleotide sequences, which are capable of affecting expression of a gene in hosts compatible with such sequences. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression may also be used, such as, for example, an inducible promoter.
  • the present disclosure also provides host cells comprising the vectors described herein.
  • the cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell.
  • Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • compositions including pharmaceutical compositions, comprising the fusion proteins are provided herein.
  • a composition can be formulated in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients (e.g., biologically-active proteins of the nanoparticles).
  • Such compositions may, in some embodiments, contain salts, buffering agents, preservatives, and optionally other therapeutic agents.
  • Pharmaceutical compositions also may contain, in some embodiments, suitable preservatives.
  • Pharmaceutical compositions may, in some embodiments, be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy.
  • compositions suitable for parenteral administration comprise a sterile aqueous or non-aqueous preparation of the nanoparticles, which is, in some embodiments, isotonic with the blood of the recipient subject.
  • This preparation may be formulated according to known methods.
  • a sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent.
  • Additional compositions include modified cells, such as modified Tregs having a plurality of the fusion proteins tethered on cell surface. This can be useful for ex vivo Treg expansion as well as in vivo expansion.
  • sorted Tregs or unsorted PBMC can be loaded with backpacks (e.g., CD3/CD28/IL-2) followed by direct infusion to patient, whereby the Tregs are expanded in vivo, without in vitro culturing.
  • loaded Tregs e.g., with IL-2 backpacks
  • FIG. 1E loaded Tregs
  • the fusion proteins and compositions containing such fusions have numerous therapeutic utilities, including, e.g., the treatment of conditions or diseases where the immune system displays an excessive or overactive response.
  • the present disclosure provides, inter alia, methods for reducing or suppressing an immune response in a subject with conditions in which the immune system is overactive, such as autoimmune diseases, allergies and/or bone marrow or organ transplant.
  • Exemplary methods comprise administering to the subject a therapeutically effective amount of any of the fusion proteins and/or modified cells (e.g., Treg-targeted fusion proteins) described herein to provide, e.g., an immunosuppressive therapy.
  • Examplary applications of immunosuppressive therapy include allo-immune diseases, auto-immune diseases, allergy, and other inflammatory diseases ( FIG. 1H ).
  • Allo-immune diseases include organ transplant rejection, graft versus host disease (GVHD) (e.g., post allogeneic hematopoietic stem cell transplant, HSCT) and GVHD post allogeneic stem cell transplantation (SCT).
  • GVHD graft versus host disease
  • HSCT post allogeneic hematopoietic stem cell transplant
  • SCT GVHD post allogeneic stem cell transplantation
  • Autoimmune diseases are diseases in which the immune system attacks its own proteins, cells, and tissues, or in which immune effector T cells are autoreactive to endogenous self peptides and cause destruction of tissue. Thus an immune response is mounted against a subject's own antigens, referred to as self antigens.
  • self antigens A comprehensive listing and review of autoimmune diseases can be found in The Autoimmune Diseases (Rose and Mackay, 2014, Academic Press).
  • Autoimmune diseases are generally considered to be Th1 biased.
  • Th2 immune response or Th2 like cytokines can be beneficial.
  • Such cytokines include IL-4, IL-5 and IL-10.
  • Examplary auto-immune diseases that can be treated include Type 1 diabetes, Multiple Sclerosis and alopecia. These diseases have functional defects in Tregs, and decreased number of Tregs.
  • Autoimmune diseases include but are not limited to rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis (e.g., crescentic glomerulonephritis
  • Inflammatory diseases can be used to broadly define a vast array of disorders and conditions that are characterized by inflammation. Examples include allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury and transplant rejection.
  • inflammatory diseases can include Inflammatory Bowel Disease, Rheumatoid Arthritis and Lupus.
  • Inflammatory Bowel Disease IBD includes two major types, namely Crohn's Disease (CD) and Ulcerative Colitis (UC). These diseases have increased Treg number, but decreased Treg function. Tregs have been shown to control/prevent these diseases in mouse models.
  • CD and UC as inflammatory disorders have long been considered as a breakdown in immunoregulation in the tissues of the intestinal mucosa, representing the most immunologically active sites of the human body.
  • the interaction between luminal flora and the adaptive immune system is considered critical to disease pathogenesis.
  • compositions and cells e.g., modified and/or expanded Tregs
  • the fusion proteins or pharmaceutical composition can be administered to the subject parenterally.
  • the modified cells can be administered to the subject intravenously, subcutaneously, intratumorally, intranodally, intramuscularly, intradermally, or intraperitoneally.
  • the cells are administered, e.g., injected, directly into a tumor or lymph node.
  • the cells are administered as an infusion (e.g., as described in Rosenberg et al., New Eng. J. of Med. 319:1676, 1988) or an intravenous push.
  • the cells are administered as an injectable depot formulation.
  • the subject is a mammal.
  • the subject is a human, monkey, pig, dog, cat, cow, sheep, goat, rabbit, rat, or mouse.
  • the subject is a human.
  • the subject is a pediatric subject, e.g., less than 18 years of age, e.g., less than 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less years of age.
  • the subject is an adult, e.g., at least 18 years of age, e.g., at least 19, 20, 21, 22, 23, 24, 25, 25-30, 30-35, 35-40, 40-50, 50-60, 60-70, 70-80, or 80-90 years of age.
  • the fusion proteins disclosed herein can be used in combination with a second therapeutic agent or procedure.
  • the FM is administered in conjunction with a cell therapy, e.g., a cell therapy chosen from an adoptive cell therapy, CAR-T cell therapy, engineered TCR T cell therapy, a tumor infiltrating lymphocyte therapy, an antigen-trained T cell therapy, or an enriched antigen-specific T cell therapy.
  • a cell therapy e.g., a cell therapy chosen from an adoptive cell therapy, CAR-T cell therapy, engineered TCR T cell therapy, a tumor infiltrating lymphocyte therapy, an antigen-trained T cell therapy, or an enriched antigen-specific T cell therapy.
  • the FM and the second therapeutic agent or procedure are administered/performed after a subject has been diagnosed with a condition in which the immune system is overactive, e.g., before the condition has been eliminated from the subject.
  • the FM and the second therapeutic agent or procedure are administered or performed simultaneously or concurrently.
  • the delivery of one treatment is still occurring when the delivery of the second commences, e.g., there is an overlap in administration of the treatments.
  • the FM and the second therapeutic agent or procedure are administered or performed sequentially. For example, the delivery of one treatment ceases before the delivery of the other treatment begins.
  • combination therapy can lead to more effective treatment than monotherapy with either agent alone.
  • the combination of the first and second treatment is more effective (e.g., leads to a greater reduction in symptoms and/or cancer cells) than the first or second treatment alone.
  • the combination therapy permits use of a lower dose of the first or the second treatment compared to the dose of the first or second treatment normally required to achieve similar effects when administered as a monotherapy.
  • the combination therapy has a partially additive effect, wholly additive effect, or greater than additive effect.
  • FMs comprising multiple formats: (i) FMs comprising an IgG, Fab fragment, or scFv fragment, (ii) fusion to the N- or C-terminus of the antibody or antibody fragment; (iii) fusion of either IL-15 or the IL-15R ⁇ sushi domain to the antibody or antibody fragment (e.g., FIG. 1 and FIGS. 2A-2C ); and (iv) FMs comprising varied linker composition between the antibody and cytokine.
  • FMs comprising IL-15 and mAb clone BC8, which targets human CD45, an abundant receptor on the surface of immune cells (Cyster et al., EMBO Journal, Vol 10, no4, 893-902, 1991).
  • the FMs comprised three different antibody formats: scFv, Fab, and full-length IgG.
  • wild-type IL-15 was genetically fused to the C-terminus of the Fab or IgG light-chain using an amino acid linker consisting of (GGGGS)3 (SEQ ID NO: 36) or to the C-terminus of the scFv using the amino acid linker RSGSGGGGSLQ (SEQ ID NO: 39).
  • the Fab and IgG antibody formats comprised human constant regions and variable domains from the mouse monoclonal antibody BC8 (e.g. chBC8 Fab or chBC8 IgG).
  • the scFv comprised BC8 heavy- and light-chain variable domains genetically fused with a (GGGGS)3 linker.
  • the IL-15/antibody fusions were co-expressed with IL-15R ⁇ -sushi; the high-affinity interaction between IL-15 and sushi resulted in an IL-15-antibody complex comprising a noncovalent association with IL-15R ⁇ -sushi. Together, this resulted in three FM variants: chBC8-IL15/sushi scFv, chBC8-IL15/sushi Fab, and chBC8-IL15/sushi IgG ( FIG. 2A ; see Protein Variants section for more details).
  • the effects of the FMs on CD8 T cell expansion were evaluated using a pulse bioassay in which the cytokine is incubated with cells for a fixed amount of time followed by removal of unbound cytokine by washing.
  • the pulse bioassay provides better characterization of the persistence of a biological effect. It also accommodates confounding factors associated with ligand-depletion, which is a well-appreciated challenge for quantitative analysis of high-affinity receptor/ligand interactions (Hulme, Tevethick, Ligand binding assays at equilibrium: validation and interpretation Br J Pharm 2010, 161:1219-1237).
  • the first two FMs were constructed by genetically fusing IL-15 to the N-terminus of chBC8 Fab or chBC8 IgG light-chains and the antibody fusions were co-expressed with IL-15R ⁇ to result in the FMs: IL15-chBC8/sushi Fab and IL15-chBC8/sushi IgG ( FIGS. 3A-3B ; see “Protein Variants” section for more details).
  • chBC8-IL15 in Example 1A above and IL15-chBC8 here to refer to fusion of IL-15 to the antibody C- or N-terminus, respectively.
  • the four additional FMs were constructed by fusing wild-type IL-15R ⁇ -sushi to the N- or C-terminus of the chBC8 Fab or IgG light-chain, and co-expressed these antibody fusions with IL-15 N72D ( FIGS.
