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WO2018144514A2 - Polypeptides de fusion de la superfamille du tnf - Google Patents

Polypeptides de fusion de la superfamille du tnf Download PDF

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Publication number
WO2018144514A2
WO2018144514A2 PCT/US2018/016100 US2018016100W WO2018144514A2 WO 2018144514 A2 WO2018144514 A2 WO 2018144514A2 US 2018016100 W US2018016100 W US 2018016100W WO 2018144514 A2 WO2018144514 A2 WO 2018144514A2
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domain
amino acid
seq
another embodiment
acid residues
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PCT/US2018/016100
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WO2018144514A3 (fr
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Birgit M. Schoeberl
Eric M. Tam
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Merrimack Pharmaceuticals, Inc.
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Publication of WO2018144514A2 publication Critical patent/WO2018144514A2/fr
Publication of WO2018144514A3 publication Critical patent/WO2018144514A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • T cells recognize peptide antigens in the context of the major histocompatibility complex (MHC) molecule complexes depending on the specificity of their T-cell antigen receptor (TCR).
  • MHC major histocompatibility complex
  • T cells require an additional co-stimulatory signal to ensure full activation, clonal expansion and concomitant effector differentiation.
  • This co- stimulatory signal can also be found at the interface of the antigen presenting cells (APC) and the T cells and involve several members of TNF ligand/receptor superfamily.
  • APC antigen presenting cells
  • 4-lBB also known as CD137 or "tumor necrosis factor superfamily, member 9"
  • TNFRSF9 TNFRSF9
  • TNFRSF9 TNFRSF9
  • B cells B cells
  • NK cells NK cells
  • dendritic cells see, e.g., Chester C, et al., Cancer Immunol. Immunother. 2016 Oct;65(10): 1243-8).
  • 4-lBB signaling is critical for formation of immunological memory and T cell proliferation.
  • 4-lBB signaling induces maturation of dendritic cells and production of inflammatory cytokines.
  • CD27 (TNFRSF7) binds to ligand CD70 and plays a key role in regulating B-cell activation and immunoglobulin synthesis (see, e.g., van de Ven K, Borst J., Immunotherapy, 2015;7(6):655-67).
  • CD27 promotes proliferation of naive CD4+ and CD8+ T cells and the generation of effector cells.
  • CD27 is expressed on naive T, B and NK cells and is upregulated upon activation.
  • Glucocorticoid-induced TNFR-related protein a co-stimulatory molecule also known as TNFRSF18, AITR, CD357, and GITR-D
  • GITR a co-stimulatory molecule also known as TNFRSF18, AITR, CD357, and GITR-D
  • Other related members of the TNF receptor family include CD40, CD27, 4- IBB, and OX40.
  • GITR expression is low in naive CD4+ and CD8+ cells, it is constitutively expressed in regulatory T cells (Tone et al., PNAS 2003;100: 15059-64).
  • GITR engagement promotes their activation, proliferation, and cytokine production (Watts, Annual Reviews in Immunology 2005;23:23-68).
  • Tregs CD4+CD25+ regulatory T cells
  • Shimizu reported that GITR engagement suppresses their function (Shimizu et al, Nature Immunology 2002;3: 135-42) using a mixed culture suppression assay.
  • subsequent work by Stephans et al. JI 2004 15; 173(8):5008-20
  • T eff Teffector
  • OX40 (TNFRSF4), also known as ACT35, IMD16, TXGP1L, and CD134, is a 50-kD type I transmembrane glycoprotein in the TNFSFR family of costimulatory receptors expressed on activated CD4+ T cells, natural killer cells and natural killer T cells (see, e.g., Croft M, et al., Immunological reviews. 2009;229(1): 173-191).
  • OX40-expressing activated T cells are found in tumor infiltrating lymphocytes.
  • OX40 and its ligand, OX40L play a crucial role in inducing and maintaining T-cell responses.
  • OX40 signaling plays a role in the proliferation and differentiation into effector T cells (Teff cells) and augments production of cytokines.
  • CD40 (TNFRSF5) is a stimulatory receptor that has been found to be essential in mediating a broad variety of immune and inflammatory responses, including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation.
  • CD40 also regulates activity of dendritic cells, macrophages and B cells (see, e.g., Beatty GL, et al., Expert Rev. Anticancer Ther. 2017 Feb; 17(2): 175-186).
  • CD40 signals can promote survival in these cell types and induce production of inflammatory cytokines in macrophages and DCs.
  • CD40 can also upregulate molecules involved in antigen presentation and T cell stimulation.
  • CD40 is constitutively expressed on B cells, dendritic cells and macrophages.
  • TNFRSF14 HVEM functions in signal transduction pathways that activate inflammatory and inhibitory T-cell immune response.
  • the protein encoded by this gene is a member of the tumor necrosis factor (TNF) ligand family.
  • TNF tumor necrosis factor
  • Activation of TNFRSF14 by its ligand, LIGHT (TNFSF) has been shown to stimulate the proliferation of T cells, and trigger apoptosis of various tumor cells.
  • fusion polypepetides which comprise a 4-1BBL moiety, a CD70 moiety, a GITRL moiety, an OX40L moiety, a CD40L moiety, and a LIGHT moiety.
  • fusion polypeptides comprising a 4- 1BBL moiety (e.g., which agonizes 4-1BB signaling to B and T cells).
  • exemplary fusion polypeptides include a 4-1BBL moiety (e.g., 4-1BBL monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-4-lBBL fusion), Fab-Fc (Fab-Fc -4-1BBL fusion), Fab fragment (4-lBBL-antibody Fab fragment fusion), antibody (4-lBBL-antibody fusion) or albumin (4-lBBL-albumin fusion).
  • the 4- 1BBL polypeptides described herein are therapeutically advantageous based on their increased half-life in circulating blood (e.g., in a human patient).
  • the polypeptide comprises an antibody Fc region or a fragment thereof and one or more 4-lBBL domains.
  • the 4-lBBL moiety comprises one, two, three, four, or five 4-lBBL domains.
  • the 4-lBBL moiety comprises three 4- 1BBL domains.
  • the 4-lBBL moiety is C-terminal to the antibody Fc region or fragment thereof.
  • the antibody Fc region is selected from the group consisting of a human IgGl Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, and variants thereof.
  • the polypeptide is a Fc-4-lBBL fusion polypeptide comprising a human IgG Fc moiety, or fragment thereof, bound to a set of three human 4-lBBL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first 4-lBBL domain, an inter-domain linker, a second 4-lBBL domain, a second inter-domain linker, and a third 4-lBBL domain.
  • the Fc4-1BBL fusion polypeptide comprises two separate Fc-4- 1BBL fusion polypeptides.
  • the two separate Fc-4-lBBL fusion polypeptides are dimerized by at least one inter-Fc disulfide bond.
  • the Fc-4-lBBL fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide-bound to a set of three human 4-lBBL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first 4-lBBL domain, an inter-domain linker, a second 4-lBBL domain, a second inter-domain linker, and a third 4-lBBL domain, wherein each linker consists of 15-20 amino acids and each of the two inter-4-lBBL monomer linkers comprises 3 G4S motifs.
  • the Fc-4-lBBL fusion polypeptide comprises the amino acid sequences set for in SEQ ID NO: SEQ ID NO: 8 or a sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 8.
  • the polypeptide is a Fab-Fc-4-lBBL fusion polypeptide.
  • the polypeptide is a 4-lBBL-antibody Fab fragment fusion polypeptide.
  • the polypeptide is an antibody-4-lBBL fusion polypeptide.
  • the polypeptide is a 4-lBBL-albumin fusion polypeptide (e.g., an HSA-4-1BBL fusion).
  • the polypeptide comprises one or more 4-1BBL domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • each 4-1BBL domain independently, comprises amino acid residues 50-254 of SEQ ID NO: SEQ ID NO:4, or a sequence at least 85% identical thereto (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each 4-1BBL domain independently, comprises amino acid residues 71-254 of SEQ ID NO:4, or a sequence at least 85% identical thereto (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each 4-1BBL domain independently, comprises amino acid residues 85-254 of SEQ ID NO:4, or a sequence at least 85% identical thereto (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each 4-1BBL domain independently, consists of amino acid residues 50-254 of SEQ ID NO:4. In another embodiment, each 4-1BBL domain, independently, consists of amino acid residues 71-254 of SEQ ID NO:4. In another embodiment, each 4-1BBL domain, independently, consists of amino acid residues 50-254 of SEQ ID NO:4.
  • CD70 Fusion Polypetides Provided herein are fusion polypeptides comprising a CD70 moiety (e.g., which agonizes CD27 signaling).
  • exemplary fusion polypeptides include a CD70 moiety (e.g., CD70 monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-CD70 fusion), Fab-Fc (Fab-Fc-CD70 fusion), Fab fragment (CD70-antibody Fab fragment fusion), antibody (CD70-antibody fusion) or albumin (CD70-albumin fusion).
  • the CD70 polypeptides described herein are therapeutically advantageous based on their increased half-life in circulating blood (e.g., in a human patient).
  • the polypeptide comprises an antibody Fc region or a fragment thereof and one or more CD70 domains.
  • the CD70 moiety comprises one, two, three, four, or five CD70 domains.
  • the CD70 moiety comprises three CD70 domains.
  • the CD70 moiety is C-terminal to the antibody Fc region or fragment thereof.
  • the antibody Fc region is selected from the group consisting of a human IgGl Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, and variants thereof.
  • the polypeptide is a Fc-CD70 fusion polypeptide comprising a human IgG Fc moiety, or fragment thereof, bound to a set of three human CD70 domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD70 domain, an inter-domain linker, a second CD70 domain, a second inter-domain linker, and a third CD70 domain.
  • the FcCD70 fusion polypeptide comprises two separate Fc-CD70 fusion polypeptides.
  • the two separate Fc-CD70 fusion polypeptides are dimerized by at least one inter-Fc disulfide bond.
  • the Fc-CD70 fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide- bound to a set of three human CD70 domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD70 domain, an inter-domain linker, a second CD70 domain, a second inter-domain linker, and a third CD70 domain, wherein each linker consists of 15-20 amino acids and each of the two inter-CD70 monomer linkers comprises 3 G4S motifs.
  • the Fc-CD70 fusion polypeptide comprises the amino acid sequences set for in SEQ ID NO: 18 or a sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 18.
  • the polypeptide is a Fab-Fc-CD70 fusion polypeptide.
  • the polypeptide is a CD70-antibody Fab fragment fusion polypeptide.
  • the polypeptide is an antibody-CD70 fusion polypeptide.
  • polypeptide is a CD70-albumin fusion polypeptide (e.g., HSA-
  • the polypeptide comprises one or more CD70 domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • CD70 domains linked e.g., fused
  • each CD70 domain independently, comprises amino acid residues 39-193 of SEQ ID NO: 16. In another embodiment, each CD70 domain, independently, comprises an amino acid sequence at least 85% identical to amino acid residues 39-193 of SEQ ID NO: 16 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, each CD70 domain, independently, consists of amino acid residues 39-193 of SEQ ID NO: 16.
  • GITRL fusion polypeptides comprising a GITRL moiety (e.g., which agonizes GITR signaling).
  • exemplary fusion polypeptides include a GITRL moiety (e.g., GITRL monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-GITRL fusion), Fab-Fc (Fab-Fc -GITRL fusion), Fab fragment (GITRL-antibody Fab fragment fusion), antibody (GITRL-antibody fusion) or albumin (GITRL-albumin fusion).
  • the GITRL polypeptides described herein are therapeutically advantageous based on their increased half-life in circulating blood (e.g., in a human patient).
  • the polypeptide comprises an antibody Fc region or a fragment thereof and one or more GITRL domains.
  • the GITRL moiety comprises one, two, three, four, or five GITRL domains.
  • the GITRL moiety comprises three GITRL domains.
  • the GITRL moiety is C-terminal to the antibody Fc region or fragment thereof.
  • the antibody Fc region is selected from the group consisting of a human IgGl Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, and variants thereof.
  • the polypeptide is a Fc-GITRL fusion polypeptide comprising a human IgG Fc moiety, or fragment thereof, bound to a set of three human GITRL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first GITRL domain, an inter-domain linker, a second GITRL domain, a second inter-domain linker, and a third GITRL domain.
  • the Fc-GITRL fusion polypeptide comprises two separate Fc-GITRL fusion polypeptides.
  • the two separate Fc-GITRL fusion polypeptides are dimerized by at least one inter-Fc disulfide bond.
  • the Fc-GITRL fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide- bound to a set of three human GITRL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first GITRL domain, an inter-domain linker, a second GITRL domain, a second inter-domain linker, and a third GITRL domain, wherein each linker consists of 15-20 amino acids and each of the two inter-GITRL monomer linkers comprises 3 G4S motifs.
  • the Fc-GITRL fusion polypeptide comprises the amino acid sequences set for in SEQ ID NO: 32 or a sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 32.
  • the polypeptide is a Fab-Fc -GITRL fusion polypeptide.
  • the polypeptide is a GITRL-antibody Fab fragment fusion polypeptide.
  • the polypeptide is an antibody-GITRL fusion polypeptide.
  • polypeptide is a GITRL-albumin fusion polypeptide (e.g., HSA-
  • the polypeptide comprises one or more GITRL domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • GITRL domains linked e.g., fused
  • each GITRL domain independently, comprises amino acid residues 72-199 of SEQ ID NO: 29.
  • each GITRL domain independently, comprises an amino acid sequence at least 85% identical to amino acid residues 47-261 of SEQ ID NO: 29 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each GITRL domain independently, consists of amino acid residues 72-199 of SEQ ID NO: 29.
  • each GITRL domain independently, comprises amino acid residues 74-199 of SEQ ID NO: 29.
  • each GITRL domain independently, comprises an amino acid sequence at least 85% identical to amino acid residues 74-199 of SEQ ID NO: 29 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, each GITRL domain, independently, consists of amino acid residues 74-199 of SEQ ID NO: 29.
  • OX40L Fusion Polypeptides comprising an OX40Lmoiety (e.g., which agonizes OX40 signaling).
  • exemplary fusion polypeptides include an OX40Lmoiety (e.g., OX40L monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-OX40L fusion), Fab-Fc (Fab-Fc-OX40L fusion), Fab fragment (OX40L-antibody Fab fragment fusion), antibody (OX40L-antibody fusion) or albumin (OX40L-albumin fusion).
  • the OX40L polypeptides described herein are therapeutically advantageous based on their increased half-life in circulating blood (e.g., in a human patient).
  • the polypeptide comprises an antibody Fc region or a fragment thereof and one or more OX40L domains.
  • the OX40L moiety comprises one, two, three, four, or five OX40L domains.
  • the OX40L moiety comprises three OX40L domains.
  • the OX40L moiety is C-terminal to the antibody Fc region or fragment thereof.
  • the antibody Fc region is selected from the group consisting of a human IgGl Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, and variants thereof.
  • the polypeptide is a Fc-OX40L fusion polypeptide comprising a human IgG Fc moiety, or fragment thereof, bound to a set of three human OX40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first OX40L domain, an inter-domain linker, a second OX40L domain, a second inter-domain linker, and a third OX40L domain.
  • the FcOX40L fusion polypeptide comprises two separate Fc-OX40L fusion polypeptides.
  • the two separate Fc-OX40L fusion polypeptides are dimerized by at least one inter-Fc disulfide bond.
  • the Fc-OX40L fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide- bound to a set of three human OX40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first OX40L domain, an inter-domain linker, a second OX40L domain, a second inter-domain linker, and a third OX40L domain, wherein each linker consists of 15-20 amino acids and each of the two inter-OX40L domain linkers comprises 3 G4S motifs.
  • the Fc-OX40L fusion polypeptide comprises the amino acid sequences set for in SEQ ID NO:41 or a sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical toSEQ ID NO:41.
  • the polypeptide is a Fab-Fc-OX40L fusion polypeptide.
  • the polypeptide is an OX40L-antibody Fab fragment fusion polypeptide.
  • the polypeptide is an antibody-OX40L fusion polypeptide.
  • polypeptide is a OX40L-albumin fusion polypeptide (e.g., HSA-
  • the polypeptide comprises one or more OX40L domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • each OX40L domain independently, comprises amino acid residues 51-183 of SEQ ID NO:39.
  • each OX40L domain independently, comprises an amino acid sequence at least 85% identical to amino acid residues 51-183 of SEQ ID NO:39 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each OX40L domain independently, consists of amino acid residues 51-183 of SEQ ID NO:39.
  • CD40L Fusion Polypeptides comprising a CD40L moiety (e.g., which agonizes CD40 signaling).
  • exemplary fusion polypeptides include a CD40L moiety (e.g., CD40L monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-CD40L fusion), Fab-Fc (Fab-Fc-CD40L fusion), Fab fragment (CD40L-antibody Fab fragment fusion), antibody (CD40L-antibody fusion) or albumin (CD40L-albumin fusion).