  • FIGS. 4A-4B and FIGS. 5A-5B We observed a trend in which fusion of IL-15 or sushi to the C-terminus of the chBC8 IgG or Fab resulted in greater CD8 T cell expansion than fusion to the antibody N-terminus ( FIGS. 4A-4B and FIGS. 5A-5B ).
  • each of the FM variants (including N-terminal fusion to the IgG or Fab) displayed greater potency than the control protein, IL15-N72D/sushiL77I-Fc, which does not contain CD45 receptor binding functionality ( FIGS. 3C -3D and FIGS. 4A-4B ).
  • chimeric BC8 or the parental BC8 mouse mAb antibodies alone did not induce T cell expansion ( FIGS. 4A-4B and FIGS.
  • Wild-type IL-15 was genetically fused to the C-terminus of the chBC8 heavy-chain (HC) and this construct was co-expressed with IL15R ⁇ -sushi to yield the FM variant chBC8(HC)-IL15/sushi IgG ( FIG. 6A ).
  • Activated human CD8 T cells were pulsed with seven serial five-fold dilutions spanning 400 nM to 25.6 pM of chBC8(HC)-IL15/sushi IgG or chBC8-IL15(LC)/sushi IgG for 1 hr at 37° C., and then washed three times will full media and plated at 500,000 cells/mL. Serial dilution of media only, the parental BC8 antibody (which does not contain a fusion with IL-15), or IL-15 WT /sushi-Fc (which is devoid of immune-targeting function) were used as controls. Following 5 days of culture we measured CD8 T cell expansion using CellTiter Blue.
  • the FM with the functional BC8 region comprised a Fab antibody fragment with humanized BC8 variable domain, in which CDRs from the parental BC8 antibody were grafted onto human variable domains, and a fusion of IL-15 WT to the humanized BC8 Fab light-chain.
  • This complex was co-expressed with IL-15R ⁇ -sushi to result in the FM variant hBC8(23)-IL15/sushi Fab (see “Protein Variants” section for more details).
  • the humanized BC8 antibody (hBC8(23)) possesses similar binding affinity as the chimeric BC8 antibody ( FIG. 10A ).
  • the FM with the nonfunctional BC8 region (hBC8-null-IL15/sushi Fab) comprised the same Fab fragment, with the exception of five amino acids of CDR3 in the antibody heavy-chain, which were replaced with the residues SGGGS. These amino acid substitutions resulted in a humanized BC8 antibody that did not exhibit detectable binding to cellular CD45 for the concentrations tested ( FIG. 10A ).
  • Each of these proteins were pulsed with activated human CD8 T cells (seven serial five-fold dilutions spanning a concentration range of 400 nM to 25.6 pM), cells were then washed and plated at 500,000 cells/mL in 96-well plates. Following three days at 37° C.
  • CD8 T cells at a density of 5 ⁇ 10 7 cells/mL were pulsed with 0.75 mg/mL hBC8(23)-IL15/sushi Fab, IL-15/sushi-Fc2Da, or IL-15 N72D /sushiL77I-Fc for 1 hr in a 1:1 mixture of PBS and HBSS, and then washed three times with full media.
  • Sushi-Fc2Da is a fusion of wild-type IL-15R ⁇ -sushi to a variant of the human IgG2-Fc domain.
  • Cells were then stained for IL-15 using a fluorescently-labeled anti-IL-15 antibody or for IgG domains (Fab or Fc fragments) using a fluorescently-labeled polyclonal antibody that recognizes human IgG light and heavy chains.
  • Fab or Fc fragments fluorescently-labeled polyclonal antibody that recognizes human IgG light and heavy chains.
  • the hBC8(23)-IL15/sushi Fab protein yielded substantially higher levels of IL-15 staining than the IL15/sushi-Fc2Da or IL15-N72D/sushiL77I-Fc proteins indicating a greater abundance of IL-15 on the cell surface ( FIG. 8A ).
  • FIG. 8A We observed consistent results for IgG staining in which the FMs resulted in a higher levels of staining than the IL15/sushi-Fc constructs ( FIG. 8A ).
  • IL-15 detection remained above background for hBC8(23)-IL15/sushi Fab, while both of the IL15/sushi-Fc variants were indistinguishable from background one day after the pulse incubation ( FIG. 8B ).
  • the pulse of hBC8(23)-IL15/sushi Fab further resulted in similar cell expansion to the saturating amount of the IL15/sushi-Fc variant ( FIG. 8C ).
  • IL-15 is presented on the dendritic cell surface via its interaction with IL-15R ⁇ , and this antibody has been used previously to detect such a presentation of IL-15 on dendritic cells (Ferlazzo, Pack, Thomas, Paludan, et al PNAS 2004). Consistent with these observations, we are able to detect hBC8(23)-IL15/sushi Fab and IL15/sushi-Fc variants on the T cell surface using this antibody ( FIGS. 8A-8B ; each protein variant comprises an association between IL-15R ⁇ -sushi and IL-15).
  • the antibody's neutralizing activity may therefore derive from competitive binding with one of the other two cell surface receptors for IL-15, IL-2/IL-15R ⁇ or the common ⁇ chain.
  • detection of IL-15 with this antibody may reflect an excess loading of IL-15 onto the T cell surface beyond the levels that saturate its cell surface receptors, e.g. IL-2/IL-15R ⁇ or the common y chain.
  • This may also explain the low levels of cell surface IL-15 detected for the IL-15/sushi-Fc constructs ( FIGS. 8A-8B ), which would only interact with T cells through the IL-15 receptors, though the dimeric IL-15 that results from association with the sushi-Fc fusion could result in a slight abundance of the cytokine.
  • FMs both facilitate greater initial loading of IL-15 on the T cell surface and support elevated levels of biologically available IL-15 on the cell surface over time.
  • linker compositions including a flexible eight amino acid linker ((GGGS)2; Linker-2, SEQ ID NO: 37) and a moderately flexible 12 amino acid linker derived from the human IgG1 hinge region (DKTHT S PP S PAP; Linker-3, SEQ ID NO: 38, underlined positions denote residues of the IgG1 hinge region that were mutated from cysteine to serine for this linker).
  • IL-15 WT was fused to the C-terminus of the chBC8 Fab fragment light-chain using Linker-2 or Linker-3 and was co-expressed with IL-15R ⁇ -sushi to generate chBC8-L2-IL15/sushi Fab and chBC8-L3-IL15/sushi Fab, respectively ( FIG. 9A ).
  • this FM variant comprises a (GGGGS)3 linker (Linker-1, SEQ ID NO: 36) between IL-15 and the chBC8 light-chain; for consistency we refer to this construct here as chBC8-L1-IL15/sushi Fab ( FIG. 9A ).
  • Protein expression Proteins were produced from suspension adapted HEK 293 cells in serum-free media. An exception was the parental mouse monoclonal antibody BC8, which was purified from the BC8 hybridoma culture obtained from American Type Cell Culture (ATCC, cat. no. HB-10507). Proteins were then purified by either protein A resin (for Fc fusions and full-length (IgG) antibodies), KappaSelect resin (GE Healthcare; for fusions to antibody Fab fragments), or histidine affinity resin (e.g. nickel-nitrilotriacetic acid, Ni-NTA; for 6-His-tagged scFv fusions) as appropriate. Purified proteins were buffer exchanged into phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • anti-CD45 antibodies other than BC8 are capable of generating FMs with improved potency
  • two additional anti-CD45 monoclonal antibodies (mAb clones 9.4 and 4B2).
  • the chimeric antibodies each comprise mouse variable domains and human constant region (human constant kappa domain and human IgG4 containing an S228P mutation to stabilize the IgG4 hinge region).
  • chBC8, hBC8(23), and a non-binding variant hBC8-null are examples of anti-CD45 monoclonal antibodies.
  • the humanized BC8 antibody consists of variable domains containing CDR regions grafted from BC8, and the hBC8-null variant contains an SGGGS substitution in the heavy-chain CDR3, which ablates binding to CD45 ( FIG. 10A ).
  • the ch4B2 and ch9.4 antibodies exhibited the highest affinity to cellular CD45, with apparent dissociation constants (K D,app ) of 0.44 and 0.84 nM, respectively, while the chBC8 and hBC8(23) antibodies exhibited K D,app of 6.68 and 7.8 nM, respectively ( FIG. 10A ).
  • the hBC8-null antibody did not exhibit significant binding at the highest concentration examined (500 nM; FIG. 10A ).
  • K D,app apparent dissociation constant
  • Pulse bioassay The pulse bioassay was conducted as described in Example 1.
  • Protein expression Proteins were produced as described in Example 1.
  • FMs targeting cell surface receptors other than CD45 comprising antibodies targeting additional cell surface receptors.
  • the alternative receptors can either be abundant receptors on the cell surface or exhibit persistence at the protein level (e.g. display slow receptor internalization or strong recycling back to the cell surface upon receptor internalization). Ideally the receptor is both abundant and persistent.
  • FMs targeting CD8, CD11a, or CD18 for evaluation.
  • CD8 is a co-receptor for the T cell receptor that is expressed on cytotoxic T cells. CD8 can also be expressed in cortical thymocytes, dendritic cells, and natural killer cells.
  • CD8 helps maintain the activated T cell receptor/MHC complex on the cell surface (Wooldridge, van den berg, Glick, et al. JBC 2005), and once the T cell receptor is internalized the CD8 co-receptor is preferentially maintained on the cell surface in comparison to the T cell receptor (1989 J Imm Boyer, Auphan, Gabert, Schmitt-Verhulst et al Comparison of phosphorylation and internalization of the antigen receptor/CD3 complex, CD8, and Class I MHC-encoded proteins on T cells).
  • Lymphocyte function-associated antigen 1 is a heterodimeric cell surface receptor composed of integrin alpha-L (CD11a) and integrin beta-2 (CD18) and is involved in intercellular adhesion and lymphocyte costimulation. Integrins generally display persistence at the protein level: they typically either exhibit slow internalization from the cell surface or are recycled back to the cell surface upon internalization (2012 J Cell Sci Integrins at a Glance and 1989 EMBO J Endocytosis and recycling of the Fn receptor in CHO).
  • LFA-1 is present at high levels on several different types of leukocytes and has been shown to exhibit slow internalization in multiple different cell types, including monocytic U937 cells, the promyelocytic leukemia cell line HL-60, and the lymphoblastoid cell line JY (1992 EMBO J Circulating Integrins Bretscher).
  • preclinical studies of the CD11a antibody efalizumab demonstrated that CD11a exhibits persistence at the protein level in human and mouse T cells isolated from blood; efalizumab can, however, induce receptor internalization and degradation in the presence of a secondary cross-linking antibody (2004 Coffey, Pippig et al J Pharm Exp Ther).
  • FMs targeting CD8 (antibody clone OKT8), CD11a (antibody clone MHM24), or CD18 (antibody clone 1B4) by genetically fusing IL-15 to the C-terminus of the respective antibody fragment light-chain.
  • the IL-15-antibody fusions were co-expressed with IL-15R ⁇ -sushi to generate three FMs: chMHM24-IL15/sushi Fab, ch1B4-IL15/sushi Fab, and chOKT8-IL15/sushi Fab ( FIG. 11A ).
  • Each Fab fragment was composed of chimeric human and mouse regions with mouse variable region and human constant domains (kappa and CH1).
  • the potency of the FMs on CD8 T cells was analyzed in the pulse bioassay as described in Example 1; after three days in culture following the pulse incubation cells were analyzed for proliferation using CellTiter Blue.
  • the three FMs each supported similar levels of T cell proliferation to that of chBC8-IL15/sushi Fab ( FIG. 11B ).
  • FIGS. 2A-2C, 4A-4B, 5A-5D, 6A-6B, and 8A-8C demonstrates that additional cell surface receptors are capable of generating FMs with improved biological persistence.
  • CD8-targeted FM was evaluated whether cell surface receptor targeting can facilitate improved potency towards individual cell types.
  • CD4 or CD8 cells were treated with chOKT8-IL15/sushi Fab in the pulse bioassay format, and cell proliferation was measured three days after the pulse incubation.
  • the CD8-targeted FM more potently stimulated CD8 T cells compared to CD4 T cells ( FIGS. 12A-12B ), supporting the ability to target specific cell types based on the antibody of the FM.
  • Pulse bioassay on CD4 or CD8 T cells Primary human CD3 T cells, which contain a mixture of cells expressing CD4, CD8, or CD4 and CD8 coreceptors, were activated using CD3/CD28 activation beads in the presence of 10 ng/mL IL-2 as described in Example 1. Following three days of activation the beads were removed and cells were rested overnight in full RPMI media. We then purified individual populations of CD4 and CD8 T cells using magnetic activated cell sorting (MACS) using (cat. no. 130-045-101, and no. 130-045-201, Miltenyi Biotec, Inc.) according the manufacturer's protocol. CD4 or CD8 T cells were then analyzed in the pulse bioassay as described in Example 1.
  • MCS magnetic activated cell sorting
  • Drug-loaded nanoparticles also referred to herein as “backpacks” hold potential for supporting cell viability and function.
  • backpacks Drug-loaded nanoparticles
  • hBC8(23)-IL15/sushi Fab into a protein nanogel using a reversible, amine-reactive, homobifunctional cross-linker.
  • Protein nanogels comprising IL15 N72D /sushi L77I -Fc were used as a comparator. Both of these proteins efficiently crosslinked into protein nanogels: by SEC analysis the protein nanogels traveled with a faster elution time than the free protein, consistent with a larger species ( FIGS.
  • FIGS. 13A-13B Comparison of HPLC traces of the protein nanogel with the free (noncrosslinked) protein ( FIGS. 13A-13B ) revealed a minimal amount of free protein remaining in the cross-linked nanogels, suggesting high conversion of the proteins into nanogels.
  • the protein nanogels were coated with a co-block polymer of polyethylene glycol and polylysine and incubated with CD8 T cells. Both protein nanogels supported long term cell expansion in comparison to non-crosslinked protein and unstimulated controls ( FIG. 13C ).
  • Protein nanogel synthesis Protein nanogels comprising a crosslinked protein nanoparticle were formed as follows.
  • the chBC8-IL15/sushi Fab fusion protein was crosslinked into protein nanogels by incubating the protein at a concentration of 17 mg/mL with a 27-fold molar excess of a degradable crosslinker (Formula I) in the presence of a final concentration of 2.5% polyethylene glycol with an average molecular weight of 400 Dalton (PEG-400, Spectrum Chemical Mfg. Corp.) and 10% glycerol (Sigma).
  • the IL15 N72D /sushiL77I-Fc protein was crosslinked into protein nanogels by incubating the protein at a concentration of 17 mg/mL with a 27-fold molar excess of a degradable crosslinker (Formula I).
  • the cross-linker used in this study Bis[2-(N-succinimidyl-oxycarbonyloxy)ethyl] disulfide, contains two N-hydroxysuccinimide (NHS) ester groups joined together by a flexible disulfide-containing linker as shown in Formula I.
  • DPBS Dulbecco's phosphate buffered saline
  • Linker leaving groups which comprise molecular fragments of the linker that are removed as part of the cross-linking reaction
  • unreacted linker by buffer exchange into DPBS using a Zeba column (40,000 MW cut-off, Thermo-Fisher).
  • Zeba columns were used according to the manufacturer's instructions, including equilibrating the column in DPBS by three consecutive washes with DPBS to facilitate buffer exchange, followed by application of the reaction products.
  • Buffer-exchanged protein nanogels were analyzed by size exclusion chromatography (SEC) using a BioSepTM SEC-s4000 column (Phenomenex Inc.) with PBS (pH 7.2) as eluent (flow rate 0.5 mL/min) on a Prominence HPLC system equipped with a photodiode array (Shimadzu Corp.).
  • SEC size exclusion chromatography
  • Protein nanogels at a cytokine concentration of approximately 1-1.5 mg/mL were conjugated with a polyethylene glycol-polylysine (PEG-polyK) block co-polymer: PEGSk-polyK30 (Alamanda Polymers cat. no. 050-KC030), which is a block co-polymer comprising a polyethylene glycol polymer of 5 kiloDalton (kD) average molecular weight and a 30 amino acid polylysine polymer (polylysine30 or polyK30).
  • PEG-polyK polyethylene glycol-polylysine
  • PEG5k-polyK30 or were reconstituted to 1 mg/mL in DPBS and added to 1-1.5 mg/mL of protein nanogels at a final block copolymer concentration of 50 ug/mL and incubated at room temperature for 30 min.
  • Protein nanogels comprising PEG5k-polyK30 at a protein concentration of 1-1.5 mg/mL were mixed in equal volume with 1 ⁇ 10 6 CD8 T-cells in HBSS at a cell concentration of 100 ⁇ 10 6 cells/mL and incubated at 37° C. for 1 hr. T-cells were then washed three times with RPMI media containing 10% FBS, penicillin/streptomycin, and Glutamax (all from ThermoFisher Scientific, Inc.), seeded at a cell density of approximately 1 ⁇ 10 6 cells/mL, and cultured for 9 days in 24-well tissue culture plates. Cells were split into fresh medium at a ratio of 1:5 on three days after cell attachment of backpacks. Cell proliferation was measured by live-dead cell stain (7-AAD) and counting beads (CountBright Absolute Counting Beads, Thermo Fisher Scientific, Inc.) by flow cytometry on Days 0, 1, 2, 3, 6, and 9.
  • FIG. 14 illustrates various combinations of immunotargeting moieties (e.g., antibodies against various cell surface receptors) and cytokine molecules.
  • Multiple receptors can facilitate cytokine immunotargeting.
  • tethered fusions can be made with various cytokines tethered to antibodies against CD11a, CD18, CD45 and/or CD2 which may be present on multiple cell types.
  • Cytokines can also be immunotargeted to specific cells via cell type-specific surface receptors such as CD4, CD8 or CD56.
  • CD8-targeted IL-7 delivers selective loading and expansion of CD8 T cells.
  • FIGS. 15A-15B shows cell-specific targeting, in which CD8-targeted-IL-7 selectively targets CD8 T cells.
  • FIG. 15C shows selective expansion of CD8 T cells, but not CD4 T cells, in cultures of activated total CD3 human T cells by pulse incubation with CD8-targeted IL-7. Briefly, activated human T cells were prepared by stimulating total CD3 human T cells (comprising both CD4 and CD8 T cells) for three days using CD3/CD28 Dynabead Activator beads (Thermo) according to the manufacturer's instructions and in the presence of in the presence of 20 ng/mL IL-7 and 100 ng/mL IL-21.
  • CD3/CD28 Dynabead Activator beads Thermo
  • CD3/CD28 beads were then removed and cells were incubated in full medium (RPMI 1640 containing 10% FBS) overnight in the presence of 20 ng/mL IL-7 and 100 ng/mL IL-21 at 37 C and 5% CO2. Cells were then washed and incubated with 500 nM of a CD8-targeted IL-7 construct (chOKT8Fab-IL7). After 1 hr at 37 C cells were washed two times with full medium, plated at a density of 200,000 cells/mL, and incubated at 37 C and 5% CO2.
  • cells were analyzed for loading onto CD8 and CD4 T cells by staining the T cells with DyLight 650-conjugated anti-human IgG polyclonal antibody (Thermo cat. no. SA5-10129; DyLight 650 fluorophore can be read on the APC channel for flow cytometry, see FIG. 15B ), FITC-conjugated anti-CD4 antibody (BioLegend cat. no. 344604), and APC/Cy7-conjugated anti-CD8 antibody (BioLegend cat. no. 344715).
  • the anti-human IgG polyclonal antibody detects the Fab region of the CD8-targeted FM.
  • the anti-CD4 and anti-CD8 antibodies can differentiate the CD4 and CD8 T cell populations ( FIG. 15A ).