  • the CD40L polypeptides described herein are therapeutically advantageous based on their increased half-life in circulating blood (e.g., in a human patient).
  • the polypeptide comprises an antibody Fc region or a fragment thereof and one or more CD40L domains.
  • the CD40L moiety comprises one, two, three, four, or five CD40L domains.
  • the CD40L moiety comprises three CD40L domains.
  • the CD40L moiety is C-terminal to the antibody Fc region or fragment thereof.
  • the antibody Fc region is selected from the group consisting of a human IgGl Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, and variants thereof.
  • the polypeptide is a Fc-CD40L fusion polypeptide comprising a human IgG Fc moiety, or fragment thereof, bound to a set of three human CD40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD40L domain, an inter-domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain.
  • the FcCD40L fusion polypeptide comprises two separate Fc-CD40L fusion polypeptides.
  • the two separate Fc-CD40L fusion polypeptides are dimerized by at least one inter-Fc disulfide bond.
  • the Fc-CD40L fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide- bound to a set of three human CD40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD40L domain, an inter-domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain, wherein each linker consists of 15-20 amino acids and each of the two inter-CD40L monomer linkers comprises 3 G4S motifs.
  • the Fc-CD40L fusion polypeptide comprises the amino acid sequences set for in SEQ ID NO:52 or a sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 8.
  • the polypeptide is a Fab-Fc-CD40L fusion polypeptide.
  • the polypeptide is a CD40L-antibody Fab fragment fusion polypeptide.
  • the polypeptide is an antibody-CD40L fusion polypeptide.
  • the polypeptide is a CD40L-albumin fusion polypeptide (e.g., HSA-
  • the polypeptide comprises one or more CD40L domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • CD40L domains linked e.g., fused
  • each CD40L domain independently, comprises amino acid residues 47-261 of SEQ ID NO:48.
  • each CD40L domain independently, comprises an amino acid sequence at least 85% identical to amino acid residues 47-261 of SEQ ID NO:48 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each CD40L domain independently, consists of amino acid residues 47-261 of SEQ ID NO:48.
  • each CD40L domain independently, comprises amino acid residues 1 16-261 of SEQ ID NO:48.
  • each CD40L domain independently, comprises an amino acid sequence at least 85% identical to amino acid residues 1 16-261 of SEQ ID NO:48 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each CD40L domain independently, consists of amino acid residues 1 16-261 of SEQ ID NO:48.
  • each CD40L domain independently, comprises amino acid residues 108-261 of SEQ ID NO:48.
  • each CD40L domain independently, comprises an amino acid sequence at least 85% identical to amino acid residues 108-261 of SEQ ID NO:48 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each CD40L domain independently, consists of amino acid residues 108-261 of SEQ ID NO:48.
  • each CD40L domain independently, comprises amino acid residues 1 13-261 of SEQ ID NO:48.
  • each CD40L domain independently, comprises an amino acid sequence at least 85% identical to amino acid residues 1 13-261 of SEQ ID NO:48 (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each CD40L domain independently, consists of amino acid residues 1 13-261 of SEQ ID NO:48.
  • LIGHT Fusion Polypeptides comprising a LIGHT moiety (e.g., which agonizes TNFRSF 14 signaling to B and T cells).
  • exemplary fusion polypeptides include a LIGHT moiety (e.g., a LIGHT monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-LIGHT fusion), Fab-Fc (Fab-Fc-LIGHT fusion), Fab fragment (LIGHT-antibody Fab fragment fusion), antibody (LIGHT -antibody fusion) or albumin (LIGHT-albumin fusion).
  • LIGHT-polypeptides described herein are therapeutically advantageous based on their increased half-life in circulating blood (e.g., in a human patient).
  • the polypeptide comprises an antibody Fc region or a fragment thereof and one or more LIGHT domains.
  • the LIGHT moiety comprises one, two, three, four, or five LIGHT domains.
  • the LIGHT moiety comprises three LIGHT domains.
  • the LIGHT moiety is C-terminal to the antibody Fc region or fragment thereof.
  • the antibody Fc region is selected from the group consisting of a human IgGl Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, and variants thereof.
  • the polypeptide is a Fc- LIGHT fusion polypeptide comprising a human IgG Fc moiety, or fragment thereof, bound to a set of three human LIGHT domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first LIGHT domain, an inter-domain linker, a second LIGHT domain, a second inter-domain linker, and a third LIGHT domain.
  • the Fc-LIGHT fusion polypeptide comprises two separate Fc- LIGHT fusion polypeptides.
  • the two separate Fc- LIGHT fusion polypeptides are dimerized by at least one inter-Fc disulfide bond.
  • the Fc- LIGHT fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide- bound to a set of three human LIGHT domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first LIGHT domain, an inter-domain linker, a second LIGHT domain, a second inter-domain linker, and a third LIGHT domain, wherein each linker consists of 15-20 amino acids and each of the two inter- LIGHT monomer linkers comprises 3 G4S motifs.
  • the Fc- LIGHT fusion polypeptide comprises the amino acid sequences set for in SEQ ID NO:63 or a sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:63.
  • the polypeptide is a Fab-Fc- LIGHT fusion polypeptide.
  • the polypeptide is a LIGHT -antibody Fab fragment fusion polypeptide.
  • the polypeptide is an antibody- LIGHT fusion polypeptide.
  • the polypeptide is a LIGHT -albumin fusion polypeptide (e.g., an HSA- LIGHT fusion).
  • the polypeptide comprises one or more LIGHT domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • LIGHT domains linked e.g., fused
  • each LIGHT domain independently, comprises amino acid residues 59-240 of SEQ ID NO:58, or a sequence at least 85% identical thereto (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each LIGHT domain independently, comprises amino acid residues 74-240 of SEQ ID NO:58, or a sequence at least 85% identical thereto (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each LIGHT domain independently, comprises amino acid residues 74-240 of SEQ ID NO:58, or a sequence at least 85% identical thereto (e.g., 90%, 95%, 96%, 97%, 98%, or 99% identical thereto).
  • each LIGHT domain independently, consists of amino acid residues 59-240 of SEQ ID NO:58. In another embodiment, each LIGHT domain, independently, consists of amino acid residues 74-240 of SEQ ID NO:58. In another embodiment, each LIGHT domain, independently, consists of amino acid residues 59-240 of SEQ ID NO:58.
  • Individual 4-1BBL domains can be linked together and/or to an antibody Fc region, antibody, or albumin via any suitable means.
  • an Fc region or albumin can be separated from the 4-1BBL moiety by a linker.
  • each 4-1BBL domain of the 4-1BBL moiety can be separated by an inter-domain linker.
  • Individual CD70 domains can be linked together and/or to an antibody Fc region, antibody, or albumin via any suitable means.
  • an Fc region or albumin can be separated from the CD70 moiety by a linker.
  • each CD70 domain of the CD70 moiety can be separated by an inter-domain linker.
  • Individual GITRL domains can be linked together and/or to an antibody Fc region, antibody, or albumin via any suitable means.
  • an Fc region or albumin can be separated from the GITRL moiety by a linker.
  • each GITRL domain of the GITRL moiety can be separated by an inter-domain linker.
  • Individual OX40L domains can be linked together and/or to an antibody Fc region (or fragment thereof), antibody, or albumin via any suitable means.
  • an Fc region (or fragment thereof) or albumin can be separated from the OX40L moiety by a linker.
  • each OX40L domain of the OX40L moiety can be separated by an inter-domain linker.
  • Individual CD40L domains can be linked together and/or to an antibody Fc region, antibody, or albumin via any suitable means.
  • an Fc region or albumin can be separated from the CD40L moiety by a linker.
  • each CD40L domain of the CD40L moiety can be separated by an inter-domain linker.
  • Individual LIGHT domains can be linked together and/or to an antibody Fc region, antibody, or albumin via any suitable means.
  • an Fc region or albumin can be separated from the LIGHT moiety by a linker.
  • each LIGHT domain of the LIGHT moiety can be separated by an inter-domain linker.
  • each linker or inter-domain linker comprises 5-25 amino acids.
  • the linker or inter-domain linker comprises 5-10, 5-15, 5-20, 5-25, 10-15, 10-20, 10-25, 15-20, 15-25, or 20-25 amino acids.
  • the linker or inter-domain linker comprises 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.
  • the linker or inter-domain linker comprises 15-20 amino acids.
  • the linker or inter-domain linker comprises at least one, two, or three G4S motifs.
  • a G4S motif comprises four glycine residues followed by one serine residue (i. e., amino acid sequence GGGGS [SEQ ID NO: 12).
  • the linker or inter-domain linker comprises three G4S motifs.
  • compositions comprising any of the polypeptides described herein and a carrier.
  • Such compositions can be administered by any suitable means, including, but not limited to, intravenous injection.
  • methods for treating a disease (e.g., cancer or an autoimmune disease) in a human patient by administering the 4-1BBL polypeptides described herein are provided.
  • methods for treating a disease (e.g., cancer or an autoimmune disease) in a human patient by administering the CD70 polypeptides described herein are provided.
  • methods for treating a disease (e.g., cancer or an autoimmune disease) in a human patient by administering the GITRL polypeptides described herein are provided.
  • methods for treating a disease (e.g., cancer or an autoimmune disease) in a human patient by administering the OX40L polypeptides described herein are provided.
  • methods for treating a disease e.g., cancer or an autoimmune disease
  • methods for treating a disease e.g., cancer or an autoimmune disease in a human patient by administering the CD40L polypeptides described herein are provided.
  • methods for treating a disease e.g., cancer or an autoimmune disease in a human patient by administering the LIGHT polypeptides described herein are provided.
  • the polypeptides can be administered alone (e.g., as a monotherapy) or in combination with one or more additional therapeutic agents.
  • the methods described herein comprise administering a 4-1BBL polypeptide (e.g., Fc-41BBL fusion polypeptide) in combination with one or more other antineoplastic agents (e.g., other chemotherapeutics or other small molecule drugs).
  • the methods described herein comprise administering a CD70 polypeptide (e.g., Fc-CD70 fusion polypeptide) in combination with one or more other antineoplastic agents (e.g., other chemotherapeutics or other small molecule drugs).
  • the methods described herein comprise administering a GITRL polypeptide (e.g., Fc-GITRL fusion polypeptide) in combination with one or more other antineoplastic agents (e.g., other chemotherapeutics or other small molecule drugs).
  • a GITRL polypeptide e.g., Fc-GITRL fusion polypeptide
  • other antineoplastic agents e.g., other chemotherapeutics or other small molecule drugs.
  • the methods described herein comprise administering an OX40L polypeptide (e.g., Fc-OX40L fusion polypeptide) in combination with one or more other antineoplastic agents (e.g., other chemotherapeutics or other small molecule drugs).
  • the methods described herein comprise administering a CD40L polypeptide (e.g., Fc-CD40L fusion polypeptide) in combination with one or more other antineoplastic agents (e.g., other chemotherapeutics or other small molecule drugs).
  • the methods described herein comprise administering an LIGHT polypeptide (e.g., Fc- LIGHT fusion polypeptide) in combination with one or more other antineoplastic agents (e.g., other chemotherapeutics or other small molecule drugs).
  • an LIGHT polypeptide e.g., Fc- LIGHT fusion polypeptide
  • antineoplastic agents e.g., other chemotherapeutics or other small molecule drugs
  • Adjunctive or combined administration includes simultaneous administration of any of the polypeptides described herein and one or more agents in the same or different dosage form, or separate administration of the polypeptide and one or more agents (e.g. , sequential administration). Such concurrent or sequential administration preferably results in both the polypeptide and the one or more agents being simultaneously present in treated patients.
  • no more than three other antineoplastic agents are administered within the treatment cycle. In another embodiment, no more than two other antineoplastic agents are administered within the treatment cycle. In another embodiment, no more than one other antineoplastic agent is administered within the treatment cycle. In another embodiment, no other antineoplastic agent is administered within the treatment cycle.
  • kits for modifying an immune response in a subject comprising administering to the subject the polypeptides described herein, such that the immune response in the subject is modified.
  • the response is enhanced, stimulated or up-regulated.
  • methods of stimulating (activating) immune cells for cancer therapy by administering the polypeptides described herein to a patient are provided.
  • methods of maintaining T cells for adoptive cell transfer therapy are provided.
  • methods of stimulating proliferation of T cells for adoptive cell transfer therapy are provided.
  • T cells that can be enhanced stimulated with the polypeptides described herein include CD4+ T cells and CD8+ T cells.
  • the T cells can be T e ff cells, e.g., CD4+ T e ff cells, CD8+ T e ff cells, Thelper (Th) cells and T cytotoxic (T c ) cells.
  • kits comprising the polypeptides and compositions described herein.
  • the kit comprises instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS
  • Fig. 1A shows a homodimer of a 4-1BBL moiety (grey) fused to the C terminus of human IgGl Fc (white). The disulfide bonds of the hinge region and the GS linkers connecting 4-1BBL domains are also shown.
  • Fig. IB shows a homodimer of a CD70 moiety (grey) fused to the C terminus of human IgGl Fc (white). The disulfide bonds of the hinge region and the GS linkers connecting CD70 domains are also shown.
  • Fig. 1C shows a homodimer of a GITRL moiety (grey) fused to the C terminus of human IgGl Fc (white). The disulfide bonds of the hinge region and the GS linkers connecting GITRL domains are also shown.
  • Fig. ID shows a homodimer of an OX40Lmoiety (grey) fused to the C terminus of human IgGl Fc (white). The disulfide bonds of the hinge region and the GS linkers connecting OX40L domains are also shown.
  • Fig. IE shows a homodimer of a CD40L moiety (grey) fused to the C terminus of human IgGl Fc (white). The disulfide bonds of the hinge region and the GS linkers connecting CD40L domains are also shown.
  • Fig. IF shows a homodimer of a LIGHT moiety (grey) fused to the C terminus of human IgGl Fc (white). The disulfide bonds of the hinge region and the GS linkers connecting CD40L domains are also shown.
  • Fig. 2 is a graph showing the activity of Fc-sc41BBL, recombinant 41BBL, as compared to the corresponding recombinant ligand an agonistic antibody (utomilumab).
  • Fig. 3 is a graph showing antitumor activity of murine Fc-sc41BBL in the MC38 syngeneic mouse model.
  • Fig. 4 is a graph showing the activity of Fc -GITRL, recombinant GITRL, as compared to the corresponding recombinant ligand and an agonistic antibody (TRX518).
  • Fig. 5 is a graph showing the activity of Fc-OX40L, recombinant OX40L, as compared to the corresponding recombinant ligand an agonistic antibody (Medi6469).
  • Fig. 6 is a graph showing the activity of Fc-CD40L, recombinant CD40L, as compared to the corresponding recombinant ligand an agonistic antibody (CP-870893).
  • fusion polypeptides comprising a 4-1BBL moiety (e.g., which agonizes 4-1BB signaling to B and T cells).
  • exemplary fusion polypeptides include a 4-1BBL moiety (e.g., 4- 1BBL monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-4-lBBL fusion), Fab-Fc (Fab-Fc-4-lBBL fusion), Fab fragment (4-lBBL-antibody Fab fragment fusion), antibody (4-lBBL-antibody fusion) or albumin (4-lBBL-albumin fusion).
  • the polypeptide comprises one or more 4-1BBL domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • an albumin e.g., HSA
  • fusion polypeptides comprising a CD70 moiety (e.g., which agonizes CD27 signaling).
  • exemplary fusion polypeptides include a CD70 moiety (e.g., CD70 monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-CD70 fusion), Fab-Fc (Fab-Fc-CD70 fusion), Fab fragment (CD70 -antibody Fab fragment fusion), antibody (CD70 -antibody fusion) or albumin (CD70 -albumin fusion).
  • the polypeptide comprises one or more CD70 domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • CD70 domains linked e.g., fused
  • methods of treating disease e.g. , cancer or autoimmune disease
  • fusion polypeptides comprising a GITRL moiety (e.g., which agonizes GITR signaling).
  • exemplary fusion polypeptides include a GITRL moiety (e.g., GITRL monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-GITRL fusion), Fab-Fc (Fab- Fc-GITRL fusion), Fab fragment (GITRL-antibody Fab fragment fusion), antibody (GITRL-antibody fusion) or albumin (GITRL-albumin fusion).
  • the polypeptide comprises one or more GITRL domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • GITRL domains linked e.g., fused
  • fusion polypeptides comprising an OX40L moiety (e.g., which agonizes OX40 signaling).