  • FIG. 15B shows that the CD8-targeted IL-7 construct selectivey loaded to greater levels on CD8 T cells when incubated with a mixed population of CD4 and CD8 T cells.
  • FIG. 15C shows selective expansion of the CD8 T cells by the CD8-targeted IL-7 construct.
  • FIG. 15D shows strong selective loading onto CD8 T cells for each construct.
  • FIG. 15E we similarly show selective loading of IL-15 onto CD8 T cells using anti-IL15 staining (Fab constructs that do not contain IL-15, chOKT8Fab and ch51.1Fab, are of course negative in this assay).
  • BC8scFv was constructed using a hexahistidine tag, which is used here as a handle for detection by flow cytometry); together with the minimal binding of chOKT8Fab and chOKT8Fab-I115/sushi to CD4 T cells, we conclude that binding of chOKT8Fab-BC8scFv-IL15/sushi to CD4 T cells is driven by the BC8scFv.
  • a dimeric form of BC8scFv results in higher affinity to T cells, likely due to the avidity affect from bivalent binding.
  • FMs can be constructed using antibody clones that are specific for different cell surface receptors to improve cellular affinity and loading.
  • one or more of the selected antibodies can be specific for a particular cell type in order to enable improved affinity while still retaining cell-selective loading. Using such a method, we show that it is further feasible to construct heterospecific antibodies with varied loading efficiencies on different cell types (e.g. in this case, stronger loading onto CD8 T cells and weaker—but nonzero—loading onto CD4 T cells).
  • IL-15 variants comprising mutations to amino acid side-chains believed to interact with the IL-15/IL-2 receptor beta (specifically, IL-15-D8N, IL-15-D61N, and IL-15N72A), and three additional IL-15 variants comprising mutations to amino acid side-chains believed to interact with the gamma chain (IL-15-K10Q, IL-15-Q101N, and IL-15-Q108N).
  • IL-15-K10Q, IL-15-Q101N, and IL-15-Q108N We constructed the IL-15 mutants as fusions to the chBC8Fab antibody fragment and evaluated their binding and activity on human T cells.
  • FIG. 16A and FIG. 16B show that three different IL-15 variants result in stronger cellular potency than wild-type IL-15 (IL-15-K10Q, IL-15-Q101N, and IL-15-N72A), two IL-15 variants resulted in reduced cellular potency (IL-15-D61N and IL-15-Q108N), and a single IL-15 variant did not display cellular activity under the range of concentrations evaluated (IL-15-D8N).
  • CD8-targeted FMs comprising the IL-15 variants IL-15-D61H, IL-15E64H, IL-15-D65H, or IL-15-N72H.
  • Activated human T cells were prepared by stimulating CD3 human T cells for three days using CD3/CD28 Dynabead Activator beads (Thermo) according to the manufacturer's instructions, beads were then removed and cells were incubated in full medium (RPMI 1640 containing 10%
  • FIG. 16D demonstrates high loading of the CD8-targeted IL-15 constructs onto CD8 T cells, and minimal loading onto CD4 T cells. The limited loading of the CD8-targeted FMs onto CD8 T cells.
  • CD4 T cells enabled a controlled evaluation of the effects of IL-15 mutations on cellular responses.
  • Expansion of CD4 T cells in reponse to incubation with CD8-targeted IL-15 constructs comprising D61H, E64H, N65H, or N72H point mutations demonstrated attenuating activity of D61H, E64H, and N65H mutations ( FIG. 16E ).
  • Each CD8-targeted construct selectively loaded to higher degrees onto CD8 T cells as compared with CD4 T cells ( FIG. 17A ).
  • each of the hisitidine mutants comprising attenuated cellular activity (D61H, E64H, and N65H) exhibited higher surface persistence one day following the pulse incubation onto mixed CD4 and CD8 T cells ( FIG. 17A ).
  • CD45-targeted IL-15 FM constructed with the same antibody configuration as the CD8-targeted IL-15 FMs (h9.4Fab-BC8scFv-IL15/sushi, which contains a Fab-scFv format consistent with the antibody format for the CD8-targeted constructs).
  • Each CD8-targeted IL-15 construct delivered selective expansion of CD8 T cells, and the CD8-targeted IL-15 constructs comprising the D61H and E64H mutations supported greater CD8 T cell expansion than the CD8-targeted construct comprising wild-type IL-15 ( FIG.
  • each of the CD8-targeted IL-15 constructs only weakly expanded the CD4 T cells ( FIG. 17B ), consistent with their design for CD8-specificity and consistent with their specificity for loading onto CD8 T cells.
  • a full expansion time course is shown for the CD8-targreted constructs comprising wild-type IL-15, IL-15-D61H, or IL-15-E64H ( FIG. 17C ).
  • CD25 is a surface marker for Tregs, but it is also expressed on activated T cells.
  • the IL-7 receptor CD127
  • Magnetic and flow cytometry-based sorting methods that enrich for the surface markers CD4 + CD25 + and deplete CD127 + cells are used to enrich and isolate Tregs from PBMCs or CD3 T cells.
  • Treg-enriched and Treg-depleted CD4 populations were sorted into Treg-enriched and Treg-depleted CD4 populations by magnetic sorting.
  • the Treg-enriched fraction was positively selected for CD4 and CD25, and negatively selected for CD127 (CD4 + /CD25 + /CD127 ⁇ ) using magnetic sorting reagents from Miltenyi and following the manufacturer's protocols.
  • Treg -enriched and -depleted cells were activated with CD3/CD28 beads and expanded with IL-2 for 5 days. After 5 days of culture, the Treg-enriched cells maintained significantly more FoxP3 + cells, and significantly fewer CD127 + cells ( FIG. 18 ). As shown in FIG. 18 , enrichment via CD4+/CD25+/CD127dim magnetic selection gives 30% FoxP3-postive cells, a 2-fold enrichment over Treg-depleted.
  • An exemplary IL-2 containing FM can have the following sequence (SEQ ID NO: 79) that can be linked to an antibody or fragment thereof at one terminus:
  • This FM was made by expression of a fusion protein comprising three proteins: the targeting antibody (e.g., anti-CD45, anti-CD4 and/or anti-CD25), human serum albumin (SEQ ID NO: 79, no underline), and IL-2 (SEQ ID NO: 80).
  • the three subunits are operably linked together via a peptide linker comprising G and S (SEQ ID NO: 79, single underline). Details of nucleic acid contruct preparation, protein expression and use can be found in Examples 1-4.
  • Treg therapies have shown promise in clinical trials for Graft vs. Host Disease, Type 1 Diabetes, and Crohn's Disease, one common failure point in all of these trials has been a rapid loss of detectable Tregs. Multiple trials have suggested that 90-95% of injected Tregs are lost within the first two weeks after reinjection into the human subject. It is widely believed that a lack of cytokine support for the in vitro-expanded Tregs leads to Activation-Induced Cell Death (AICD) and a rapid decrease in cell numbers. To support Treg survival in vivo, some studies have incorporated systemic low-dose IL-2 into the Treg adoptive cell therapy treatment regimen. IL-2 is a pan-T cell pro-proliferative and survival cytokine.
  • IL-2 aldesleukin
  • IL-2 receptor alpha subunit IL2R ⁇ or CD25
  • FMs comprising IL-2 on adoptively transferred Tregs.
  • This approach can improve Treg survival and engraftment, and promote stronger and more durable treatment of various immune-related conditions, such as recovery of gut homeostasis in Inflammatory Bowel Disease (IBD), including refractory Crohn's Disease and Ulcerative Colitis.
  • IBD Inflammatory Bowel Disease
  • cytokine is attached directly to the target cells, delivery is non-systemic and a comparable on-target dose is given with far-lower total protein administration, leading to a significantly improved safety profile. Furthermore, because it is non-genetic, the cell production time is reduced, genome insertion related concerns are eliminated, and the cell process is significantly less expensive. Together this can provide to a cell product with improved efficacy and lower cost to the patient and insurers.
  • Treg-enriched CD4 T cells were obtained as described in Example 8.
  • the Treg-enriched CD4 T cells were activated as described in Example 8.
  • Cells were then pulse incubated with an IL-2 FM (h9.4Fab-IL2 for 1 hr at 37 C, washed two times, and then plated at a density of 500,000 cells/mL in full medium (containing RPMI 1640 and 10% FBS).
  • IL-2 surface levels following pulse incubation with the IL-2 FM were analyzed by flow cytometry.
  • FIG. 20 shows IL-2 surface levels after titration of an FM comprising IL-2 pulse-loaded onto Treg-enriched CD4s.
  • the IL-2 FM shows titratable surface loading at Day 0 and persistence on the cell surface for at least 3 days.
  • the cells show dose-dependent proliferation, and all concentrations of the IL-2 FM evaluated (concentration range 1.5 -100 nM) were capable of maintaining cell viability above those of the negative control ( FIG. 20 ).
  • FMs comprising IL-2 we designed and produced multiple FMs comprising human IL-2 and a variety of human CD45-targeting moieties and linker configurations including Fab-IL2 (h9.4Fab-IL2), Fab-HSA-IL2 (h9.4Fab-HSA-IL2), scFv-HSA-IL2 (scFv-h9.4-HSA-IL2), scFv-FcgR3B-IL2 (scFv-h9.4-FcgR3B-IL2).
  • Fab-IL2 h9.4Fab-IL2
  • Fab-HSA-IL2 Fab-HSA-IL2
  • scFv-HSA-IL2 scFv-h9.4-HSA-IL2
  • scFv-FcgR3B-IL2 scFv-h9.4-FcgR3B-IL2
  • Treg-enriched cells were loaded by incubating with 100 nM tethered fusions (TF) or HBSS (mock), washed to removed unbound TF, plated, and then analyzed for cell expansion over time. After 18 hours, soluble recombinant IL2 (sIL2) was added to one of the mock wells at a concentration of 100 ng/mL to serve as a positive control for expansion by saturating levels of IL-2. As shown in FIG. 21 , left panel, at 18 hours, strong surface staining was observed for all IL-2 TFs, with intensity of Fab-IL2 >Fab-HSA-IL2 >scFv-HSA-IL2.