  • OX40L moiety e.g., OX40L monomer, dimer, or trimer
  • Fab-Fc Fab- Fc-OX40L fusion
  • Fab fragment OX40L-antibody Fab fragment fusion
  • antibody OX40L-antibody fusion
  • albumin OX40L-albumin fusion
  • the polypeptide comprises one or more OX40L domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • OX40L domains linked e.g., fused
  • fusion polypeptides comprising a CD40L moiety (e.g., which agonizes CD40 signaling).
  • exemplary fusion polypeptides include a CD40L moiety (e.g., CD40L monomer, dimer, or trimer) linked to an antibody Fc region or fragment thereof (Fc-CD40L fusion), Fab-Fc (Fab- Fc-CD40L fusion), Fab fragment (CD40L-antibody Fab fragment fusion), antibody (CD40L-antibody fusion) or albumin (CD40L-albumin fusion).
  • the polypeptide comprises one or more CD40L domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • CD40L domains linked e.g., fused
  • fusion polypeptides comprising an LIGHT moiety (e.g., which agonizes TNFSF14 signaling).
  • LIGHT moiety e.g., LIGHT monomer, dimer, or trimer
  • Fc- LIGHT fusion Fab-Fc
  • Fab fragment LIGHT-antibody Fab fragment fusion
  • antibody LIGHT-antibody fusion
  • albumin LIGHT-albumin fusion
  • the polypeptide comprises one or more LIGHT domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA). Also provided herein are methods of treating disease (e.g. , cancer or autoimmune disease) in a human patient by administering to the patient an effective amount of the Fc- LIGHT fusion polypeptides described herein.
  • disease e.g. , cancer or autoimmune disease
  • the term "subject” or “patient” is a human patient (e.g., a patient having cancer).
  • 4-1BB also known as CD137 or “tumor necrosis factor superfamily, member 9” (TNFRSF9)
  • TNFRSF9 tumor necrosis factor superfamily, member 9
  • This receptor contributes to the clonal expansion, survival, and development of T cells. It can also induce proliferation in peripheral monocytes, enhance T cell apoptosis induced by TCR/CD3 triggered activation, and regulate CD28 co- stimulation to promote Thl cell responses. The expression of this receptor is induced by lymphocyte activation.
  • 4- IBB was first identified in mice by a modified differential screening procedure (see Kwon BS, Weissman SM, Proc. Natl. Acad. Sci.
  • 4-1BB was found to map to murine chromosome 4 at the 75.5 cM position (see Cheuk A. et al., Cancer Gene Therapy (2004) 11, 215-226). In mice, the 4-1BB gene spans
  • Murine 4-1BB amino acid sequence (1-256) (NP_001070977.1) is: [00132] MGN CYNVVVIVLLLVGCEKVGAVQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIG GQPNCNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKDCRPGQELTKQGCKTCSL GTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQ VLTLFLALTSALLLALIFITLLFSVLKWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGG GGYEL (SEQ ID N0: 1)
  • the human homologue of 4-1BB was cloned from activated human T-cell leukemia virus type 1 -transformed human T-lymphocytes library (Schwarz H, et al, Gene. 1993;134:295).
  • Human 4- 1BB resides on chromosome lp3613 in a cluster of related genes including, TNFR2, CD30, OX40 and TAMP/Apo3, which have been shown to be mutated in several malignancies (see Cheuk A. et al., Cancer Gene Therapy (2004) 11, 215-226 and Schwarz H, et al, Biochem. Biophys. Res. Commun.
  • the human 4-1BB contains 255 amino acids with two potential N-linked glycosylation sites (see Alderson MR, et al , Eur. J. Immunol. 199 >4;24:2219-2227).
  • the molecular weight of the protein was calculated to be 27 kDal5 and was shown to be 60% identical to murine 4-1BB (see Cheuk A. et al, Cancer Gene Therapy (2004) 11, 215-226).
  • In the cytoplasmic domain five regions of amino acid sequences were conserved between mice and human (see Alderson MR, et al. , Eur. J. Immunol. 1994;24:2219-2227).
  • 4-1BBL (also referred to as “CD137L” or “TNFSF9”) refers to member of the TNF superfamily that binds 4- IBB.
  • the presence of a ligand for 4- IBB was first confirmed in the EL4 cell line through its binding with a fusion 4-lBB/Fc protein.
  • the 4-1BBL gene was then cloned through the screening of an ECl cDNA expression library (see Goodwin RG, et al, Eur. J. Immunol. 1993;23:2631- 2641).
  • Murine 4-1BBL consists of 309 amino acid polypeptide.
  • Murine 4-1BBL amino acid sequence (1-309) (NP_033430) is:
  • 4-lBBL binds to its receptor 4-1BB (CD 137/TNFRSF9) on activated CD8+ and CD4+ T cells, B cells, NK cells, and dendritic cells.
  • 4-1BB CD 137/TNFRSF9
  • 4-lBBL has been shown to reactivate anergic T lymphocytes and to promote T lymphocyte proliferation.
  • 41BBL-receptor signaling induces maturation of dendritic cells and production of inflammatory cytokines.
  • 41BBL is expressed on carcinoma cell lines and 4-1BB signaling is thought to be a therapeutic target for the treatment of various diseases (e.g. , cancer and autoimmune disease).
  • the 4-lBBL fusion polypeptide is an Fc-4-lBBL fusion polypeptide.
  • the 4-lBBL fusion polypeptide is a Fab-Fc-4-lBBL fusion polypeptide.
  • the 4-lBBL fusion polypeptide is an HSA-4-1BBL fusion polypeptide.
  • Suitable human serum albumin (HSA) moieties for use in such an HSA-41BBL fusion polypeptide include native and mutant HSAs disclosed in U.S. patent Nos. 8,927,694 and 8,877,687.
  • CD27 refers to a receptor that is a member of the TNF-receptor superfamily, which binds to ligand CD70 (Ranheim, E.A. et al. (1995) Blood, 85(12):3556-65).
  • CD27 typically exists as a glycosylated, type I transmembrane protein, frequently in the form of homodimers with a disulfide bridge linking the two monomers.
  • the disulfide bridge is in the extracellular domain close to the membrane (Camerini et al. (1991) J. Immunol., 147:3165-69).
  • CD27 may also be expressed in a soluble form (see, e.g., van Oers, M.H. et al. (1993) Blood 82(11):3430-6 and Loenen, W.A. et al. (1992) Eur. J. Immunol, 22:447).
  • CD27 is expressed on mature thymocytes, on most CD4+ and CD8+ peripheral blood T cells, natural killer cells and B cells (Kobata, T. et al. (1995) Proc. Natl. Acad. Sci. USA, 92(24): 11249-53). CD27 is also highly expressed on B cell non-Hodgkin's lymphomas and B cell chronic lymphocytic leukemias (Ranheim, E.A. et al. (1995) Blood, 85(12):3556-65).
  • CD27 protein soluble CD27 protein
  • CMV cytomegalovirus
  • sarcoidosis sarcoidosis
  • multiple sclerosis multiple sclerosis
  • B-cell chronic lymphocytic leukemia Loenen, W.A. et al. (1992) Eur. J. Immunol, 22:447).
  • CD27 includes any variants or isoforms of CD27 which are naturally expressed by cells (e.g., human CD27 deposited with GENBANK® having accession no. AAH12160.1). CD27 or any variants and isoforms thereof, may either be isolated from cells or tissues that naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.
  • the murine CD27 amino acid sequence (isoform a) (1-250) (NP_001028298) is:
  • the human CD27 amino acid sequence (1-260) (NP_001233) is:
  • CD70 also referred to as “CD70 molecule”, “CD27L”,
  • CD27LG refers to the ligand for CD27 (see, for example, Bowman MR et al., J.
  • CD70 is a type II transmembrane protein that belongs to the tumor necrosis factor (TNF) ligand family. It is a surface antigen on activated T and B lymphocytes that induces proliferation of co-stimulated T cells, enhances the generation of cytolytic T cells, and contributes to T cell activation. It has also been suggested that CD70 plays a role in regulating B-cell activation, cytotoxic function of natural killer cells, and immunoglobulin synthesis (Hintzen RQ et al. , J. Immunol. 1994 Feb 15; 152(4): 1762-73). [00151] The murine CD70 amino acid sequence (1-195) (NP_035747) is:
  • the human CD70 amino acid sequence (1-193) (NP_001243) is:
  • the CD70 fusion polypeptide is an Fc-CD70 fusion polypeptide.
  • the CD70 fusion polypeptide is a Fab-Fc-CD70 fusion polypeptide.
  • the CD70 fusion polypeptide is an HSA-CD70 fusion polypeptide.
  • Suitable human serum albumin (HSA) moieties for use in such an HSA-CD70 fusion polypeptide include native and mutant HSAs disclosed in U.S. patent Nos. 8,927,694 and 8,877,687.
  • GITR GITR ligand
  • GITR is also referred to as tumor necrosis factor receptor superfamily, member 18 (TNFRSF18), AITR and CD357 (see, e.g., Knee DA, Hewes B, Brogdon JL, Eur. J. Cancer. 2016 Nov;67: 1-10).
  • TNFRSF18 tumor necrosis factor receptor superfamily, member 18
  • AITR AITR
  • CD357 see, e.g., Knee DA, Hewes B, Brogdon JL, Eur. J. Cancer. 2016 Nov;67: 1-10).
  • GITR includes any variants or isoforms of GITR which are naturally expressed by cells. GITR or any variants and isoforms thereof, may either be isolated from cells or tissues which naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.
  • Murine GITR isoform 1 (Accession No. NP 33426.1; SEQ ID NO: 22):
  • Murine GITR isoform 2 (Accession No. NP 68820.1; SEQ ID NO: 23):
  • Variant 1 (Accession No. NP_004186; SEQ ID NO: 25) consists of 241 amino acids and represents the longest transcript. It contains an extra coding segment that leads to a frame shift, compared to variant 2. The resulting protein (isoform 1) contains a distinct and shorter C-terminus, as compared to isoform 2.
  • Variant 2 (Accession No. NP_683699; SEQ ID NO: 26) encodes the longest protein (isoform 2), consisting of 255 amino acids, and is soluble.
  • Variant 3 (Accession No.
  • NP_683700; SEQ ID NO: 27 contains an extra coding segment that leads to a frame shift, compared to variant 2.
  • the resulting protein (isoform 3) contains a distinct and shorter C- terminus, as compared to isoform 2, and consists of 234 amino acids.
  • Human GITR isoform 1 (Accession No. NP 004186; SEQ ID NO: 22; encoded by the nucleotide sequence having Accession No. NM_004195.2):
  • Human GITR isoform 2 (Accession No. NP 683699.1; SEQ ID NO: 23; encoded by the nucleotide sequence having Accession No. NM_148901.1):
  • Human GITR isoform 3 (Accession No. NP 683700.1; SEQ ID NO: 24; encoded by the nucleotide sequence having Accession No. NM_148902.1):
  • the signal sequence of isoforms 1-3 corresponds to amino acids 1-25.
  • the mature isoforms 1, 2 and 3 consist of amino acids 26 to 241, 255 or 234, respectively.
  • the extracellular domain of mature GITR consists of amino acids 26-162 and has the amino acid sequence:
  • GNKTHNAVCVPGSPPAEP (SEQ ID NO: 28)
  • GITRL also referred to as "GITR ligand," “GITR7L,” or “CD 154” refers to the ligand for GITR (see, for example, Schonbeck and Libby (2001) Cell Mol Life Sci, 58(1):4— 43). GITRL is primarily expressed on activated T cells and is a member of the TNF superfamily of molecules. It binds to GITR on antigen-presenting cells (APC), which leads to many effects depending on the target cell type (Parham, Peter (2004). The Immune System (2 nd ed.). Garland Science. Pp. 169-173).
  • GITR-L is expressed at low levels in antigen-presenting cells (e.g., B cells, dendritic cells), but is transiently upregulated in these cells upon activation, e.g., by viral infection (Suvas et al, J Virol. 2005;79: 11935-42).
  • antigen-presenting cells e.g., B cells, dendritic cells
  • Murine GITR-L protein sequence (Accession No. NP_899247; SEQ ID NO: 24):
  • the GITRL fusion polypeptide is an Fc-GITRL fusion polypeptide.
  • the GITRL fusion polypeptide is a Fab-Fc-GITRL fusion polypeptide.
  • the GITRL fusion polypeptide is an HSA-GITRL fusion polypeptide.
  • Suitable human serum albumin (HSA) moieties for use in such an HSA-GITRL fusion polypeptide include native and mutant HSAs disclosed in U.S. patent Nos. 8,927,694 and 8,877,687.
  • OX40 and its binding partner, OX40L (CD252), are members of the TNFR/TNF superfamily and are expressed on activated CD4 and CD8 T cells as well as a number of other lymphoid and non-lymphoid cells (see Michael Croft et al, Immunol. Rev. 2009 May; 229(1): 173-191).
  • OX40 is also referred to as tumor necrosis factor receptor superfamily, member 4 (TNFRSF4), ACT35, IMD16, TXGP1L, and CD134.
  • Costimulatory signals from OX40 to a conventional T cell promote division and survival, augmenting the clonal expansion of effector and memory populations as they are being generated to antigen.
  • OX40 additionally suppresses the differentiation and activity of Treg, further amplifying this process.
  • OX40 and OX40L also regulate cytokine production from T cells, antigen- presenting cells, NK cells, and NKT cells, and modulate cytokine receptor signaling.
  • the term "OX40" includes any variants or isoforms of OX40 which are naturally expressed by cells. OX40 or any variants and isoforms thereof, may either be isolated from cells or tissues which naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.
  • Murine OX40 protein sequence (Accession No. NP_035789; SEQ ID NO:36):
  • OX40L The ligand of OX40 (also known as "OX40L”, “CD252”, and “TNFSF4") belongs to the TNF superfamily and was first identified as a protein on HTLV-I transformed cells (see Tanaka Y, et al, Int. J. Cancer, 1985;36:549-55 and Miura S, et al., Mol. Cell Biol, 1991; 11: 1313-25)) and later found to bind OX40 (see Godfrey WR, et al, J. Exp. Med. 1994; 180:757-62 and Baum PR, et al, EMBO J. , 1994; 13:3992-4001).).
  • OX40L is also not constitutively expressed, but can be induced on professional antigen-presenting cells (APC) such as B cells (Stuber E, et al, Immunity, 1995;2:507-21), dendritic cells (Ohshima Y, et al, J. Immunol, 1997; 159:3838-48) and macrophages (Weinberg AD, et al, J. Immunol, 1999; 162: 1818-26), in line with its action in controlling the extent of T cell priming following recognition of antigen (Gramaglia I, et al, J. Immunol, 1998; 161 :6510-7 and Gramaglia I, et al, J. Immunol. 2000;165:3043-50).
  • APC professional antigen-presenting cells
  • B cells Ster E, et al, Immunity, 1995;2:507-21
  • dendritic cells Ohshima Y, et al, J. Immunol, 1997; 159:
  • T cells can also express OX40L (Takasawa N, et al, Jpn J. Cancer Res. , 2001;92:377-82) that is functional during T cell-T cell interactions, creating an additional mechanism to further amplify T cell responsiveness (Soroosh P, et al, J. Immunol. 2006; 176:5975-87).
  • Murine OX40-L protein sequence (Accession No. NP_033478; SEQ ID NO:37):
  • the OX40L fusion polypeptide is an Fc-OX40L fusion polypeptide.
  • the OX40L fusion polypeptide is a Fab-Fc-OX40L fusion polypeptide.
  • the OX40L fusion polypeptide is an HSA-OX40L fusion polypeptide.
  • Suitable human serum albumin (HSA) moieties for use in such an HSA-OX40L fusion polypeptide include native and mutant HSAs disclosed in U.S. patent Nos. 8,927,694 and 8,877,687.
  • CD40 refers to a receptor that is a member of the TNF -receptor superfamily, which binds to ligand CD40L (also referred to as CD154).
  • CD40 is mediates a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, and memory B cell development.
  • the term "CD40” includes any variants or isoforms of CD40 which are naturally expressed by cells (e.g., human CD40 isoform 1 deposited with GENBANK® having accession no. NP_001241). CD40 or any variants and isoforms thereof, may either be isolated from cells or tissues that naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.
  • Murine CD40 amino acid sequence (isoform 1) (1-289) (NP_035741) is:
  • CD40L refers to the ligand for CD40 (see, for example, Schonbeck and Libby (2001) CellMol Life Sci, 58(1):4— 43). CD40L is primarily expressed on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen-presenting cells (APC), which leads to many effects depending on the target cell type (Parham, Peter (2004). The Immune System (2 nd ed.). Garland Science. Pp. 169-173).
  • APC antigen-presenting cells
  • Murine CD40L amino acid sequence (1-260) (NP_035746.2) is:
  • the CD40L fusion polypeptide is an Fc-CD40L fusion polypeptide.