  • CD4 cells were pulsed with vehicle or IL-2 FMs and cultured for 9 days.
  • a separate mock-treated condition was supplemented with constant incubation with 100 ng/mL recombinant IL-2 to serve as a positive control.
  • the IL-2 TFs expanded Treg-enriched CD4 T cells and increased viability over time ( FIG. 22 ).
  • Cells loaded with each of the TF constructs showed equal or better expansion compared to constant stimulation with sIL2 through 6 days post-pulse incubation; Fab-IL2 had the highest surface stain and best growth and survival effects.
  • Mock pulsed cells showed reduced viability and cell number by day 3, and retained only 5% viability by day 6.
  • IL-2 TFs supported sustained cell expansion and had>75% viability 6 days post-BP.
  • multiple configurations of IL-2 TFs are capable of promoting expansion and survival of Treg-enriched CD4s for at least 9 days in vitro.
  • tethered fusion-pulsed cells were injected into NOD scid gamma (NSG) mice.
  • 5 million human CD4 T cells were loaded with Fab-HSA-IL2 as described above and injected into NSG mice (d0).
  • 1 million activated human CD8s were injected on d3 in order to mimic the clinical application of HSCT and measure off-target effects.
  • Blood was drawn on d4, 7, 10, 13, 20, 27, 30, etc. to measure Treg-enriched CD4s and CD8s.
  • adoptively transferred cells were detectable in the blood starting d7, and loading of Fab-HSA-IL2 significantly increased cell numbers at early and late time points.
  • FIG. 24 shows that the CD4:CD8 ratio was higher in the mice treated with the FM-pulsed CD4 cells. This indicates that there were greater effects of the IL-2 on pulsed cells than on bystander CD8s, and that pulse loading of an IL-2 FM provides pro-survival and proliferative signals preferentially to the pulsed cells.
  • TFs show strong binding to unsorted CD3s or Treg-enriched CD4s. Binding is concentration-dependent and saturates between 25 and 100 nM for all constructs. All TFs promote survival and expansion similar to soluble IL-2 at saturating concentrations in vitro. In vivo, Fab-HSA-IL2 promotes early expansion and prolonged engraftment of Treg-enriched CD4s (5 fold at d10).
  • TFs cytokine fusions
  • the next step is to reduce these off-target effects, giving maximal selectivity of IL-2 activity for the Tregs.
  • This can be accomplished by engineering TFs for (a) reduced IL-2 affinity for its receptor and (b) enhanced strength, stability and selectivity of Treg binding. Because Tregs have extremely high expression of the IL-2 receptor and the cytokine will be held in close proximity to the receptor via antibody targeting, reduced affinity IL-2 will still drive signaling on loaded cells without off target effects on nearby cells or in the circulation.
  • IL-2 affinity modulated mutants can be engineered to increase Treg-specific signaling. This can increase specificity of activation by reducing paracrine off-target IL-2 signaling on non-loaded cells.
  • Reduced affinity human and mouse IL-2 molecules can be made based on structure of receptor binding and analogous work with IL-15 (see Example 7).
  • Optimal mutant can be selected in vitro using mixed loaded/non-loaded cell assay and maximizing ratio of on-target to off-target activity. Reduced off-target effects of affinity modulated IL-2 in vivo can be validated.
  • Potency and Treg selectivity of antibody binding motifs can also be optimized This can increase specificity and strength of IL-2 tethering to Tregs to reduce off target effects, and enable selective loading of stabler and more active Treg subsets.
  • Affinity matured, humanized CD45 antibody and humanized anti-CD4 (OKT4) can be produced.
  • Antibody campaigns for CD25 (general Treg) and CD39 (active subset specific) can also be conducted.
  • Optimal antibody binding motifs can be selected using the following criteria: (a) Stable and selective binding to Tregs; (b) Maximized stability of binding to Tregs.
  • Treg selectivity is less important since clinical flow-based sorting and ex vivo expansion results in a 90-95% pure Treg population; and (c) Treg selectivity is more important if tethered fusions come off the target cell and enter circulation. In that case, Treg-specificity will prevent re-binding and activation of off-target cell types.
  • Optimized lead molecules can include bi-specific antibody tethering (e.g., in a design similar to FIG. 3E ), which allows for increased persistence and cell type selectivity, and/or complex surface marker selectivity.
  • Human leads and mouse surrogates can then be validated using in vitro binding, expansion and off-target mixed cell assays. Efficacy, off-target cell effects and toxicity can be validated using mouse surrogates in Treg-controllable disease models, e.g., Naive T cell model of colitis (IBD model). Expansion, survival, engraftment and homing of final human molecule can be validated in the NSG human intestinal graft model.
  • IBD model Naive T cell model of colitis
  • IL-2 signaling occurs primarily on loaded cells (1).
  • trans signaling (2), dissociation and soluble signaling (3) and rebinding and signaling (4) result in low levels of off-target cell activation.
  • FIG. 25 lower panel, by increasing antibody targeting potency and specificity (e.g., by using a bi-valent or multi-specific binding moiety), as well as reducing IL-2 affinity for its receptor, loaded cells can continue to receive maximal IL-2 signal while eliminating off-target effects. This can result in specific survival, expansion and engraftment of transferred Tregs, leading to improved outcomes for patients.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022178067A1 (en) * 2021-02-19 2022-08-25 Janssen Biotech, Inc Materials and methods for targeting regulatory t cells for enhancing immune surveillance
US11471490B2 (en) 2017-07-03 2022-10-18 Torque Therapeutics, Inc. T cells surface-loaded with immunostimulatory fusion molecules and uses thereof
US11642417B2 (en) 2020-05-13 2023-05-09 Bonum Therapeutics, Inc. Compositions of protein complexes and methods of use thereof
US20230416394A1 (en) * 2020-11-27 2023-12-28 Elpiscience (Suzhou) Biopharma, Ltd. Novel conjugate molecules targeting cd39 and tgfbeta
WO2024097307A1 (en) * 2022-11-01 2024-05-10 Lupagen, Inc. Devices and methods for extracorporeal cell treatment
US12024559B2 (en) 2020-10-23 2024-07-02 Asher Biotherapeutics, Inc. Fusions with CD8 antigen binding molecules for modulating immune cell function
US12122827B2 (en) 2021-05-19 2024-10-22 Asher Biotherapeutics, Inc. IL-21 polypeptides and targeted constructs
WO2024220537A3 (en) * 2023-04-17 2025-01-30 ZAG Bio, Inc. Tissue-directed antibodies and methods of using the same
WO2025072623A1 (en) * 2023-09-28 2025-04-03 The Trustees Of The University Of Pennsylvania Compositions and methods for targeting immune cells
US12465640B2 (en) 2019-02-25 2025-11-11 The University Of Chicago Methods and compositions for treating inflammatory and autoimmune conditions with ECM-affinity peptides linked to anti-inflammatory agents
WO2026015637A1 (en) * 2024-07-10 2026-01-15 The Trustees Of Dartmouth College Treg targeted il-2 fusion proteins and therapeutic use thereof

Families Citing this family (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI797097B (zh) 2016-11-28 2023-04-01 日商中外製藥股份有限公司 包含抗原結合域與運送部分的多胜肽
WO2018097308A1 (ja) 2016-11-28 2018-05-31 中外製薬株式会社 リガンド結合活性が調整可能なリガンド結合分子
BR112019018915A2 (pt) 2017-03-15 2020-04-14 Pandion Therapeutics Inc imunotolerância direcionada
JP2020521452A (ja) 2017-05-24 2020-07-27 パンディオン・セラピューティクス・インコーポレイテッド 標的化免疫寛容
AU2018308088B2 (en) 2017-07-25 2025-05-29 Truebinding, Inc. Treating cancer by blocking the interaction of TIM-3 and its ligand
DK3661954T3 (da) 2017-08-03 2022-04-19 Amgen Inc Interleukin-21-muteiner og fremgangsmåder til behandling
CN111315395A (zh) 2017-09-06 2020-06-19 耶鲁大学 白细胞介素-18变体和使用方法
CN111836828B (zh) 2017-11-28 2024-12-20 中外制药株式会社 包括抗原结合结构域和运送部分的多肽
CN111630062A (zh) 2017-11-28 2020-09-04 中外制药株式会社 具有可调节的配体结合活性的配体结合分子
USRE50550E1 (en) 2017-12-06 2025-08-26 Pandion Operations, Inc. IL-2 muteins and uses thereof
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
US10174091B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
EP3737692A4 (en) 2018-01-09 2021-09-29 Elstar Therapeutics, Inc. CALRETICULIN AND MODIFIED T-LYMPHOCYTES BINDING CONSTRUCTIONS FOR THE TREATMENT OF DISEASES
TW201930344A (zh) 2018-01-12 2019-08-01 美商安進公司 抗ｐｄ-１抗體及治療方法
US12152073B2 (en) 2018-03-14 2024-11-26 Marengo Therapeutics, Inc. Multifunctional molecules that bind to calreticulin and uses thereof
CA3100050A1 (en) 2018-05-11 2019-11-14 Lupagen, Inc. Systems and methods for closed loop, real-time modifications of patient cells
CA3100349A1 (en) * 2018-05-17 2019-11-21 The Board Of Trustees Of The Leland Stanford Junior University Receptor inhibition by phosphatase recruitment
EP3818083A2 (en) 2018-07-03 2021-05-12 Elstar Therapeutics, Inc. Anti-tcr antibody molecules and uses thereof
US20220380440A1 (en) * 2018-12-31 2022-12-01 Merus N.V. Truncated multivalent multimers
AU2020214796A1 (en) 2019-01-30 2021-07-29 Truebinding, Inc. Anti-Gal3 antibodies and uses thereof
CA3128518A1 (en) * 2019-02-01 2020-08-06 Actinium Pharmaceuticals, Inc. Molecules and their derivatives directed against cd45
US20220125836A1 (en) * 2019-02-08 2022-04-28 Biontech Cell & Gene Therapies Gmbh Treatment involving car-engineered t cells and cytokines
WO2020165374A1 (en) * 2019-02-14 2020-08-20 Ose Immunotherapeutics Bifunctional molecule comprising il-15ra
US12006345B2 (en) 2019-02-21 2024-06-11 Xencor, Inc. Untargeted and targeted IL-10 Fc-fusion proteins
JP7710373B2 (ja) 2019-02-21 2025-07-18 マレンゴ・セラピューティクス，インコーポレーテッド Ｔ細胞関連のがん細胞に結合する多機能性分子およびその使用
AU2020224681A1 (en) 2019-02-21 2021-09-16 Marengo Therapeutics, Inc. Antibody molecules that bind to NKp30 and uses thereof
SG11202110810WA (en) * 2019-03-29 2021-10-28 Torque Therapeutics Inc Immunotherapeutic compositions and use thereof
EP3969119A1 (en) 2019-05-17 2022-03-23 Xencor, Inc. Il-7-fc-fusi0n proteins
WO2020236875A1 (en) * 2019-05-20 2020-11-26 Pandion Therapeutics, Inc. Madcam targeted immunotolerance
US20220235133A1 (en) * 2019-06-24 2022-07-28 Nanjing GenScript Biotech Co., Ltd. Monoclonal antibody-cytokine fusion protein dimer and application thereof
WO2021001289A1 (en) * 2019-07-02 2021-01-07 F. Hoffmann-La Roche Ag Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody
WO2021009694A1 (en) * 2019-07-17 2021-01-21 National University Of Singapore Functional binders synthesized and secreted by immune cells
CA3147757A1 (en) 2019-07-26 2021-02-04 Visterra, Inc. Interleukin-2 agents and uses thereof
US20220289838A1 (en) * 2019-08-19 2022-09-15 Nantong Yichen Biopharma. Co. Ltd. Immunocytokine, Preparation for the Same, and Uses Thereof
CN110731968A (zh) * 2019-09-10 2020-01-31 南京鼓楼医院 Icos+cxcr3+调节性t细胞在制备预防重症肺炎药物中的应用
EP3795583A1 (en) * 2019-09-19 2021-03-24 Ecole Polytechnique Federale de Lausanne (EPFL) Il10/fc fusion proteins useful as enhancers of immunotherapies
RU2753282C2 (ru) * 2019-09-19 2021-08-12 Закрытое Акционерное Общество "Биокад" ИММУНОЦИТОКИН, ВКЛЮЧАЮЩИЙ ГЕТЕРОДИМЕРНЫЙ БЕЛКОВЫЙ КОМПЛЕКС НА ОСНОВЕ IL-15/IL-15Rα, И ЕГО ПРИМЕНЕНИЕ
EP4051711A4 (en) * 2019-11-01 2024-02-21 Magenta Therapeutics, Inc. ANTI-CD45 ANTIBODIES AND THEIR CONJUGATES
US10792360B1 (en) * 2019-11-21 2020-10-06 Chemocentryx, Inc. Compositions and methods for treating inflammatory bowel disease using CCR9 inhibitor and anti-TNF-alpha blocking antibodies
KR20220109436A (ko) 2019-12-03 2022-08-04 에보텍 인터내셔널 게엠베하 인터페론-결합된 항원 결합 단백질 및 이의 용도
CA3160759A1 (en) * 2019-12-05 2021-06-10 Vycellix, Inc. Modulators of the immune escape mechanism for universal cell therapy
BR112022012112A2 (pt) 2019-12-20 2022-09-06 Regeneron Pharma Agonistas de il2 e métodos de uso dos mesmos
CN116234829A (zh) 2020-01-03 2023-06-06 马伦戈治疗公司 抗tcr抗体分子及其用途
US11981715B2 (en) 2020-02-21 2024-05-14 Pandion Operations, Inc. Tissue targeted immunotolerance with a CD39 effector
WO2021188835A1 (en) * 2020-03-18 2021-09-23 City Of Hope Multivalent chemokine receptor binding complexes
WO2021242776A2 (en) 2020-05-26 2021-12-02 Truebinding, Inc. Methods of treating inflammatory diseases by blocking galectin-3
WO2021242989A1 (en) * 2020-05-29 2021-12-02 Health Research, Inc. Compositions and methods for inducing cas9 protein expression by use of autonomous pd-1 promoter activity
TW202217002A (zh) * 2020-07-13 2022-05-01 法商傳斯堅公司 免疫抑制之治療
WO2022040577A1 (en) * 2020-08-20 2022-02-24 City Of Hope Compositions and uses of cd45 targeted chimeric antigen receptor modified t cells
JP2023548746A (ja) 2020-10-13 2023-11-21 クリヤ セラピューティクス インコーポレイテッド サイトカインをコードする核酸を送達するためのウイルスベクターコンストラクトおよびがんを処置するためのその使用
EP4255466A1 (en) 2020-12-04 2023-10-11 Visterra, Inc. Methods of using interleukin-2 agents
CN116829194A (zh) * 2020-12-09 2023-09-29 亚设生物治疗公司 用于工程化细胞疗法的靶向细胞因子构建体
TW202241508A (zh) 2021-01-29 2022-11-01 美商艾歐凡斯生物治療公司 細胞介素相關之腫瘤浸潤性淋巴球組合物及方法
IL304916A (en) * 2021-02-05 2023-10-01 Salubris Biotherapeutics Inc Il-15 fusion proteins and methods of making and using same
KR20230147619A (ko) * 2021-02-26 2023-10-23 난징 레전드 바이오테크 씨오., 엘티디. 외인적으로 도입된 사이토카인을 발현하는 조작된 면역이펙터 세포
IL306074A (en) * 2021-03-24 2023-11-01 Alkermes Inc UPAR antibodies and fusion proteins with them
EP4314029A4 (en) * 2021-03-31 2025-02-26 The Regents of University of California BISPECIFIC BINDER-LIGAND FUSIONS FOR TARGET PROTEIN DEGRADATION
KR102604849B1 (ko) * 2021-05-11 2023-11-22 가톨릭대학교 산학협력단 항-cd25 항체, il-10 및 cxcr3를 발현하는 미니서클 벡터
TW202317623A (zh) 2021-06-14 2023-05-01 美商再生元醫藥公司 基於il2之治療劑及其使用方法
CN113444744A (zh) * 2021-06-28 2021-09-28 北京鼎成肽源生物技术有限公司 一种用于基因编辑NK细胞的mRNA模板及构建方法、mRNA的体外转录方法和应用
JP2024527582A (ja) 2021-07-19 2024-07-25 中外製薬株式会社 融合ポリペプチドを用いるプロテアーゼ媒介性の標的特異的サイトカイン送達
CN118434759A (zh) * 2021-10-10 2024-08-02 安立玺荣生医(香港)有限公司 用抗-pd-l1/il-10融合蛋白治疗疾病的方法
US20250041339A1 (en) * 2021-11-19 2025-02-06 The Trustees Of The University Of Pennsylvania Engineered Pan-Leukocyte Antigen CD45 to Facilityate CAR T Cell Therapy
WO2023102463A1 (en) * 2021-12-01 2023-06-08 Visterra, Inc. Methods of using interleukin-2 agents
CN116376942A (zh) * 2021-12-31 2023-07-04 广州国家实验室 mRNA疫苗
US20230279130A1 (en) * 2022-01-14 2023-09-07 Qilu Puget Sound Biotherapeutics Corporation Anti-ccr8 antibodies
WO2023141601A1 (en) * 2022-01-20 2023-07-27 Repertoire Immune Medicines, Inc. Cd8-targeted il-12 fusion proteins
WO2023147488A1 (en) 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Cytokine associated tumor infiltrating lymphocytes compositions and methods
WO2023183927A2 (en) * 2022-03-24 2023-09-28 Actinium Pharmaceuticals, Inc. Humanized anti-cd45 antibodies
WO2023201008A1 (en) * 2022-04-13 2023-10-19 President And Fellows Of Harvard College Compositions and methods for localized delivery of cytokines for adoptive cell therapy
JP2025515470A (ja) * 2022-04-27 2025-05-15 アシャ バイオセラピューティクス，インク． サイトカイン融合タンパク質とｃｄ８抗原結合分子の併用
CN116854821A (zh) * 2022-08-15 2023-10-10 海徕科(北京)生物技术有限公司 IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途
WO2024055040A2 (en) * 2022-09-09 2024-03-14 Actinium Pharmaceuticals, Inc. Humanized anti-cd45 antibodies
TW202417511A (zh) * 2022-10-12 2024-05-01 大陸商蘇州創勝醫藥集團有限公司 包含抗ｖｅｇｆｒ２抗體的製劑
EP4634216A1 (en) 2022-12-16 2025-10-22 Repertoire Immune Medicines, Inc. T cell receptors binding hpv-16 epitopes
WO2024178414A2 (en) * 2023-02-24 2024-08-29 The Wistar Institute Of Anatomy And Biology Epitope editing and multi-specific immunce-cell engager therapy and methods of use
WO2024178305A1 (en) * 2023-02-24 2024-08-29 Modernatx, Inc. Compositions of mrna-encoded il-15 fusion proteins and methods of use thereof for treating cancer
WO2024254514A1 (en) * 2023-06-08 2024-12-12 The University Of North Carolina At Chapel Hill Engineered antibody fusion proteins with modulable binding constants and their applications
WO2025080338A1 (en) * 2023-10-11 2025-04-17 Massachusetts Institute Of Technology Cell-surface anchored cytokine therapy