  • the CD40L fusion polypeptide is a Fab-Fc-CD40L fusion polypeptide.
  • the CD40L fusion polypeptide is an HSA-CD40L fusion polypeptide.
  • Suitable human serum albumin (HSA) moieties for use in such an HSA-CD40L fusion polypeptide include native and mutant HSAs disclosed in U.S. patent Nos. 8,927,694 and 8,877,687.
  • TNFRSF14 and its ligand, LIGHT (TNFSF14), are secreted proteins of the TNF superfamily. LIGHT has been shown to stimulate the proliferation of T cel ls, and trigger a poptosis of va rious tu mor cel ls.
  • Extracellular domain of mouse LIGHT (TNFSF 14) residues 59-239 of full length is:
  • Extracellar domain of mouse LIGHT (TNFSF14), residues 72-239 of full length is: [00208] DGGKGSWEKLIQDQRSHQANPAAHLTGANASLIGIGGPLLWETRLGLAFLRGLTYH
  • NSSRVWWDSSFLGGVVHLEAGEEVVVRVPGNRLVRPRDGTRSYFGAFMV SEQ ID NO:60.
  • Extracellular domain of human LIGHT (TNFSF14) (59-240) of full length is:
  • RVWWD S SFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRS YFGAFMV SEQ ID NO:61.
  • Extracellular domain of human LIGHT (TNFSF14) (residues 74-240) of full length is:
  • RVWWD S SFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRS YFGAFMV SEQ ID NO:62.
  • VRVLDERLVRLRDGTRSYFGAFMV (SEQ ID NO: 63).
  • Mouse IgG2a Fc D265A/N297A Fc and mouse single chain LIGHT amino acid sequences 1- 781 is:
  • Human single chain LIGHT amino acid sequence 1-5 13 is:
  • Mouse single chain LIGHT amino acid sequence 1-534 is:
  • GGVVHLEAGEEVVVRVPGNRLVRPRDGTRSYFGAFMV (SEQ ID NO: 66).
  • the LIGHT fusion polypeptide is an Fc-LIGHT fusion polypeptide.
  • the LIGHT fusion polypeptide is a Fab-Fc-LIGHT fusion polypeptide.
  • the LIGHT fusion polypeptide is an HSA-LIGHT fusion polypeptide.
  • Suitable human serum albumin (HSA) moieties for use in such an HSA-LIGHT fusion polypeptide include native and mutant HSAs disclosed in U.S. patent Nos. 8,927,694 and 8,877,687.
  • Peptide refers to any peptide comprising two or more amino acids joined by peptide bonds or modified peptide bonds (e.g., peptide isosteres).
  • Peptides can contain amino acids other than the 20 naturally occurring nucleic acid encoded amino acids, and include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given peptide. Also, a given polypeptide can contain many types of modifications.
  • Polypeptides can be branched as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural posttranslational processes or can be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • isolated protein or "isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • variant as used herein is defined as a modified or altered form of a wildtype sequence, e.g. where one or more amino acids may be replaced by other amino acid(s) or non-amino acid(s) which do not substantially affect function.
  • the variant may contain an altered side chain for at least one amino acid residue.
  • antigen as used herein is defined as an entity which elicits an immune system response.
  • the term herein may be abbreviated to "Ag.”
  • immune cell refers to cells that play a role in the immune response, including lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • An "immune response” refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them.
  • An immune response is mediated by the action of a cell of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8
  • an “immunomodulator” or “immunoregulator” refers to an agent, e.g., a component of a signaling pathway, that may be involved in modulating, regulating, or modifying an immune response.
  • “Modulating,” “regulating,” or “modifying” an immune response refers to any alteration in a cell of the immune system or in the activity of such cell (e.g., an effector T cell).
  • Such modulation includes stimulation or suppression of the immune system which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes which can occur within the immune system.
  • Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in a tumor microenvironment.
  • the immunomodulator is located on the surface of a T cell.
  • immunomodulatory target or “immunoregulatory target” is an immunomodulator that is targeted for binding by, and whose activity is altered by the binding of, a substance, agent, moiety, compound or molecule.
  • Immunomodulatory targets include, for example, receptors on the surface of a cell
  • immunomodulatory receptors and receptor ligands
  • immunomodulatory ligands receptor ligands
  • T cell-mediated response refers to a response mediated by T cells, including effector T cells (e.g. , CD8 + cells) and helper T cells (e.g. , CD4 + cells).
  • T cell mediated responses include, for example, T cell cytotoxicity and proliferation.
  • cytotoxic T lymphocyte (CTL) response refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8 + T cells.
  • inhibitor means to reduce by a measurable amount.
  • Inhibitors and “antagonists,” or “activators” and “agonists,” refer to inhibitory or activating molecules, respectively, e.g. , for the activation of, e.g. , a ligand, receptor, cofactor, a gene, cell, tissue, or organ.
  • a modulator of, e.g. , a gene, a receptor, a ligand, or a cell is a molecule that alters an activity of the gene, receptor, ligand, or cell, where activity can be activated, inhibited, or altered in its regulatory properties.
  • the modulator may act alone, or it may use a cofactor, e.g. , a protein, metal ion, or small molecule.
  • Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate, e.g. , a gene, protein, ligand, receptor, or cell.
  • Activators are compounds that increase, activate, facilitate, enhance activation, sensitize, or up regulate, e.g. , a gene, protein, ligand, receptor, or cell.
  • An inhibitor may also be defined as a compound that reduces, blocks, or inactivates a constitutive activity.
  • An "agonist” is a compound that interacts with a target to cause or promote an increase in the activation of the target (e.g., a polypeptide which agonizes (promotes) 4-1BB signaling to B and T cells).
  • An "antagonist” is a compound that opposes the actions of an agonist.
  • An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist.
  • An antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g. , a target receptor, even where there is no identified agonist.
  • % identical refers to two or more nucleic acid or polypeptide sequences or subsequences that are the same (100% identical) or have a specified percentage of nucleotide or amino acid residues that are the same, when the two sequences are aligned for maximum correspondence and compared. To align for maximum correspondence, gaps may be introduced into one of the sequences being compared. The amino acid residues or nucleotides at corresponding positions are then compared and quantified. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the sequences are identical at that position.
  • the two sequences are the same length.
  • the determination that one sequence is a measured % identical with another sequence can be determined using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm utilized for such comparison of two sequences is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • comparing sequences for % identity for purposes of this specification utilize the ALIGN program e.g. , for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additional algorithms for sequence analysis are well known in the art and many are available online.
  • the 4-lBBL polypeptides which comprise a 4-lBBL moiety.
  • the 4-lBBL moiety comprises one 4-lBBL domain (monomer).
  • the 4-lBBL moiety comprises two 4-lBBL domains (dimer).
  • the moiety comprises three 4-lBBL domains (trimer).
  • the moiety comprises the amino acid sequence of SEQ ID NO: 10.
  • the polypeptide comprises a 4-lBBL moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • the 4-lBBL domain comprises full-length human 4-lBBL (i.e., amino acid residues 1-254 of SEQ ID NO:4).
  • the 4-lBBL domain comprises a portion of the amino acid sequence set forth in SEQ ID NO:4.
  • the 4-lBBL domain comprises amino acid residues 30-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of amino acid residues 30-254 of SEQ ID NO:4.
  • the 4-lBBL domain comprises amino acid residues 50-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of amino acid residues 50-254 of SEQ ID NO:4.
  • the 4-lBBL domain comprises amino acid residues 71-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of amino acid residues 71-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain comprises amino acid residues 85-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of amino acid residues 85-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 31-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 32-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 33-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 34-254 of SEQ ID NO:4. In another embodiment, the 4- 1BBL domain consists of or comprises amino acid residues 35-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 36-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 37-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 38- 254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 39-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 40-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 41-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 42-254 of SEQ ID NO:4.
  • the 4- 1BBL domain consists of or comprises amino acid residues 43-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 44-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 45-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 46- 254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 47-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 48-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 49-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 50-254 of SEQ ID NO:4. In another embodiment, the 4- 1BBL domain consists of or comprises amino acid residues 51-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 52-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 53-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 54- 254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 55-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 56-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 57-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 58-254 of SEQ ID NO:4. In another embodiment, the 4- 1BBL domain consists of or comprises amino acid residues 59-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 60-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 61-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 62- 254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 63-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 64-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 65-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 66-254 of SEQ ID NO:4.
  • the 4- 1BBL domain consists of or comprises amino acid residues 67-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 68-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 69-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 70- 254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 71-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 72-254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 73-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 74-254 of SEQ ID NO:4. In another embodiment, the 4- 1BBL domain consists of or comprises amino acid residues 75-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 76-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 77-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 78- 254 of SEQ ID NO:4.
  • the 4-lBBL domain consists of or comprises amino acid residues 79-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 80-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 81-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 82-254 of SEQ ID NO:4. In another embodiment, the 4- 1BBL domain consists of or comprises amino acid residues 83-254 of SEQ ID NO:4. In another embodiment, the 4-lBBL domain consists of or comprises amino acid residues 84-254 of SEQ ID NO:4.
  • the 4-lBBL domain comprises or consists of a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N-terminus at any one of amino acid residues 31-84 of SEQ ID NO:4 and a C terminus at any one of amino acid residues 234-254 of SEQ ID NO:4.
  • the 4-lBBL domain comprises no more than about 200 amino acid residues, preferably no more than about 150 amino acid residues, and more preferably no more than about 100 amino acid residues. In another embodiment, the 4-lBBL domain consists of no more than about 200 amino acid residues, preferably no more than about 150 amino acid residues, and more preferably no more than about 100 amino acid residues. [00241] In another embodiment, the 4-lBBL domain comprises the amino acid sequence of SEQ ID NO: 5 or a portion thereof. In another embodiment, the 4-lBBL domain consists of the amino acid sequence of SEQ ID NO: 5. In another embodiment, the 4-lBBL domain comprises the amino acid sequence of 6 or a portion thereof.
  • the 4-lBBL domain consists of the amino acid sequence of SEQ ID NO:6. In another embodiment, the 4-lBBL domain comprises the amino acid sequence of SEQ ID NO: 7 or a portion thereof. In another embodiment, the 4-lBBL domain consists of the amino acid sequence of SEQ ID NO: 7.
  • the 4-lBBL domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein.
  • the 4-lBBL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-254 of SEQ ID NO:4.
  • the 4-lBBL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 30-254, 50-254, 71-254, or 85-254 of SEQ ID NO:4.
  • the 4-lBBL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, 6, or 7.
  • the 4-lBBL domain comprises an amino acid sequence at least 95% identical to residues 30-254, 50-254, 71-254, or 85-254 of SEQ ID NO:4.
  • the 4-lBBL domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 5, 6, or 7.
  • the fusion polypeptide comprises a set of three human 4-lBBL domains to form a single-chain 4-lBBL trimer.
  • the single-chain 4-lBBL trimer comprises, in amino- to carboxyl-terminal order, a first 4-lBBL domain, an inter-domain linker, a second 4-lBBL domain, a second inter-domain linker, and a third 4-lBBL domain.
  • each linker consists of 15-20 amino acids.
  • each of the two inter-41BBL monomer linkers comprises 3 G4S domains.
  • the 4-lBBL moiety binds to its receptor 4-lBB.
  • the 4-lBBL moiety binds to 4-lBB on activated CD8+ and CD4+ T cells, B cells, NK cells, and dendritic cells.
  • the 4-lBBL moiety activates or agonizes 4-lBB signaling.
  • the 4-lBBL moiety reactivates anergic T lymphocytes and/or promotes T lymphocyte proliferation.
  • the 4-lBBL moiety induces maturation of dendritic cells.
  • the 4-lBBL moiety induces production of inflammatory cytokines.
  • the 4-lBBL moiety induces clonal expansion, survival, and/or development of T Cells.
  • the 4-lBBL moiety induces proliferation in peripheral monocytes.
  • the 4-lBBL moiety regulates CD28 co-stimulation to promote Thl cell responses.
  • CD70 polypeptides which comprise a CD70 moiety.
  • the CD70 moiety comprises one CD70 domain (monomer). In another embodiment, the CD70 moiety comprises two CD70 domains (dimer). In another embodiment, the moiety comprises three CD70 domains (trimer). In another embodiment, the moiety comprises the amino acid sequence of SEQ ID NO:20. In another embodiment, the polypeptide comprises a CD70 moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • the CD70 domain comprises full-length human CD70 (i.e., amino acid residues 1-193 of SEQ ID NO: 16). In another embodiment, the CD70 domain comprises a portion of the amino acid sequence set forth in SEQ ID NO: 16. In another embodiment, the CD70 domain comprises amino acid residues 20-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of amino acid residues 20-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain comprises amino acid residues 39-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of amino acid residues 39-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain comprises amino acid residues 60-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of amino acid residues 60-193 of SEQ ID NO: 16.
  • the CD70 domain consists of or comprises amino acid residues 21- 193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 22-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 23-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 24-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 25-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 26-193 of SEQ ID NO: 16.
  • the CD70 domain consists of or comprises amino acid residues 27-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 28-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 29- 193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 30-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 31-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 32-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 33-193 of SEQ ID NO: 16.
  • the CD70 domain consists of or comprises amino acid residues 34-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 35-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 36-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 37- 193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 38-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 39-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 40-193 of SEQ ID NO: 16.
  • the CD70 domain consists of or comprises amino acid residues 41-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 42-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 43-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 44-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 45- 193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 46-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 47-193 of SEQ ID NO: 16.
  • the CD70 domain consists of or comprises amino acid residues 48-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 49-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 50-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 51-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 52-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 53- 193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 54-193 of SEQ ID NO: 16.
  • the CD70 domain consists of or comprises amino acid residues 55-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 56-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 57-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 58-193 of SEQ ID NO: 16. In another embodiment, the CD70 domain consists of or comprises amino acid residues 59-193 of SEQ ID NO: 16.
  • the CD70 domain comprises or consists of a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N- terminus at any one of amino acid residues 21-59 of SEQ ID NO: 16 and a C terminus at any one of amino acid residues 173-193 of SEQ ID NO: 16.
  • the CD70 domain comprises no more than about 175 amino acid residues, preferably no more than about 150 amino acid residues, more preferably no more than about 125 amino acid residues, and even more preferably no more than about 100 amino acid residues.
  • the CD70 domain consists of no more than about 175 amino acid residues, preferably no more than about 150 amino acid residues, more preferably no more than about 125 amino acid residues, and even more preferably no more than 100 amino acid residues.
  • the CD70 domain comprises the amino acid sequence of SEQ ID NO: 17 or a portion thereof. In another embodiment, the CD70 domain consists of the amino acid sequence of SEQ ID NO: 17.
  • the CD70 domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein.
  • the CD70 domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-193 of SEQ ID NO: 16.
  • the CD70 domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 39-193 of SEQ ID NO: 16.
  • the CD70 domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.
  • the CD70 domain comprises an amino acid sequence at least 95% identical to residues 39-193 of SEQ ID NO: 16.
  • the CD70 domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 17.
  • the fusion polypeptide comprises a set of three human CD70 domains to form a single-chain CD70 trimer.
  • the single-chain CD70 trimer comprises, in amino- to carboxyl-terminal order, a first CD70 domain, an inter-domain linker, a second CD70 domain, a second inter-domain linker, and a third CD70 domain.
  • each linker consists of 15-20 amino acids.
  • each of the two inter-CD70 monomer linkers comprises 3 G4S domains.
  • the CD70 moiety binds to its receptor CD27. In another embodiment, the CD70 moiety binds to CD27 on naive T cells, B cells, and NK cells. In another embodiment, the CD70 moiety activates or agonizes CD27 signaling. In another embodiment, the CD70 moiety promotes proliferation of naive CD4+ and CD8+ T cells and the generation of effector cells. In another embodiment, the CD70 moiety reactivates anergic T lymphocytes and/or promotes T lymphocyte proliferation. In another embodiment, the CD70 moiety induces maturation of dendritic cells. In another embodiment, the CD70 moiety induces production of inflammatory cytokines. In another embodiment, the CD70 moiety induces clonal expansion, survival, and/or development of T Cells.
  • GITRL polypeptides which comprise a GITRL moiety.
  • the GITRL moiety comprises one GITRL domain (monomer).
  • the GITRL moiety comprises two GITRL domains (dimer).
  • the moiety comprises three GITRL domains (trimer).
  • the moiety comprises the amino acid sequence of SEQ ID NO: 34.
  • the polypeptide comprises a GITRL moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • the GITRL domain comprises full-length human GITRL (i. e., amino acid residues 1-199 of SEQ ID NO: 29). In another embodiment, the GITRL domain consists of full- length human GITRL (i.e., amino acid residues 1-199 of SEQ ID NO: 29). In another embodiment, the GITRL domain comprises a portion of the amino acid sequence set forth in SEQ ID NO: 29. In another embodiment, the GITRL domain comprises amino acid residues 50-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of amino acid residues 50-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain comprises amino acid residues 72-199 of SEQ ID NO: 29.
  • the GITRL domain consists of amino acid residues 72-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain comprises amino acid residues 74-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of amino acid residues 74-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain comprises of amino acid residues 90-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of amino acid residues 90-199 of SEQ ID NO: 29.
  • the GITRL domain consists of or comprises amino acid residues 51- 199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 52-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 53-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 54-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 55-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 56-199 of SEQ ID NO: 29.
  • the GITRL domain consists of or comprises amino acid residues 57-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 58-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 59- 199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 60-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 61-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 62-199 of SEQ ID NO: 29.
  • the GITRL domain consists of or comprises amino acid residues 63-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 64-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 65-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 66-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 67- 199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 68-199 of SEQ ID NO: 29.
  • the GITRL domain consists of or comprises amino acid residues 69-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 70-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 71-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 72-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 73-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 74-199 of SEQ ID NO: 29.
  • the GITRL domain consists of or comprises amino acid residues 75- 199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 76-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 77-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 78-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 79-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 80-199 of SEQ ID NO: 29.
  • the GITRL domain consists of or comprises amino acid residues 81-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 82-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 83- 199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 84-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 85-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 86-199 of SEQ ID NO: 29.
  • the GITRL domain consists of or comprises amino acid residues 87-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 88-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain consists of or comprises amino acid residues 89-199 of SEQ ID NO: 29.
  • the GITRL domain comprises or consists of a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N- terminus at any one of amino acid residues 51-89 of SEQ ID NO: 29 and a C terminus at any one of amino acid residues 179-199 of SEQ ID NO: 29.
  • the GITRL domain comprises no more than about 150 amino acid residues, preferably no more than about 125 amino acid residues, and more preferably no more than about 100 amino acid residues. In another embodiment, the GITRL domain consists of no more than about 150 amino acid residues, preferably no more than about 125 amino acid residues, and more preferably no more than about 100 amino acid residues.
  • the GITRL domain comprises the amino acid sequence of SEQ ID NO: 30 or a portion thereof. In another embodiment, the GITRL domain consists of the amino acid sequence of SEQ ID NO: 30. In another embodiment, the GITRL domain comprises the amino acid sequence of SEQ ID NO: 31 or a portion thereof. In another embodiment, the GITRL domain consists of the amino acid sequence of SEQ ID NO: 31.
  • the GITRL domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein.
  • the GITRL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-199 of SEQ ID NO: 29.
  • the GITRL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 72-199 of SEQ ID NO: 29.
  • the GITRL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 74-199 of SEQ ID NO: 29. In another embodiment, the GITRL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30 or 10. In another embodiment, the GITRL domain comprises an amino acid sequence at least 95% identical to amino acid residues 72-199 or amino acid residues 74-199 of SEQ ID NO: 29. In a particular embodiment, the GITRL domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 29.
  • the fusion polypeptide comprises a set of three human GITRL domains to form a single-chain GITRL trimer.
  • the single-chain GITRL trimer comprises, in amino- to carboxyl-terminal order, a first GITRL domain, an inter-domain linker, a second GITRL domain, a second inter-domain linker, and a third GITRL domain.
  • each linker consists of 15-20 amino acids.
  • each of the two inter-GITRL monomer linkers comprises 3 G4S domains.
  • the GITRL moiety binds to its receptor GITR. In another embodiment, the GITRL moiety binds to GITR on regulatory T cells (Tregs), memory T cells, NK cells, macrophages, and/or dendritic cells. In another embodiment, the GITRL moiety activates or agonizes GITR signaling (e.g., which in turn inhibits Treg suppression function and promotes effector T cell (Teff) resistance to Treg suppression). In another embodiment, the GITRL moiety stimulates an antigen-specific T cell response. In another embodiment, the GITRL moiety activates or co-stimulates a T cell.
  • the GITRL moiety increases IL-2 and/or IFN- ⁇ production in and/or proliferation of a T cell. In another embodiment, the GITRL moiety reduces of depletes the number of T regulatory cells in a tumor of a subject.
  • OX40L polypeptides which comprise an OX40Lmoiety.
  • the OX40L moiety comprises one OX40L domain (monomer).
  • the OX40L moiety comprises two OX40L domains (dimer).
  • the moiety comprises three OX40L domains (trimer).
  • the moiety comprises the amino acid sequence of SEQ ID NO:43.
  • the polypeptide comprises an OX40Lmoiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • the OX40L domain comprises full-length human OX40L (i. e., amino acid residues 1-183 of SEQ ID NO:39). In another embodiment, the OX40L domain consists of full-length human OX40L (i.e., amino acid residues 1-183 of SEQ ID NO:39). In another embodiment, the OX40L domain comprises a portion of the amino acid sequence set forth in SEQ ID NO:39. In another embodiment, the OX40L domain comprises amino acid residues 30-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of amino acid residues 30-183 of SEQ ID NO:39.
  • the OX40L domain comprises amino acid residues 51-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of amino acid residues 51-183 of SEQ ID NO:39. In another embodiment, the OX40L domain comprises amino acid residues 70-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of amino acid residues 70-183 of SEQ ID NO:39
  • the OX40L domain consists of or comprises amino acid residues 31- 183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 32-183 of SEQ ID NO: 39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 33-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 34-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 35-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 36-183 of SEQ ID NO:39.
  • the OX40L domain consists of or comprises amino acid residues 37-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 38-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 39- 183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 40-183 of SEQ ID NO: 39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 41-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 42-183 of SEQ ID NO:39.
  • the OX40L domain consists of or comprises amino acid residues 43-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 44-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 45-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 46-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 47- 183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 48-183 of SEQ ID NO: 39.
  • the OX40L domain consists of or comprises amino acid residues 49-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 40-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 51-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 52-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 53-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 54-183 of SEQ ID NO:39.
  • the OX40L domain consists of or comprises amino acid residues 55- 183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 56-183 of SEQ ID NO: 39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 57-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 58-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 59-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 60-183 of SEQ ID NO:39.
  • the OX40L domain consists of or comprises amino acid residues 61-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 62-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 63- 183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 64-183 of SEQ ID NO: 39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 65-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 66-183 of SEQ ID NO:39.
  • the OX40L domain consists of or comprises amino acid residues 67-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 68-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 69-183 of SEQ ID NO:39. In another embodiment, the OX40L domain consists of or comprises amino acid residues 70-183 of SEQ ID NO:39.
  • the OX40L domain comprises or consists of a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N-terminus at any one of amino acid residues 31-70 of SEQ ID NO:39 and a C terminus at any one of amino acid residues 163-183 ofSEQ ID NO:39.
  • the OX40L domain comprises no more than about 150 amino acid residues, preferably no more than about 125 amino acid residues, and more preferably no more than about 100 amino acid residues. In another embodiment, the OX40L domain consists of no more than about 150 amino acid residues, preferably no more than about 125 amino acid residues, and more preferably no more than about 100 amino acid residues.
  • the OX40L domain comprises the amino acid sequence of SEQ ID NO: 40 or a portion thereof. In another embodiment, the OX40L domain consists of the amino acid sequence of SEQ ID NO: 40.
  • the OX40L domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein.
  • the OX40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-183 of SEQ ID NO:39.
  • the OX40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 51-183 of SEQ ID NO:39.
  • the OX40L domain comprises an amino acid sequence at least 95% identical to amino acid residues 51-183 of SEQ ID NO:39. In a particular embodiment, the OX40L domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 40.
  • the fusion polypeptide comprises a set of three human OX40L domains to form a single-chain OX40L trimer.
  • the single-chain OX40L trimer comprises, in amino- to carboxyl-terminal order, a first OX40L domain, an inter-domain linker, a second OX40L domain, a second inter-domain linker, and a third OX40L domain.
  • each linker consists of 15-20 amino acids.
  • each of the two inter-OX40L monomer linkers comprises 3 G4S domains.
  • the OX40L moiety binds to its receptor OX40. In another embodiment, the OX40L moiety binds to OX40 on activated T cells, NK cells, and NK T cells. In another embodiment, the OX40L moiety activates or agonizes OX40 signaling. In another embodiment, the OX40L moiety stimulates and/or augments production of cytokines. In another embodiment, the OX40L moiety stimulates proliferation of T cells.
  • CD40L polypeptides which comprise a CD40L moiety.
  • the CD40L moiety comprises one CD40L domain (monomer).
  • the CD40L moiety comprises two CD40L domains (dimer).
  • the moiety comprises three CD40L domains (trimer).
  • the moiety comprises the amino acid sequence of SEQ ID NO:54.
  • the polypeptide comprises a CD40L moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • the CD40L domain comprises full-length human CD40L (i. e., amino acid residues 1-261 of SEQ ID NO:48). In another embodiment, the CD40L domain comprises a portion of the amino acid sequence set forth in SEQ ID NO:48. In another embodiment, the CD40L domain comprises amino acid residues 30-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of amino acid residues 30-261 of SEQ ID NO:48. In another embodiment, the CD40L domain comprises amino acid residues 47-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of amino acid residues 47-261 of SEQ ID NO:48.
  • the CD40L domain comprises amino acid residues 1 16-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of amino acid residues 1 16-261 of SEQ ID NO:48. In another embodiment, the CD40L domain comprises amino acid residues 108-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of amino acid residues 108-261 of SEQ ID NO:48. In another embodiment, the CD40L domain comprises amino acid residues 1 13-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of amino acid residues 1 13-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 31- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 32-261 of SEQ ID NO: 48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 33-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 34-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 35-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 36-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 37-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 38-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 39- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 40-261 of SEQ ID NO: 48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 41-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 42-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 43-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 44-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 45-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 46-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 47- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 48-261 of SEQ ID NO: 48.
  • the CD40L domain consists of or comprises amino acid residues 49-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 50-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 51-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 52-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 53-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 54-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 55- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 56-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 57-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 58-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 59-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 60-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 61-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 62-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 63- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 64-261 of SEQ ID NO: 48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 65-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 66-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 67-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 68-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 69-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 70-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 71- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 72-261 of SEQ ID NO: 48.
  • the CD40L domain consists of or comprises amino acid residues 73-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 74-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 75-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 76-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 77-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 78-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 79- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 80-261 of SEQ ID NO: 48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 81-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 82-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 83-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 84-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 85-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 86-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 87- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 88-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 89-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 90-261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 91-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 92-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 93-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 94-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 95- 261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 96-261 of SEQ ID NO: 48.
  • the CD40L domain consists of or comprises amino acid residues 97-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 98-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 99-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 100-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 101-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 102- 261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 103-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 104-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 105-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 106-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 107-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 108- 261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 109-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 110-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 111-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 112-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 113-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 114- 261 of SEQ ID NO:48.
  • the CD40L domain consists of or comprises amino acid residues 115-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 116-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 117-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 118-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 119-261 of SEQ ID NO:48. In another embodiment, the CD40L domain consists of or comprises amino acid residues 120- 261 of SEQ ID NO:48.
  • the CD40L domain comprises or consists of a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N- terminus at any one of amino acid residues 31-120 of SEQ ID NO:48 and a C terminus at any one of amino acid residues 240-261 of SEQ ID NO:48.
  • the CD40L domain comprises no more than about 250 amino acid residues, preferably no more than about 200 amino acid residues, more preferably no more than about 150 amino acid residues, and even more preferably no more than 100 amino acid residues. In another embodiment, the CD40L domain consists of no more than about 250 amino acid residues, preferably no more than about 200 amino acid residues, more preferably no more than about 150 amino acid residues, and even more preferably no more than 100 amino acid residues.
  • the CD40L domain comprises the amino acid sequence of SEQ ID NO: 49 or a portion thereof. In another embodiment, the CD40L domain consists of the amino acid sequence of SEQ ID NO: 49. In another embodiment, the CD40L domain comprises the amino acid sequence of SEQ ID NO: 50 or a portion thereof. In another embodiment, the CD40L domain consists of the amino acid sequence of SEQ ID NO: 50. In another embodiment, the CD40L domain comprises the amino acid sequence of SEQ ID NO: 51 or a portion thereof. In another embodiment, the CD40L domain consists of the amino acid sequence of SEQ ID NO: 51. In another embodiment, the CD40L domain comprises the amino acid sequence of SEQ ID NO:56 or a portion thereof.
  • the CD40L domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-261 of SEQ ID NO:48.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 47-261 of SEQ ID NO:48.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 116-261 of SEQ ID NO:48.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 108- 261 of SEQ ID NO:48.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 113-261 of SEQ ID NO:48.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:49, 50, or 51.
  • the CD40L domain comprises an amino acid sequence at least 95% identical to residues 47-261, 113-261, 116-261, or 108-261 of SEQ ID NO:48.
  • the CD40L domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:49, 50, 751, or 56.
  • the fusion polypeptide comprises a set of three human CD40L domains to form a single-chain CD40L trimer.
  • the single-chain CD40L trimer comprises, in amino- to carboxyl-terminal order, a first CD40L domain, an inter-domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain.
  • each linker consists of 15-20 amino acids.
  • each of the two inter-CD40L monomer linkers comprises 3 G4S domains.
  • the CD40L moiety binds to its receptor CD40. In another embodiment, the CD40L moiety binds to CD40 on B cells, dendritic cells, and /or macrophages. In another embodiment, the CD40L moiety activates or agonizes CD40 signaling. In another embodiment, the CD40L moiety regulates the activity of B cells, dendritic cells, and /or macrophages. In another embodiment, the CD40L moiety regulates the activity of B cells, dendritic cells, and /or macrophages. In another embodiment, the CD40L moiety induces production of inflammatory cytokines. In another embodiment, the CD40L moiety unregulates molecules involved in antigen presentation and T cell stimulation.
  • LIGHT polypeptides which comprise a LIGHT moiety.
  • the LIGHT moiety comprises one LIGHT domain (monomer).
  • the LIGHT moiety comprises two LIGHT domains (dimer).
  • the moiety comprises three LIGHT domains (trimer).
  • the moiety comprises the amino acid sequence of SEQ ID NO:65.
  • the polypeptide comprises a LIGHT moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • the LIGHT domain comprises full-length human LIGHT (i. e., amino acid residues 1-240 of SEQ ID NO:58). In another embodiment, the LIGHT domain consists of full- length human LIGHT (i. e., amino acid residues 1-240 of SEQ ID NO:58). In another embodiment, the LIGHT domain comprises a portion of the amino acid sequence set forth in SEQ ID NO:58. In another embodiment, the LIGHT domain comprises amino acid residues 59-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of amino acid residues 59-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain comprises amino acid residues 58-240 of SEQ ID NO:58.
  • the LIGHT domain consists of amino acid residues 58-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain comprises amino acid residues 74-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of amino acid residues 74-240 of SEQ ID NO:58
  • the LIGHT domain consists of or comprises amino acid residues 59- 240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 59-240 of SEQ ID NO: 58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 60-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 61-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 62-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 63-240 of SEQ ID NO:58.
  • the LIGHT domain consists of or comprises amino acid residues 64-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 65-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 66- 240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 67-240 of SEQ ID NO: 58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 68-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 69-240 of SEQ ID NO:58.
  • the LIGHT domain consists of or comprises amino acid residues 70-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 71-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 72-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 73-240 of SEQ ID NO:58. In another embodiment, the LIGHT domain consists of or comprises amino acid residues 74- 240 of SEQ ID NO:58.
  • the LIGHT domain comprises or consists of a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N- terminus at any one of amino acid residues 59-240 of SEQ ID NO: 58 and a C terminus at any one of amino acid residues 220-240 of SEQ ID NO:58.
  • the LIGHT domain comprises no more than about 180 amino acid residues, preferably no more than about 170 amino acid residues. In another embodiment, the LIGHT domain consists of no more than about 180 amino acid residues, preferably no more than about 170.
  • the LIGHT domain comprises the amino acid sequence of SEQ ID NO: 61 or a portion thereof. In another embodiment, the LIGHT domain consists of the amino acid sequence of SEQ ID NO : 61.
  • the LIGHT domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein.
  • the LIGHT domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-240 of SEQ ID NO:58.
  • the LIGHT domain comprises an amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 59-240 of SEQ ID NO:58.
  • the LIGHT domain comprises an amino acid sequence at least 95% identical to amino acid residues 59-240 of SEQ ID NO:58.
  • the LIGHT domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:61.
  • the fusion polypeptide comprises a set of three human LIGHT domains to form a single-chain LIGHT trimer.
  • the single-chain LIGHT trimer comprises, in amino- to carboxyl-terminal order, a first LIGHT domain, an inter-domain linker, a second LIGHT domain, a second inter-domain linker, and a third LIGHT domain.