Family Cites Families (113)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5650150A (en) 1990-11-09 1997-07-22 Gillies; Stephen D. Recombinant antibody cytokine fusion proteins
US5405940A (en) 1992-08-31 1995-04-11 Ludwig Institute For Cancer Research Isolated nonapeptides derived from MAGE genes and uses thereof
EP1489100B1 (en) 1997-12-08 2016-06-15 Merck Patent GmbH Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
RU2229305C2 (ru) 1998-04-15 2004-05-27 Лексиген Фармасьютикэлс Корпорейшн Усиление иммунных ответов, опосредованных белками, слитыми из антитела и цитокина, посредством совместного введения ингибитора ангиогенеза
US7550143B2 (en) 2005-04-06 2009-06-23 Ibc Pharmaceuticals, Inc. Methods for generating stably linked complexes composed of homodimers, homotetramers or dimers of dimers and uses
US7534866B2 (en) 2005-10-19 2009-05-19 Ibc Pharmaceuticals, Inc. Methods and compositions for generating bioactive assemblies of increased complexity and uses
US7527787B2 (en) 2005-10-19 2009-05-05 Ibc Pharmaceuticals, Inc. Multivalent immunoglobulin-based bioactive assemblies
US8119101B2 (en) 1999-05-10 2012-02-21 The Ohio State University Anti-CD74 immunoconjugates and methods of use
US8383081B2 (en) 1999-05-10 2013-02-26 Immunomedics, Inc. Anti-CD74 immunoconjugates and methods of use
JP4793971B2 (ja) 1999-08-09 2011-10-12 メルク パテント ゲーエムベーハー 複合サイトカイン−抗体複合体
DE60129695T2 (de) 2000-06-29 2008-06-05 Merck Patent Gmbh Steigerung von durch antikörper-zytokin-fusionsproteine mediierten immunantworten durch eine kombinierte behandlung mit mitteln zur erhöhung der immunzytokinaufnahme
US20030171546A1 (en) 2001-04-30 2003-09-11 City Of Hope Chimeric immunoreceptor useful in treating human cancers
PT1454138E (pt) * 2001-12-04 2012-03-28 Merck Patent Gmbh Imunocitoquinas com seletividade modulada
WO2003061695A1 (en) * 2002-01-17 2003-07-31 Amgen Inc. Methods of regulating cytokine receptor signaling
US8287864B2 (en) 2002-02-14 2012-10-16 Immunomedics, Inc. Structural variants of antibodies for improved therapeutic characteristics
US20070003514A1 (en) 2002-04-05 2007-01-04 The Regents Of The University Of California Mono-and bi-functional antibody conjugates as effective adjuvants of protein vaccination
US7906118B2 (en) 2005-04-06 2011-03-15 Ibc Pharmaceuticals, Inc. Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (DNL) technology
US7615223B2 (en) 2002-07-15 2009-11-10 Board Of Regents, The University Of Texas System Selected immunoconjugates for binding to aminophospholipids
EP1418184A1 (en) 2002-11-08 2004-05-12 Cell Center Cologne GmbH Recombinant fusion protein consisting of the p40 and p35 subunits of IL-12 and a ScFv and use thereof
KR20060041205A (ko) 2003-07-01 2006-05-11 이뮤노메딕스, 인코오포레이티드 양특이성 항체들의 다가 담체들
NZ547157A (en) 2003-12-10 2009-07-31 Medarex Inc Interferon Alpha Antibodies and their uses
US20110020273A1 (en) 2005-04-06 2011-01-27 Ibc Pharmaceuticals, Inc. Bispecific Immunocytokine Dock-and-Lock (DNL) Complexes and Therapeutic Use Thereof
US8034352B2 (en) 2005-04-06 2011-10-11 Ibc Pharmaceuticals, Inc. Tetrameric cytokines with improved biological activity
US8652484B2 (en) 2004-02-13 2014-02-18 Immunomedics, Inc. Delivery system for cytotoxic drugs by bispecific antibody pretargeting
US9550838B2 (en) 2004-02-13 2017-01-24 Ibc Pharmaceuticals, Inc. Dock-and-lock (DNL) complexes for therapeutic and diagnostic use
US8067006B2 (en) 2005-04-06 2011-11-29 Immunomedics, Inc. Polymeric carriers of therapeutic agents and recognition moieties for antibody-based targeting of disease sites
US9931413B2 (en) 2005-04-06 2018-04-03 Ibc Pharmaceuticals, Inc. Tetrameric cytokines with improved biological activity
AU2006232310B9 (en) 2005-04-06 2011-07-21 Ibc Pharmaceuticals, Inc. Improved stably tethered structures of defined compositions with multiple functions or binding specificities
US20120237442A1 (en) 2005-04-06 2012-09-20 Ibc Pharmaceuticals, Inc. Design and Construction of Novel Multivalent Antibodies
US9623115B2 (en) 2005-04-06 2017-04-18 Ibc Pharmaceuticals, Inc. Dock-and-Lock (DNL) Complexes for Disease Therapy
ATE459648T1 (de) 2005-05-11 2010-03-15 Philogen Spa Fusionsprotein von antikörper l19 gegen fibronectin ed-b und interleukin 12
US7612181B2 (en) 2005-08-19 2009-11-03 Abbott Laboratories Dual variable domain immunoglobulin and uses thereof
US20100226884A1 (en) 2009-01-20 2010-09-09 Immunomedics, Inc. Novel Class of Monospecific and Bispecific Humanized Antibodies that Target the Insulin-like Growth Factor Type I Receptor (IGF-1R)
CN101374546B (zh) 2005-12-16 2016-03-23 Ibc医药公司 基于免疫球蛋白的多价生物活性装配体
BRPI0620648B1 (pt) 2005-12-30 2022-12-20 Merck Patent Gesellschaft Mit Beschrãnkter Haftung Variante de il-12 p40, proteínas il-12, il-23 e de fusão, composição farmacêutica e uso da mesma
US8450112B2 (en) * 2008-04-09 2013-05-28 Maxcyte, Inc. Engineering and delivery of therapeutic compositions of freshly isolated cells
US9283184B2 (en) 2008-11-24 2016-03-15 Massachusetts Institute Of Technology Methods and compositions for localized agent delivery
EP3912643B8 (en) 2009-02-13 2023-08-23 Immunomedics, Inc. Immunoconjugates with an intracellularly-cleavable linkage
EP3009455A1 (en) 2009-09-16 2016-04-20 Immunomedics Inc. Class i anti-cea antibodies and uses thereof
US20110165122A1 (en) * 2009-11-10 2011-07-07 The Regents Of The University Of California Method for targeted and sustained antiviral therapy
WO2011066342A2 (en) 2009-11-24 2011-06-03 Amplimmune, Inc. Simultaneous inhibition of pd-l1/pd-l2
WO2011090492A1 (en) 2010-01-19 2011-07-28 Immunomedics, Inc. Novel class of monospecific and bispecific humanized antibodies that target the insulin-like growth factor type i receptor (igf-1r)
CN101829322A (zh) * 2010-03-05 2010-09-15 孙嘉琳 细胞因子-超抗原融合蛋白在制备抗实体瘤药物的应用
US8617557B2 (en) 2010-03-12 2013-12-31 The Regents Of The University Of California Antibody fusion with IL-12 proteins with disrupted heparin-binding activity
DK2560993T3 (da) 2010-04-20 2024-10-14 Genmab As Heterodimeric antibody fc-containing proteins and methods for production thereof
BR112012029611A2 (pt) * 2010-05-21 2017-07-25 Merrimack Pharmaceuticals Inc proteína de fusão biespecífica, composição farmacêutica, método de tratamento de dano ao tecido em um indivíduo, método de promoção da regeração ou sobrevivência do tecido em um indivíduo e molécula de ácido nucleico
WO2012019157A2 (en) * 2010-08-06 2012-02-09 The Regents Of The University Of California Expression and purification of fusion protein with multiple mbp tags
CN107880136B (zh) * 2010-09-21 2021-11-12 阿尔托生物科学有限公司 多聚体il-15可溶性融合分子与其制造与使用方法
RU2013120302A (ru) * 2010-10-01 2014-11-20 Модерна Терапьютикс, Инк. Сконструированные нуклеиновые кислоты и способы их применения
CA2819074C (en) * 2010-12-06 2020-04-14 Shell Internationale Research Maatschappij B.V. Process for removing mercaptans from a gas stream
EP2723380B1 (en) 2011-06-24 2019-08-21 Stephen D. Gillies Light chain immunoglobulin fusion proteins and methods of use thereof
EP2736535B1 (en) 2011-07-27 2018-10-17 Philogen S.p.A. Il-12 immunoconjugate
EP2764362B1 (en) 2011-10-03 2018-09-05 Immunomedics, Inc. Dye conjugated peptides for fluorescent imaging
SG10201603411WA (en) * 2011-10-28 2016-07-28 Teva Pharmaceuticals Australia Pty Ltd Polypeptide constructs and uses thereof
WO2013082254A1 (en) 2011-12-02 2013-06-06 Ibc Pharmaceuticals, Inc. Multivalent antibody complexes targeting igf-1r show potent toxicity against solid tumors
ES2800426T3 (es) * 2012-03-03 2020-12-30 Immungene Inc Moléculas de fusión anticuerpos-mutante de interferón modificadas
EP3878964A1 (en) 2012-11-27 2021-09-15 Ajou University Industry-Academic Cooperation Foundation Ch3 domain variant pair inducing formation of heterodimer of heavy chain constant region of antibody at high efficiency, method for preparing same, and use thereof
EP2970486B1 (en) 2013-03-15 2018-05-16 Xencor, Inc. Modulation of t cells with bispecific antibodies and fc fusions
EP2988784A1 (en) 2013-04-25 2016-03-02 Philogen S.p.A. Antibody-drug conjugates
WO2014190441A1 (en) 2013-05-31 2014-12-04 Zymeworks Inc. Heteromultimers with reduced or silenced effector function