  • each linker consists of 15-20 amino acids.
  • each of the two inter- LIGHT monomer linkers comprises 3 G4S domains.
  • the LIGHT moiety binds to its receptor TNFRSF14. In another embodiment, the LIGHT moiety binds to TNFRSF14 on activated T cells, NK cells, and NK T cells. In another embodiment, the LIGHT moiety activates or agonizes TNFRSF14 signaling. In another embodiment, the LIGHT moiety stimulates and/or augments production of cytokines. In another embodiment, the LIGHT moiety stimulates proliferation of T cells.
  • Fc-4-lBBL Fusion Polypeptides In one embodiment, a 4-1BBL moiety is linked to an Fc region or fragment thereof.
  • Fc-CD70 Fusion Polypeptides In one embodiment, a CD70 moiety is linked to an Fc region or fragment thereof.
  • Fc-GITRL Fusion Polypeptides In one embodiment, a GITRL moiety is linked to an Fc region or fragment thereof.
  • an OX40L moiety is linked to an Fc region or fragment thereof.
  • Fc-CD40L Fusion Polypeptides In one embodiment, a CD40L moiety is linked to an Fc region or fragment thereof.
  • Fc-LIGHT Fusion Polypeptides In one embodiment, a LIGHT moiety is linked to an Fc region or fragment thereof.
  • an "Fc region” fragment crystallizable region or “Fc domain” or “Fc” refers to the C- terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system.
  • an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CHI or CL).
  • the Fc region comprises two identical protein fragments, derived from the second (Cm) and third (Cm) constant domains of the antibody's two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
  • the Fc region comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy2.
  • the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position C226 or P230 (or amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, wherein the numbering is according to the EU index as in Kabat.
  • the Cm domain of a human IgG Fc region extends from about amino acid 231 to about amino acid 340, whereas the Cm domain is positioned on C-terminal side of a Cm domain in an Fc region, / ' . e. , it extends from about amino acid 341 to about amino acid 447 of an IgG.
  • the Fc region may be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc).
  • Fc may also refer to this region in isolation or in the context of an Fc-comprising protein polypeptide such as a "binding protein comprising an Fc region," also referred to as an "Fc fusion protein” (e.g., an antibody or immunoadhesin).
  • the Fc-4-lBBL fusion polypeptide comprises a native sequence Fc region.
  • the Fc-CD70 fusion polypeptide comprises a native sequence Fc region.
  • the Fc-GITRL fusion polypeptide comprises a native sequence Fc region.
  • the Fc-OX40L fusion polypeptide comprises a native sequence Fc region.
  • the Fc-CD40L fusion polypeptide comprises a native sequence Fc region.
  • the LIGHT fusion polypeptide comprises a native sequence Fc region.
  • a "native sequence Fc region" or "native sequence Fc” comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature.
  • Native sequence human Fc regions include a native sequence human IgGl Fc region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • Native sequence Fc include the various allotypes of Fes (see, e.g., Jefferis et al. (2009) mAbs 1 : 1).
  • the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and/or biological activity.
  • a variant Fc region e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and/or biological activity.
  • Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable.
  • the variant Fc region may include two, three, four, five, etc substitutions therein, e.g. of the specific Fc region positions identified herein.
  • a variant Fc region may also comprise a sequence alteration wherein amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even when cysteine residues are removed, single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.
  • the Fc region may be modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc region, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase.
  • one or more glycosylation sites within the Fc domain may be removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).
  • sites involved in interaction with complement such as the Clq binding site, may be removed from the Fc region. For example, one may delete or substitute the EKK sequence of human IgGl .
  • sites that affect binding to Fc receptors may be removed, preferably sites other than salvage receptor binding sites.
  • an Fc region may be modified to remove an ADCC site.
  • ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgGl .
  • Specific examples of variant Fc domains are disclosed for example, in WO 97/34631 and WO 96/32478.
  • the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of Fc is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody.
  • one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
  • the Fc region may be modified to increase antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity for an Fey receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430
  • Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E.
  • Exemplary variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T.
  • Fc modifications that increase binding to an Fey receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (WO00/42072).
  • Fc modifications that can be made to Fes are those for reducing or ablating binding to FcyR and/or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.
  • Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index.
  • Exemplary substitutions include but are not limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index.
  • An Fc variant may comprise 236R/328R.
  • the Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publications WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/0201 14).
  • Fc variants that enhance affinity for an inhibitory receptor FcyRllb may also be used. Such variants may provide an Fc fusion protein with immunomodulatory activities related to FcyRllb + cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRllb relative to one or more activating receptors. Modifications for altering binding to FcyRllb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index.
  • Exemplary substitutions for enhancing FcyRllb affinity include but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.
  • Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y.
  • Fc variants for enhancing binding to FcyRllb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.
  • the affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • in vitro assay methods biochemical or immunological based assays
  • equilibrium methods e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)
  • kinetics e.g., BIACORE analysis
  • indirect binding assays e.g., competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophore
  • These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • the antibody is modified to increase its biological half-life.
  • this may be done by increasing the binding affinity of the Fc region for FcRn.
  • one or more of more of following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375.
  • Specific exemplary substitutions include one or more of the following: T252L, T254S, and/or T256F.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos.
  • exemplary variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M.
  • Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al, 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al.
  • hybrid IgG isotypes with particular biological characteristics may be used.
  • an IgGl/IgG3 hybrid variant may be constructed by substituting IgGl positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ.
  • hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F.
  • an IgGl/IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and/or CH3 region with amino acids from IgGl at positions where the two isotypes differ.
  • hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to an insertion of a glycine at position 236), and 327A.
  • IgGl variants with strongly enhanced binding to FcyRIIIa have been identified, including variants with S239D/I332E and S239D/I332E/A330L mutations which showed the greatest increase in affinity for FcyRIIIa, a decrease in FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al, 2006).
  • IgGl mutants containing L235V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcyRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al, 2007; Nordstrom et al, 2011).
  • Other Fc mutants that may be used include: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L,
  • an Fc-4-lBBL polypeptide chain is dimerized to a second Fc-4- 1BBL polypeptide chain (see Fig. 1A).
  • the two Fc-4-lBBL polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two Fc-4-lBBL polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two Fc-4-lBBL polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fc-41BBL fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide-bound to a set of three human 4-lBBL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl -terminal order, the Fc moiety, a linker, a first 4-lBBL domain, an inter-domain linker, a second 4-lBBL domain, a second inter-domain linker, and a third 4-lBBL domain, wherein each linker consists of 15-20 amino acids and each of the two inter-4-lBBL monomer linkers comprises 3 G4S motifs.
  • an Fc-CD70 polypeptide chain is dimerized to a second Fc-CD70 polypeptide chain (see Fig. IB).
  • the two Fc-CD70 polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two Fc-CD70 polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two Fc-CD70 polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fc-CD70 fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide-bound to a set of three human CD70 domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD70 domain, an inter- domain linker, a second CD70 domain, a second inter-domain linker, and a third CD70 domain, wherein each linker consists of 15-20 amino acids and each of the two inter-CD70 monomer linkers comprises 3 G4S motifs.
  • an Fc -GITRL polypeptide chain is dimerized to a second Fc-GITRL polypeptide chain (see Fig. 1C).
  • the two Fc-GITRL polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two Fc-GITRL polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two Fc-GITRL polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fc-GITRL fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide-bound to a set of three human GITRL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first GITRL domain, an inter- domain linker, a second GITRL domain, a second inter-domain linker, and a third GITRL domain, wherein each linker consists of 15-20 amino acids and each of the two inter-GITRL monomer linkers comprises 3 G4S motifs.
  • an Fc-OX40L polypeptide chain is dimerized to a second Fc-OX40L polypeptide chain (Fig. ID).
  • the two Fc-OX40L polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two Fc-OX40L polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two Fc-OX40L polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fc-OX40L fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide-bound to a set of three human OX40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first OX40L domain, an inter-domain linker, a second OX40L domain, a second inter-domain linker, and a third OX40L domain, wherein each linker consists of 15-20 amino acids and each of the two inter-OX40L monomer linkers comprises 3 G4S motifs.
  • an Fc-CD40L polypeptide chain is dimerized to a second Fc-CD40L polypeptide chain (see Fig. IE).
  • the two Fc-CD40L polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two Fc-CD40L polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two Fc-CD40L polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fc-CD40L fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide-bound to a set of three human CD40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD40L domain, an inter-domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain, wherein each linker consists of 15-20 amino acids and each of the two inter-CD40L monomer linkers comprises 3 G4S motifs.
  • an Fc -LIGHT polypeptide chain is dimerized to a second Fc- LIGHT polypeptide chain (Fig. IF).
  • the two Fc- LIGHT polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two Fc- LIGHT polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two Fc- LIGHT polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fc- LIGHT fusion polypeptide comprises two polypeptide chains dimerized by at least one inter-Fc disulfide bond, each chain comprising a human IgG Fc moiety peptide-bound to a set of three human LIGHT domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first LIGHT domain, an inter- domain linker, a second LIGHT domain, a second inter-domain linker, and a third LIGHT domain, wherein each linker consists of 15-20 amino acids and each of the two inter- LIGHT monomer linkers comprises 3 G4S motifs.
  • the Fc region is modified with respect to effector function, so as to enhance the effectiveness of the polypeptide in treating a disease, e.g., cancer.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing inter-chain disulfide bond formation in this region.
  • the homodimeric polypeptide thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity
  • ADCC anti-tumor activity
  • Homodimeric polypeptides with enhanced anti -tumor activity may also be prepared using heterobifunctional cross-linkers.
  • a polypeptide can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities.
  • the Fc-4-lBBL fusion polypeptide comprises a human IgG Fc moiety, or fragment thereof, bound to a set of three human 4-lBBL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first 4-lBBL domain, an inter-domain linker, a second 4-lBBL domain, a second inter-domain linker, and a third 4- 1BBL domain.
  • the Fc-4-lBBL fusion polypeptide comprises SEQ ID NO:8.
  • the Fc-4-lBBL fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human 4-1BBL.
  • the Fc-4-lBBL fusion polypeptide stimulates cell proliferation and proinflammatory cytokine production in activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells.
  • the Fc-CD70 fusion polypeptide comprises a human IgG Fc moiety, or fragment thereof, bound to a set of three human CD70 domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD70 domain, an inter-domain linker, a second CD70 domain, a second inter-domain linker, and a third CD70 domain.
  • the Fc-CD70 fusion polypeptide comprises SEQ ID NO:20.
  • the Fc-CD70 fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human CD70.
  • the Fc-CD70 fusion polypeptide activates naive CD4+ and CD8+ T cells, as well as NK cells, leading to differentiation, proliferation and cytokine release.
  • the Fc-GITRL fusion polypeptide comprises a human IgG Fc moiety, or fragment thereof, bound to a set of three human GITRL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first GITRL domain, an inter-domain linker, a second GITRL domain, a second inter-domain linker, and a third GITRL domain.
  • the Fc-GITRL fusion polypeptide comprises SEQ ID NO: 32.
  • the Fc-GITRL fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human GITRL.
  • the Fc-GITRL fusion polypeptide stimulates cell proliferation and pro-inflammatory cytokine production in activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells.
  • the Fc-OX40L fusion polypeptide comprises a human IgG Fc moiety, or fragment thereof, bound to a set of three human OX40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first OX40L domain, an inter-domain linker, a second OX40L domain, a second inter-domain linker, and a third OX40L domain.
  • the Fc-OX40L fusion polypeptide comprises SEQ ID NO:41.
  • the Fc-OX40L fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human OX40L.
  • the Fc-OX40L fusion polypeptide stimulates cell proliferation and proinflammatory cytokine production in activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells.
  • the Fc-CD40L fusion polypeptide comprises a human IgG Fc moiety, or fragment thereof, bound to a set of three human CD40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first CD40L domain, an inter-domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain.
  • the Fc-CD40L fusion polypeptide comprises SEQ ID NO:52.
  • the Fc-CD40L fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human CD40L.
  • the Fc-CD40L fusion polypeptide activates dendritic cells (DCs) and leads to increased priming and proliferation of antigen-specific CD4+ and CD8+ T cells.
  • the Fc-CD40L fusion polypeptide induces macrophage differentiation and antibody production in activated B cells.
  • the Fc -LIGHT fusion polypeptide comprises a human IgG Fc moiety, or fragment thereof, bound to a set of three human LIGHT domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the Fc moiety, a linker, a first LIGHT domain, an inter-domain linker, a second LIGHT domain, a second inter-domain linker, and a third LIGHT domain.
  • the Fc-LIGHT fusion polypeptide comprises SEQ ID NO: 63.
  • the Fc-LIGHT fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human LIGHT.
  • the Fc-LIGHT fusion polypeptide stimulates cell proliferation and pro-inflammatory cytokine production in activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells.
  • the Fc-4-lBBL fusion polypeptides described herein further comprise an antibody Fab region, or fragment thereof (e.g., Fab-Fc-4-lBBL fusion polypeptide).
  • Fab refers to the antigen binding portion of an antibody, comprising two chains: a first chain that comprises a VH domain and a CHI domain and a second chain that comprises a VL domain and a CL domain.
  • a Fab is typically described as the N-terminal fragment of an antibody that was treated with papain and comprises a portion of the hinge region, it is also used herein as referring to a binding domain wherein the heavy chain does not comprise a portion of the hinge.
  • the 4-1BBL fusion comprises a full-length heavy and light chain, or fragment thereof.
  • the 4-1BBL fusion comprises a full-length antibody.
  • the Fab-Fc-4-lBBL fusion or the full-length heavy and light chain heavy chain 4-1BBL fusion, or fragment thereof can be dimerized to a second fusion polypeptide chain.
  • the two fusion polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two fusion polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two fusion polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fab-Fc, heavy and light chain, full-length antibody, or fragment thereof is fused to a 4-1BBL moiety with a linker.
  • the linker is an amino acid linker. Modifications can also be made within one or more of the framework or joining regions of the heavy and/or the light chain variable regions of the Fab region or antibody, so long as antigen binding affinity subsequent to these modifications is maintained.
  • the Fab-Fc-4-lBBL fusion polypeptide comprises a human Fab moiety, or fragment thereof, bound to a human Fc moiety, or fragment thereof, bound to a set of three human 4-1BBL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl- terminal order, the Fc moiety, a linker, a first 4-1BBL domain, an inter-domain linker, a second 4-1BBL domain, a second inter-domain linker, and a third 4-1BBL domain.
  • the Fab- Fc-4-lBBL fusion polypeptide comprises SEQ ID NO: 8.
  • the Fab-Fc-4-lBBL fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human 4-1BBL.
  • the Fc-CD70 fusion polypeptides described herein further comprise an antibody Fab region, or fragment thereof (e.g., Fab-Fc-CD70 fusion polypeptide).
  • Fab refers to the antigen binding portion of an antibody, comprising two chains: a first chain that comprises a VH domain and a CHI domain and a second chain that comprises a VL domain and a CL domain.
  • a Fab is typically described as the N-terminal fragment of an antibody that was treated with papain and comprises a portion of the hinge region, it is also used herein as referring to a binding domain wherein the heavy chain does not comprise a portion of the hinge.
  • the CD70 fusion comprises a full-length heavy and light chain, or fragment thereof.
  • the CD70 fusion comprises a full-length antibody.
  • the Fab-Fc-CD70 fusion or the full-length heavy and light chain heavy chain CD70 fusion, or fragment thereof can be dimerized to a second fusion polypeptide chain.
  • the two fusion polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two fusion polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two fusion polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fab-Fc, heavy and light chain, full-length antibody, or fragment thereof is fused to a CD70 moiety with a linker.
  • the linker is an amino acid linker. Modifications can also be made within one or more of the framework or joining regions of the heavy and/or the light chain variable regions of the Fab region or antibody, so long as antigen binding affinity subsequent to these modifications is maintained.
  • the Fab-Fc-CD70 fusion polypeptide comprises a human Fab moiety, or fragment thereof, bound to a human Fc moiety, or fragment thereof, bound to a set of three human CD70 domains to form a single unbranched polypeptide comprising, in amino- to carboxyl- terminal order, the Fc moiety, a linker, a first CD70 domain, an inter-domain linker, a second CD70 domain, a second inter-domain linker, and a third CD70 domain.
  • the Fab- Fc-CD70 fusion polypeptide comprises SEQ ID NO:20.
  • the Fab-Fc-CD70 fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human CD70.
  • the Fc-GITRL fusion polypeptides described herein further comprise an antibody Fab region, or fragment thereof (e.g., Fab-Fc-GITRL fusion polypeptide).
  • Fab refers to the antigen binding portion of an antibody, comprising two chains: a first chain that comprises a VH domain and a CHI domain and a second chain that comprises a VL domain and a CL domain.
  • a Fab is typically described as the N-terminal fragment of an antibody that was treated with papain and comprises a portion of the hinge region, it is also used herein as referring to a binding domain wherein the heavy chain does not comprise a portion of the hinge.
  • the GITRL fusion comprises a full-length heavy and light chain, or fragment thereof.
  • the GITRL fusion comprises a full- length antibody.
  • the Fab-Fc-GITRL fusion or the full-length heavy and light chain heavy chain GITRL fusion, or fragment thereof can be dimerized to a second fusion polypeptide chain.
  • the two fusion polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two fusion polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two fusion polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fab-Fc, heavy and light chain, full-length antibody, or fragment thereof is fused to a GITRL moiety with a linker.
  • the linker is an amino acid linker. Modifications can also be made within one or more of the framework or joining regions of the heavy and/or the light chain variable regions of the Fab region or antibody, so long as antigen binding affinity subsequent to these modifications is maintained.
  • the Fab-Fc-GITRL fusion polypeptide comprises a human Fab moiety, or fragment thereof, bound to a human Fc moiety, or fragment thereof, bound to a set of three human GITRL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl- terminal order, the Fc moiety, a linker, a first GITRL domain, an inter-domain linker, a second GITRL domain, a second inter-domain linker, and a third GITRL domain.
  • the Fab- Fc-GITRL fusion polypeptide comprises SEQ ID NO: 29.
  • the Fab-Fc-GITRL fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human GITRL.
  • the Fc-OX40L fusion polypeptides described herein further comprise an antibody Fab region, or fragment thereof (e.g., Fab-Fc-OX40L fusion polypeptide).
  • Fab refers to the antigen binding portion of an antibody, comprising two chains: a first chain that comprises a VH domain and a CHI domain and a second chain that comprises a VL domain and a CL domain.
  • a Fab is typically described as the N-terminal fragment of an antibody that was treated with papain and comprises a portion of the hinge region, it is also used herein as referring to a binding domain wherein the heavy chain does not comprise a portion of the hinge.
  • the OX40L fusion comprises a full-length heavy and light chain, or fragment thereof.
  • the OX40L fusion comprises a full- length antibody.
  • the Fab-Fc-OX40L fusion or the full-length heavy and light chain heavy chain OX40L fusion, or fragment thereof can be dimerized to a second fusion polypeptide chain.
  • the two fusion polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two fusion polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two fusion polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fab-Fc, heavy and light chain, full-length antibody, or fragment thereof is fused to an OX40Lmoiety with a linker.
  • the linker is an amino acid linker. Modifications can also be made within one or more of the framework or joining regions of the heavy and/or the light chain variable regions of the Fab region or antibody, so long as antigen binding affinity subsequent to these modifications is maintained.
  • the Fab-Fc-OX40L fusion polypeptide comprises a human Fab moiety, or fragment thereof, bound to a human Fc moiety, or fragment thereof, bound to a set of three human OX40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl- terminal order, the Fc moiety, a linker, a first OX40L domain, an inter-domain linker, a second OX40L domain, a second inter-domain linker, and a third OX40L domain.
  • the Fab- Fc-OX40L fusion polypeptide comprises SEQ ID NO:41.
  • the Fab-Fc-OX40L fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human OX40L.
  • the Fc-CD40L fusion polypeptides described herein further comprise an antibody Fab region, or fragment thereof (e.g., Fab-Fc-CD40L fusion polypeptide).
  • Fab refers to the antigen binding portion of an antibody, comprising two chains: a first chain that comprises a VH domain and a CHI domain and a second chain that comprises a VL domain and a CL domain.
  • a Fab is typically described as the N-terminal fragment of an antibody that was treated with papain and comprises a portion of the hinge region, it is also used herein as referring to a binding domain wherein the heavy chain does not comprise a portion of the hinge.
  • the CD40L fusion comprises a full-length heavy and light chain, or fragment thereof.
  • the CD40L fusion comprises a full- length antibody.
  • the Fab-Fc-CD40L fusion or the full-length heavy and light chain heavy chain CD40L fusion, or fragment thereof can be dimerized to a second fusion polypeptide chain.
  • the two fusion polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two fusion polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two fusion polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fab-Fc, heavy and light chain, full-length antibody, or fragment thereof is fused to a CD40L moiety with a linker.
  • the linker is an amino acid linker. Modifications can also be made within one or more of the framework or joining regions of the heavy and/or the light chain variable regions of the Fab region or antibody, so long as antigen binding affinity subsequent to these modifications is maintained.
  • the Fab-Fc-CD40L fusion polypeptide comprises a human Fab moiety, or fragment thereof, bound to a human Fc moiety, or fragment thereof, bound to a set of three human CD40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl- terminal order, the Fc moiety, a linker, a first CD40L domain, an inter-domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain.
  • the Fab- Fc-CD40L fusion polypeptide comprises SEQ ID NO:52.
  • the Fab-Fc-CD40L fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human CD40L.
  • the Fc -LIGHT fusion polypeptides described herein further comprise an antibody Fab region, or fragment thereof (e.g., Fab-Fc- LIGHT fusion polypeptide).
  • Fab refers to the antigen binding portion of an antibody, comprising two chains: a first chain that comprises a VH domain and a CHI domain and a second chain that comprises a VL domain and a CL domain.
  • a Fab is typically described as the N-terminal fragment of an antibody that was treated with papain and comprises a portion of the hinge region, it is also used herein as referring to a binding domain wherein the heavy chain does not comprise a portion of the hinge.
  • the LIGHT fusion comprises a full-length heavy and light chain, or fragment thereof.
  • the LIGHT fusion comprises a full-length antibody.
  • the Fab-Fc- LIGHT fusion or the full-length heavy and light chain heavy chain LIGHT fusion, or fragment thereof can be dimerized to a second fusion polypeptide chain.
  • the two fusion polypeptide chains are dimerized by at least one inter-Fc disulfide bond.
  • the two fusion polypeptide chains are dimerized by at least two inter-Fc disulfide bonds.
  • the two fusion polypeptide chains are dimerized by at least three inter-Fc disulfide bonds.
  • the Fab-Fc, heavy and light chain, full-length antibody, or fragment thereof is fused to a LIGHT moiety with a linker.
  • the linker is an amino acid linker. Modifications can also be made within one or more of the framework or joining regions of the heavy and/or the light chain variable regions of the Fab region or antibody, so long as antigen binding affinity subsequent to these modifications is maintained.
  • the Fab-Fc- LIGHT fusion polypeptide comprises a human Fab moiety, or fragment thereof, bound to a human Fc moiety, or fragment thereof, bound to a set of three human LIGHT domains to form a single unbranched polypeptide comprising, in amino- to carboxyl- terminal order, the Fc moiety, a linker, a first LIGHT domain, an inter-domain linker, a second LIGHT domain, a second inter-domain linker, and a third LIGHT domain.
  • the Fab- Fc- LIGHT fusion polypeptide comprises SEQ ID NO: 63.
  • the Fab-Fc- LIGHT fusion polypeptide comprises at least one, two, three, or four mutations not found in native wild-type human LIGHT. iii. Albumin-4-lBBL Fusion Polypeptides
  • a 4-1BBL moiety is linked to an albumin moiety (e.g., Human Serum Albumin (HSA)).
  • HSA Human Serum Albumin
  • the albumin-4-lBBL fusion polypeptide comprises one, two, or three 4-1BBL domains.
  • a single 4-1BBL fusion polypeptide chain comprises a human serum albumin moiety peptide-bound to a set of three human 4-1BBL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the albumin moiety, a linker, a first 4-1BBL domain, an inter-domain linker, a second 4-1BBL domain, a second inter-domain linker, and a third 4-1BBL domain.
  • a CD70 moiety is linked to an albumin moiety (e.g., Human Serum Albumin (HSA)).
  • HSA Human Serum Albumin
  • the albumin-CD70 fusion polypeptide comprises one, two, or three CD70 domains.
  • a single CD70 fusion polypeptide chain comprises a human serum albumin moiety peptide-bound to a set of three human CD70 domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the albumin moiety, a linker, a first CD70 domain, an inter-domain linker, a second CD70 domain, a second inter-domain linker, and a third CD70 domain.
  • a GITRL moiety is linked to an albumin moiety (e.g., Human Serum Albumin (HSA)).
  • HSA Human Serum Albumin
  • the albumin-GITRL fusion polypeptide comprises one, two, or three GITRL domains.
  • a single GITRL fusion polypeptide chain comprises a human serum albumin moiety peptide-bound to a set of three human GITRL domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the albumin moiety, a linker, a first GITRL domain, an inter-domain linker, a second GITRL domain, a second inter-domain linker, and a third GITRL domain.
  • an OX40Lmoiety is linked to an albumin moiety (e.g., Human Serum Albumin (HSA)).
  • HSA Human Serum Albumin
  • the albumin-OX40L fusion polypeptide comprises one, two, or three OX40L domains.
  • a single OX40L fusion polypeptide chain comprises a human serum albumin moiety peptide-bound to a set of three human OX40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the albumin moiety, a linker, a first OX40L domain, an inter-domain linker, a second OX40L domain, a second inter-domain linker, and a third OX40L domain.
  • a CD40L moiety is linked to an albumin moiety (e.g., Human Serum Albumin (HSA)).
  • HSA Human Serum Albumin
  • the albumin-CD40L fusion polypeptide comprises one, two, or three CD40L domains.
  • a single CD40L fusion polypeptide chain comprises a human serum albumin moiety peptide-bound to a set of three human CD40L domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the albumin moiety, a linker, a first CD40L domain, an inter-domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain.
  • a LIGHT moiety is linked to an albumin moiety (e.g., Human Serum Albumin (HSA)).
  • HSA Human Serum Albumin
  • the albumin- LIGHT fusion polypeptide comprises one, two, or three LIGHT domains.
  • a single LIGHT fusion polypeptide chain comprises a human serum albumin moiety peptide-bound to a set of three human LIGHT domains to form a single unbranched polypeptide comprising, in amino- to carboxyl-terminal order, the albumin moiety, a linker, a first LIGHT domain, an inter-domain linker, a second LIGHT domain, a second inter-domain linker, and a third LIGHT domain.
  • bispecific antibody fusions In one embodiment, the 4-1BBL moiety is fused to the c-terminus of a heavy chain of a bispecific antibody. In one embodiment, the CD70 moiety is fused to the c-terminus of a heavy chain of a bispecific antibody. In one embodiment, the GITRL moiety is fused to the c-terminus of a heavy chain of a bispecific antibody. Also provided are bispecific antibody fusions. In one embodiment, the OX40L moiety is fused to the c-terminus of a heavy chain of a bispecific antibody. Also provided are bispecific antibody fusions.
  • the CD40L moiety is fused to the c-terminus of a heavy chain of a bispecific antibody.
  • the LIGHT moiety is fused to the c-terminus of a heavy chain of a bispecific antibody.
  • Bispecific antibodies herein include at least two binding specificities for the same or different proteins which preferably bind non-overlapping or non-competing epitopes. Such bispecific antibodies can include additional binding specificities, e.g., a third protein binding specificity for another antigen, such as the product of an oncogene.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies).
  • the 4-1BBL fusion proteins, CD70 fusion proteins, GITRL fusion proteins, OX40L fusion proteins, CD40L fusion proteins, and LIGHT fusion proteins, described herein can be produced by standard recombinant techniques. Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For the expression of the binding proteins in a host cell, nucleic acids encoding the respective polypeptides are inserted into expression vectors by standard methods.
  • prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells
  • the binding protein is recovered from the cells (supernatant or cells after lysis).
  • prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells
  • General methods for recombinant production of antibodies are well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif 17 183-202 (1999); Geisse, S., et al, Protein Expr. Purif. 8 271-282 (1996); Kaufman, R.J., Mol. Biotechnol. 16 151- 161 (2000); Werner, R.G., Drug Res. 48 870-880 (1998).
  • the polypeptides may be suitably separated from the culture medium by conventional purification procedures. Purification can be performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g.
  • cation exchange (carboxylmethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta- mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g.
  • Linkers A variety of linkers can be used in the fusion polypeptides described herein. "Linked to” refers to direct or indirect linkage or connection of, in context, amino acids or nucleotides. “Linker” refers to one or more amino acids connecting two domains or regions together. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. ( 1994) Structure 2: 1 121-1 123).
  • linkers suitable for use can be found in the Registry of Standard Biological Parts at http://partsregistry.org/Protein_domains/Linker (see also, e.g., Crasto CJ and Feng JA. LINKER: a program to generate linker sequences for fusion proteins. Protein Eng 2000 May; 13(5) 309-12 and George RA and Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng 2002 Nov; 15(1 1) 871-9).
  • a linker may be 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or at least 90-100 amino acids long.
  • An Fc region or albumin can be separated from the 4-1BBL moiety by a linker.
  • each 4-1BBL domain of the 4-1BBL moiety can be separated by an inter-domain linker.
  • An Fc region or albumin can be separated from the CD70 moiety by a linker.
  • each CD70 domain of the CD70 moiety can be separated by an inter-domain linker.
  • An Fc region or albumin can be separated from the GITRL moiety by a linker.
  • each GITRL domain of the GITRL moiety can be separated by an inter-domain linker.
  • An Fc region or albumin can be separated from the OX40L moiety by a linker.
  • each OX40L domain of the OX40L moiety can be separated by an inter-domain linker.
  • each linker or inter-domain linker comprises 5-25 amino acids. In one embodiment, the linker or inter-domain linker comprises 5-10, 5-15, 5-20, 5-25, 10-15, 10-20, 10-25, 15- 20, 15-25, or 20-25 amino acids. In another embodiment, the linker or inter-domain linker comprises 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.
  • the linker or inter-domain linker comprises 15-20 amino acids.
  • the linker or inter-domain linker comprises at least one, two, or three G4S motifs.
  • a G4S motif comprises four glycine residues followed by one serine residue (i. e., amino acid sequence GGGGS).
  • the linker or inter-domain linker comprises three G4S motifs.
  • compositions comprising the polypeptides described herein are provided, as well as methods of using such compositions for diagnostic purposes or to treat a disease in a patient.
  • the compositions provided herein contain one or more of the polypeptides disclosed herein, formulated together with a carrier (e.g., a "pharmaceutically acceptable carrier").
  • a carrier e.g., a "pharmaceutically acceptable carrier”
  • the composition comprises a polypeptide comprising a 4-1BBL moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any excipient, diluent or agent is incompatible with the active compound, use thereof in the pharmaceutical
  • compositions provided herein is contemplated.
  • Supplementary active compounds e.g., additional anticancer agents
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the composition if desired, can also contain minor amounts of wetting or solubility enhancing agents, stabilizers, preservatives, or pH buffering agents.
  • isotonic agents for example, sodium chloride, sugars, polyalcohols such as mannitol, sorbitol, glycerol, propylene glycol, and liquid polyethylene glycol in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. , by injection or infusion).
  • the polypeptide may be coated in a material to protect them from the action of acids and other natural conditions that may inactivate proteins.
  • the polypeptide may be administered to a patient in an appropriate carrier, for example, in liposomes, or a diluent.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Liposomes include water-in-oil-in-water CGF emulsions, as well as conventional liposomes.
  • the composition can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • compositions may be administered alone or in combination therapy, i.e. , combined with other agents (e.g., as discussed in further detail below).
  • polypeptides, compositions, and methods described herein have numerous in vitro and in vivo utilities involving, for example, enhancement of immune response by activating 4-1BB signaling.
  • the polypeptides described herein e.g., a polypeptide comprising a 4-lBBL moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA)
  • HSA an antibody and/or an albumin
  • polypeptides, compositions, and methods described herein have numerous in vitro and in vivo utilities involving, for example, enhancement of immune response by activating CD27 signaling.
  • the polypeptides described herein e.g., a polypeptide comprising a CD70 moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA)
  • HSA an antibody and/or an albumin
  • polypeptides, compositions, and methods described herein have numerous in vitro and in vivo utilities involving, for example, enhancement of immune response by activating GITR signaling.
  • the polypeptides described herein e.g., a polypeptide comprising a GITRL moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA)
  • HSA an antibody and/or an albumin
  • polypeptides, compositions, and methods described herein have numerous in vitro and in vivo utilities involving, for example, enhancement of immune response by activating OX40 signaling.
  • the polypeptides described herein e.g., a polypeptide comprising an OX40Lmoiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA)
  • polypeptides, compositions, and methods described herein have numerous in vitro and in vivo utilities involving, for example, enhancement of immune response by activating CD40 signaling.