WO2014204762A1 (en) 2013-06-19 2014-12-24 Massachusetts Institute Of Technology In vivo targeting of cells with ligand-conjugated particles
WO2014209804A1 (en) * 2013-06-24 2014-12-31 Biomed Valley Discoveries, Inc. Bispecific antibodies
AR096891A1 (es) 2013-07-12 2016-02-03 Hanmi Pharm Ind Co Ltd Conjugado de monómero polipéptido biológicamente activo y conjugado de fragmento fc de inmunoglobulina, que muestra aclaramiento mediado por receptor reducido, y el método para la preparación del mismo
EP3049114B1 (en) 2013-09-27 2021-11-10 Massachusetts Institute of Technology Carrier-free biologically-active protein nanostructures
MY193723A (en) 2014-08-29 2022-10-27 Hoffmann La Roche Combination therapy of tumor-targeted il-2 variant immunocytokines and antibodies against human pd-l1
SG11201702295UA (en) 2014-09-22 2017-04-27 Intrexon Corp Improved therapeutic control of heterodimeric and single chain forms of interleukin-12
US20160176969A1 (en) 2014-11-26 2016-06-23 Xencor, Inc. Heterodimeric antibodies including binding to cd8
WO2016105450A2 (en) 2014-12-22 2016-06-30 Xencor, Inc. Trispecific antibodies
WO2016112983A1 (en) 2015-01-15 2016-07-21 Biontech Ag Cytokine fusion proteins
WO2016141387A1 (en) * 2015-03-05 2016-09-09 Xencor, Inc. Modulation of t cells with bispecific antibodies and fc fusions
US11022615B2 (en) 2015-05-04 2021-06-01 University Of Florida Research Foundation, Inc. Regulatory T-cells, method for their isolation and uses
EP3297684B1 (en) 2015-05-21 2020-12-23 Actinium Pharmaceuticals, Inc. Infusion administration of conjugated monoclonal antibodies
TWI833684B (zh) * 2015-06-25 2024-03-01 美商生物細胞基因治療有限公司 嵌合抗原受體（ｃａｒ）、組合物及其使用方法
US11173179B2 (en) 2015-06-25 2021-11-16 Icell Gene Therapeutics Llc Chimeric antigen receptor (CAR) targeting multiple antigens, compositions and methods of use thereof
CN108990413A (zh) 2015-08-12 2018-12-11 麻省理工学院 纳米颗粒的细胞表面偶联
SG11201802912PA (en) 2015-10-10 2018-05-30 Intrexon Corp Improved therapeutic control of proteolytically sensitive, destabilized forms of interleukin-12
GB201521383D0 (en) 2015-12-03 2016-01-20 Ucb Biopharma Sprl And Ucb Celltech Method
WO2017102010A1 (en) * 2015-12-17 2017-06-22 Biontech Rna Pharmaceuticals Gmbh Novel cytokine fusion proteins
US20190000989A1 (en) 2016-01-11 2019-01-03 STC Biologics Inc. Bispecific targeting agents and methods for their preparation
US11001631B2 (en) 2016-02-05 2021-05-11 Orionis Biosciences BV Clec9A binding agents
JP7114490B2 (ja) 2016-06-24 2022-08-08 アイセル・ジーン・セラピューティクス・エルエルシー キメラ抗体受容体（ＣＡＲｓ）の構成およびその使用方法
MA45779A (fr) 2016-07-29 2019-06-05 Juno Therapeutics Inc Polypeptides immunomdulateurs et compositions et procédés associés
WO2018030806A1 (ko) 2016-08-10 2018-02-15 아주대학교산학협력단 항체 중쇄불변부위 이종이중체에 융합된 사이토카인 및 이를 포함하는 약제학적 조성물
MX386014B (es) 2016-08-10 2025-03-18 Univ Ajou Ind Academic Coop Found Citocina fusionada a fc heterodimérico, y composición farmacéutica que comprende la misma.
JP2019532648A (ja) 2016-10-07 2019-11-14 ボード オブ リージェンツ ザ ユニヴァーシティ オブ テキサス システム がんの治療のための膜係留ｉｌ−１２を発現しているｔ細胞
US20190352335A1 (en) 2016-12-19 2019-11-21 Hanmi Pharm. Co., Ltd. Brain targeting long-acting protein conjugate
JP2020506932A (ja) 2017-02-03 2020-03-05 ハンミ ファーマシューティカル カンパニー リミテッド 持続性が増加した生理活性物質の結合体及びその用途
EP3601538A4 (en) 2017-03-27 2021-03-10 Nantcell, Inc. ANK AND IL-12 COMPOSITIONS AND METHODS
JP7285220B2 (ja) 2017-05-18 2023-06-01 モデルナティエックス インコーポレイテッド 連結したインターロイキン－１２（ｉｌ１２）ポリペプチドをコードするポリヌクレオチドを含む脂質ナノ粒子
JP2020522254A (ja) 2017-05-31 2020-07-30 エルスター セラピューティクス， インコーポレイテッド 骨髄増殖性白血病（ｍｐｌ）タンパク質に結合する多特異性分子およびその使用
CA3287539A1 (en) 2017-06-21 2026-03-02 Icell Gene Therapeutics Llc CHIMERIC ANTIGEN RECEPTORS (CARs), COMPOSITIONS AND METHODS THEREOF
US11084863B2 (en) 2017-06-30 2021-08-10 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains
US20210017247A1 (en) 2017-07-03 2021-01-21 Torque Therapeutics, Inc. Fusion Molecules Targeting Immune Regulatory Cells and Uses Thereof
CN111527109B (zh) 2017-12-26 2024-11-01 南京金斯瑞生物科技有限公司 以抗体Fc区为骨架的融合蛋白二聚体及其应用
JP2021516045A (ja) 2018-02-28 2021-07-01 ファイザー・インク Ｉｌ−１５バリアントおよびその使用
CN110396133B (zh) 2018-04-25 2021-07-23 免疫靶向有限公司 一种以白介素12为活性成分的融合蛋白型药物前体
CA3100005A1 (en) 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Activatable cytokine polypeptides and methods of use thereof
US11795203B2 (en) 2018-07-30 2023-10-24 Jinyu Zhang Protein heterodimer and use thereof
CA3109361A1 (en) 2018-08-30 2020-03-05 HCW Biologics, Inc. Single-chain and multi-chain chimeric polypeptides and uses thereof
JP2022502443A (ja) 2018-09-28 2022-01-11 ピエール、ファーブル、メディカマン 癌の処置のための新規な免疫サイトカイン
SG11202104524YA (en) 2018-11-01 2021-05-28 Gracell Biotechnologies Shanghai Co Ltd Compositions and methods for t cell engineering
CA3118943A1 (en) 2018-11-15 2020-05-22 Torque Therapeutics, Inc. Methods and compositions for cancer immunotherapy
WO2020123602A1 (en) 2018-12-11 2020-06-18 Oncosec Medical Incorporated Multigene construct for immune-modulatory protein expression and methods of use
CA3120800A1 (en) 2018-12-17 2020-06-25 Revitope Limited Twin immune cell engager
SG11202110810WA (en) 2019-03-29 2021-10-28 Torque Therapeutics Inc Immunotherapeutic compositions and use thereof
CN111848810B (zh) 2019-04-28 2024-08-23 张晋宇 一种蛋白质分子及其用途
AU2020287373B2 (en) 2019-06-05 2026-01-29 Asher Biotherapeutics, Inc. Fusions of mutant interleukin-2 polypeptides with antigen binding molecules for modulating immune cell function
WO2020255014A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Recombinant interleukin 12 construct and uses thereof
US20220235133A1 (en) 2019-06-24 2022-07-28 Nanjing GenScript Biotech Co., Ltd. Monoclonal antibody-cytokine fusion protein dimer and application thereof
WO2021001289A1 (en) 2019-07-02 2021-01-07 F. Hoffmann-La Roche Ag Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody
US20220306735A1 (en) 2019-08-30 2022-09-29 University Of Kansas Compositions including igg fc mutations and uses thereof
US20210301015A1 (en) 2020-01-17 2021-09-30 Aetio Biotherapy, Inc. Pro-antibody that reduces off-target toxicity
EP3889183A1 (en) 2020-04-01 2021-10-06 Pierre Fabre Medicament A protein complex comprising an immunocytokine

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11471490B2 (en) 2017-07-03 2022-10-18 Torque Therapeutics, Inc. T cells surface-loaded with immunostimulatory fusion molecules and uses thereof
US12465640B2 (en) 2019-02-25 2025-11-11 The University Of Chicago Methods and compositions for treating inflammatory and autoimmune conditions with ECM-affinity peptides linked to anti-inflammatory agents
US12303567B2 (en) 2020-05-13 2025-05-20 Bonum Therapeutics, Inc. Compositions of protein complexes and methods of use thereof
US11642417B2 (en) 2020-05-13 2023-05-09 Bonum Therapeutics, Inc. Compositions of protein complexes and methods of use thereof
US12024559B2 (en) 2020-10-23 2024-07-02 Asher Biotherapeutics, Inc. Fusions with CD8 antigen binding molecules for modulating immune cell function
US20230416394A1 (en) * 2020-11-27 2023-12-28 Elpiscience (Suzhou) Biopharma, Ltd. Novel conjugate molecules targeting cd39 and tgfbeta
WO2022178067A1 (en) * 2021-02-19 2022-08-25 Janssen Biotech, Inc Materials and methods for targeting regulatory t cells for enhancing immune surveillance
US12122827B2 (en) 2021-05-19 2024-10-22 Asher Biotherapeutics, Inc. IL-21 polypeptides and targeted constructs
US12247072B2 (en) 2021-05-19 2025-03-11 Asher Biotherapeutics, Inc. IL-21 polypeptides and targeted constructs
WO2024097307A1 (en) * 2022-11-01 2024-05-10 Lupagen, Inc. Devices and methods for extracorporeal cell treatment
WO2024220537A3 (en) * 2023-04-17 2025-01-30 ZAG Bio, Inc. Tissue-directed antibodies and methods of using the same
WO2025072623A1 (en) * 2023-09-28 2025-04-03 The Trustees Of The University Of Pennsylvania Compositions and methods for targeting immune cells
WO2026015637A1 (en) * 2024-07-10 2026-01-15 The Trustees Of Dartmouth College Treg targeted il-2 fusion proteins and therapeutic use thereof

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