  • the polypeptides described herein e.g., a polypeptide comprising a CD40L moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA)
  • HSA an antibody and/or an albumin
  • polypeptides, compositions, and methods described herein have numerous in vitro and in vivo utilities involving, for example, enhancement of immune response by activating TNFRSF14 signaling.
  • the polypeptides described herein e.g., a polypeptide comprising a LIGHT moiety linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA)
  • HSA an antibody and/or an albumin
  • kits for modifying an immune response in a subject comprising administering to the subject the polypeptides described herein, such that the immune response in the subject is modified.
  • the response is enhanced, stimulated or up-regulated.
  • methods of stimulating (activating) immune cells for cancer therapy by administering the polypeptides described herein to a patient are provided.
  • methods of maintaining T cells for adoptive cell transfer therapy are provided.
  • methods of stimulating proliferation of T cells for adoptive cell transfer therapy are provided.
  • T cells that can be enhanced stimulated with the polypeptides described herein include CD4+ T cells and CD8+ T cells.
  • the T cells can be T e ff cells, e.g., CD4+ T e ff cells, CD8+ T e ff cells, Thelper (TV) cells and T cytotoxic (T c ) cells.
  • the method comprises administering an additional therapeutic agent to the patient.
  • treat refers to therapeutic or preventative measures described herein.
  • treatment employ administration to a patient the polypeptides disclosed herein in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • the term "effective amount” refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disease or one or more symptoms thereof, prevent the advancement of a disease, cause regression of a disease, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disease, detect a disease, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).
  • the disease is cancer.
  • cancer as used herein is defined as a tissue of uncontrolled growth or proliferation of cells, such as a tumor.
  • the term includes pre-malignant as well as malignant cancers.
  • cancers can be cancers with solid tumors or blood malignancies (liquid tumors).
  • Non- limiting examples of cancers for treatment include squamous cell carcinoma, small-cell lung cancer, non- small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g.
  • prostate adenocarcinoma thyroid cancer
  • neuroblastoma pancreatic cancer
  • glioblastoma glioblastoma multiforme
  • cervical cancer stomach cancer
  • bladder cancer hepatoma
  • breast cancer colon carcinoma
  • head and neck cancer gastric cancer
  • gastric cancer germ cell tumor
  • pediatric sarcoma sinonasal natural killer
  • melanoma e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma
  • bone cancer skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra
  • lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cell hematologic malignancy, e.g., B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma
  • HL Hodgkin's lymphoma
  • NHL non-Hodgkin's lymphoma
  • B cell hematologic malignancy e.g., B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma
  • angiocentric (nasal) T-cell lymphoma cancer of the head or neck, renal cancer, rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma, as well as any combinations of said cancers.
  • the methods described herein may also be used for treatment of metastatic cancers, unresectable and/or refractory cancers (e.g., cancers refractory to previous immunotherapy), and recurrent cancers.
  • the disease is an autoimmune disease.
  • An "autoimmune disease” herein is a disease or disorder arising from and directed against an individual's own tissues or a co- segregate or manifestation thereof or resulting condition therefrom.
  • autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis,
  • psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails
  • dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis
  • urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RPvMS), progressive
  • glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, allergic conditions, allergic reaction, eczema including allergic or atopic eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), l
  • ANCA-associated vasculitis such as Churg-Strauss vasculitis or syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRC A), Factor VIII deficiency, hemophilia A, autoimmune neutropenia,
  • pancytopenia leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen- antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuro
  • encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE)
  • myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS)
  • sensory neuropathy multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain- Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear Ig
  • Leishmania Leishmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema,
  • autoimmune atrophic gastritis sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism ( ⁇ 0 ⁇ 4), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical
  • the disease is an infectious disease.
  • the infectious disease relates to an agent selected from the group consisting of: a virus, a bacterium, a fungus, and a protozoan parasite.
  • infectious diseases include, but are not limited to human immunodeficiency viruses (GIV), hepatitis viruses class A, B and C, human cytomegalovirus, human papilloma viruses, leishmaniasis, toxoplasmosis, cryptosporidiosis, sleeping sickness, malaria, herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus
  • GIV human immunodeficiency
  • pathogenic bacteria causing infections treatable by methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.
  • Some examples of pathogenic fungi causing infections treatable by methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • Candida albicans, krusei, glabrata, tropicalis, etc.
  • Cryptococcus neoformans Aspergillus (fumigatus, niger, etc.)
  • Genus Mucorales micor, absidia, rhizopus
  • Sporothrix schenkii Blastomyces dermatitidis
  • Paracoccidioides brasiliensis Coccidioides immitis
  • pathogenic parasites causing infections treatable by methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.
  • the polypeptides and compositions described herein can be administered alone or in combination, i.e., combined with other agents.
  • the combination therapy can include a polypeptide described herein with at least one additional therapeutic agent (e.g., an antineoplastic (anticancer) agent).
  • additional therapeutic agent e.g., an antineoplastic (anticancer) agent.
  • the polypeptides and compositions described herein can also be administered in conjunction with an anti -cancer treatment modality, such as radiation therapy and/or surgery.
  • Adjunctive or combined administration includes simultaneous administration of any of the polypeptides described herein and one or more agents in the same or different dosage form, or separate administration of the polypeptide and one or more agents (e.g. , sequential administration). Such concurrent or sequential administration preferably results in both the polypeptide and the one or more agents being simultaneously present in treated patients.
  • anti-plastic agent refers to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of antineoplastic agents.
  • the polypeptides described herein are administered in combination with an additional antineoplastic agent.
  • no more than three antineoplastic agents are administered in combination with the polypeptides described herein.
  • no more than two other antineoplastic agents are administered in combination with the polypeptides described herein.
  • no more than one other antineoplastic agent is administered in combination with the polypeptides described herein.
  • no other antineoplastic agent is administered in combination with the polypeptides described herein.
  • immune checkpoint regulators e.g., inhibitors
  • immune checkpoint regulators e.g., inhibitors
  • immune checkpoint regulators refers to a group of molecules associated with signaling pathways in cells of the immune system which down-modulate or inhibit an immune response.
  • Immune checkpoint regulators are known in the art and include, without limitation, CTLA-4, PD-1, PDL- 1, PDL-2, LAG-3, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, BTLA, and A2aR.
  • Exemplary checkpoint inhibitors include, but are not limited to ipilimumab, nivolumab, pembrolizumab, and atezolizumab).
  • the polypeptides described herein can be combined with a vaccination protocol.
  • Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition).
  • a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539- 43).
  • the polypeptides described herein can be combined with a bispecific T cell engager (e.g., blinatumomab) , adoptive T cell therapy (CAR-T cells), growth factor signaling inhibitors, targeted inhibitors, cancer vaccines, toll-like receptor agonists (e.g., TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or TLRl 1 agonist) and one or more chemotherapeutic agents.
  • a bispecific T cell engager e.g., blinatumomab
  • CAR-T cells adoptive T cell therapy
  • growth factor signaling inhibitors e.g., TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or TLRl 1 agonist
  • the polypeptides described herein can be used in combination (e.g., simultaneously or separately) with an additional treatment, such as irradiation, chemotherapy (e.g., using camptothecin (CPT-1 1), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, or camptothecin + apo21/TRAIL (a 6X combo)), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2' (bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor (e.g., INCB24360, indoximod, NLG-919, or
  • polypeptides described herein can further be used in combination with one or more antiproliferative cytotoxic agents.
  • Classes of compounds that may be used as anti -proliferative cytotoxic agents include, but are not limited to, the following: [00409] Alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide
  • Triethylenethiophosphoramine Busulfan, Carmustine, Lomustine, Streptozocin, dacarbazine, and Temozolomide.
  • Antimetabolites including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors: Methotrexate, 5-Fluorouracil, Floxuridine,
  • Cytarabine 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.
  • Suitable anti -proliferative agents for combining with the polypeptides described herein include without limitation, taxanes, paclitaxel (paclitaxel is commercially available as TAXOLTM), docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D,
  • paclitaxel paclitaxel is commercially available as TAXOLTM
  • DDM discodermolide
  • DCT dictyostatin
  • Peloruside A epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepot
  • desoxyepothilone Bl [17]-dehydrodesoxyepothilone B, [18]dehydrodesoxyepothilones B, C12,13- cyclopropyl -epothilone A, C6-C8 bridged epothilone A, trans-9, 10-dehydroepothilone D, cis-9, 10- dehydroepothilone D, 16-desmethylepothilone B, epothilone B10, discoderomolide, patupilone (EPO- 906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974, Cy ⁇ tophycins,
  • hormones and steroids including synthetic analogs, such as 17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone,
  • Fluoxymesterone Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,
  • Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEXTM can also be administered to the patient.
  • other agents used in the modulation of tumor growth or metastasis in a clinical setting such as antimimetics, can also be administered as desired.
  • chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA); the disclosure of which is incorporated herein by reference thereto.
  • PDR Physicians' Desk Reference
  • the chemotherapeutic agent(s) and/or radiation therapy can be administered according to therapeutic protocols well known in the art.
  • the administration of the chemotherapeutic agent(s) and/or radiation therapy can be varied depending on the disease being treated and the known effects of the chemotherapeutic agent(s) and/or radiation therapy on that disease.
  • the therapeutic protocols e.g., dosage amounts and times of administration
  • the therapeutic protocols can be varied in view of the observed effects of the administered therapeutic agents on the patient, and in view of the observed responses of the disease to the administered therapeutic agents.
  • kits containing the polypeptide compositions described herein and instructions for use typically include a packaged combination of reagents in predetermined amounts with instructions and a label indicating the intended use of the contents of the kit.
  • the term label or instruction includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit at any time during its manufacture, transport, sale or use. It can be in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of the manufacture, use or sale for administration to a human or for veterinary use.
  • the label or instruction can also encompass advertising leaflets and brochures, packaging materials, and audio or video instructions.
  • the kit contains the polypeptide in suitable containers and instructions for administration in accordance with the treatment regimens described herein.
  • the kit further comprises an additional antineoplastic agent.
  • the polypeptides are provided in suitable containers as a dosage unit for administration. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic.
  • the polypeptides are provided in lyophilized form, and the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use, for example, comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein.
  • a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use, for example, comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the
  • Expi293FTM cells (ThermoFisher) were grown in Expi293 expression medium (Gibco®) as suspension cultures in flasks with rotation (125 rpm). Cells were separately and singly co-transfected with a 1 ⁇ g of plasmid DNA and 2.7 ⁇ of ExpiFectamineTM transfection reagent (Gibco®) per milliliter of cell culture. The density of cells at the time of transfection was 2.5 e6 cells/ml. Transfection enhancers 1 and 2 were added to the cells 18 hours post- transfection. Six days post-transfection, cell cultures are centrifuged for 15 min at 5,000 x g to pellet the cells. The supernatant media was decanted from the cells and filtered using 0.2 ⁇ filter in preparation for purification.
  • TSKGELgel SuperSW3000 column (4.6 mm ID x 30 cm)(Tosoh Biosciences) was equilibrated with 400 mM NaC10 4 , 150 mM NaCl, pH 6.5 using a Agilent 1100 HPLC (Agilent). Fifty micrograms of protein were injected at a flow rate of 0.35 ml/min and absorbance at 280 nm was recorded over a 20 minute period.
  • GloResponseTM NFkB-luc29 Jurkat cells stably transfected with 4-1BB were thawed and aliquoted in 96-well plate (Corning).
  • GloResponseTM NFkB-luc29 U20S cells stably transfected with CD40 (Promega) were plated in 96-well plate and incubated overnight at 37 °C in CO2 incubator prior to day of treatment. Cells were then stimulated with increasing concentrations of Fc-41BBL fusion proteins, recombinant ligands, or agonist antibodies for 6 hours. Following incubation, luciferase reporter signal was measured using Bio-Glo on a SYNERGY HI plate reader (BioTek). Luminescence was normalized to untreated controls and duplicates were averaged and plotted as a function of protein concentration. Non-linear regression was fitted using a 4 parameter least square fit using PRISM software (GraphPad).
  • Fc-41BBL The activity of Fc-41BBL protein was assessed using cell lines stably transfected with the cognate TNFSF receptor and a luciferase reporter under the transcriptional control of NFkB response element.
  • Fig. 2 shows the activity of Fc-41BBL (STIM-41BBL), as compared to the corresponding recombinant ligand and agonistic antibody (utomilumbab).
  • Fc-sc41BBL The activity of Fc-sc41BBL is also assessed in vitro using cells isolated from human donors. Briefly, PBMC and isolated cell subsets, such as activated or resting T cells, B cells and NK cells, are treated with the molecules of interest and control molecules before examination for phenotypic, functional, and transcriptional changes using flow cytometry, Luminex, and ELISA. Differences between groups are assessed by single and multi-parameter statistical measures. Fc-sc41BBL is expected to stimulate cell proliferation and pro-inflammatory cytokine production in activated CD4+ and CD8+ T cells, and natural killer (NK) cells.
  • NK natural killer
  • TNF ligand-Fc fusion molecules To determine the activity of human TNF ligand-Fc fusion molecules we performed in vitro T cell activation and proliferation assays. T cells were isolated from whole blood drawn from healthy donors and labeled with CellTrace Violet to track proliferation. Labeled T cells were incubated in a 96- well plate (1 x 10 ⁇ 5 per well) pre-coated with 50ng anti-CD3 (OKT3 clone) in the presence of 2.5 nM soluble or plate-bound Fc-sc41BBL or control rhlgGl Fc.
  • TNF ligand-Fc fusion molecules were tested in vitro and in vivo.
  • T cells isolated from spleens of Balb/c mice (Pan T Cell isolation kit, Miltenyi) were labeled with CellTrace Violet and cultured in 96-well plates (1 x 10 ⁇ 5 per well) pre-coated with 50ng anti-CD3 (145-11C clone) in the presence of 2.5 nM Fc-sc41BBL.
  • Luminex for the above culture supematants according to the manufacturer's protocol.
  • CD25+ T cells were increased in the CD8+ subpopulation after treating isolated T cells with 2.5 nM of Fc- sc41BBL.
  • increased cytokine production was observed, for instance for pro-inflammatory cytokines IL-2 and GM-CSF.
  • Fc-sc41BBL Fc-sc41BBL molecule
  • STIM-41BBL Fc-sc41BBL
  • C57B1/6 mice were inoculated subcutaneously into the right flank with 1 ⁇ 10 ⁇ 6 cells. Treatment was begun when tumors reached 100 to 150 mm 3 in size (Day 6).
  • Fig. 3 shows the control group, treated with PBS, and the treatment group.
  • Expi293FTM cells (ThermoFisher) are grown in Expi293 expression medium (Gibco®) as suspension cultures in flasks with rotation (125 rpm). Cells are separately and singly co-transfected with a 1 ⁇ g of plasmid DNA and 2.7 ⁇ of ExpiFectamineTM transfection reagent (Gibco®) per milliliter of cell culture. The density of cells at the time of transfection is 2.5 x 10 6 cells/ml. Transfection enhancers 1 and 2 are added to the cells 18 hours post- transfection. Six days post-transfection, cell cultures are centrifuged for 15 min at 5,000 x g to pellet the cells. The supernatant media is decanted from the cells and filtered using 0.2 ⁇ filter in preparation for purification.
  • GloResponseTM NFkB-luc29 Jurkat cells stably transfected with CD70 are thawed and aliquoted in 96-well plate (Corning).
  • GloResponseTM NFkB-luc29 U20S cells stably transfected with CD40 are plated in 96-well plate and incubated overnight at 37 °C in CO2 incubator prior to day of treatment. The cells are then stimulated with increasing concentrations of Fc-CD70 fusion proteins, recombinant ligands, or agonist antibodies for 6 hours. Following incubation, luciferase reporter signal is measured using Bio-Glo on a SYNERGY HI plate reader (BioTek). Luminescence is normalized to untreated controls and duplicates are averaged and plotted as a function of protein concentration. Non-linear regression is fitted using a 4 parameter least square fit using PRISM software (GraphPad).
  • EXAMPLE 7 IN VITRO ACTIVITY
  • the activity of Fc-CD70 is also assessed in vitro using cells isolated from human donors. Briefly, PBMC and isolated cell subsets, such as activated or resting T cells, B cells and NK cells, are treated with the molecules of interest and control molecules before examination for phenotypic, functional, and transcriptional changes using flow cytometry, Luminex, and ELISA. Differences between groups are assessed by single and multi-parameter statistical measures. Fc-CD70 is expected to activate naive CD4+ and CD8+ T cells as well as NK cells, leading to differentiation, proliferation and cytokine release.
  • Expi293FTM cells (ThermoFisher) was grown in Expi293 expression medium (Gibco®) as suspension cultures in flasks with rotation (125 rpm). Cells were separately and singly co-transfected with a 1 ⁇ g of plasmid DNA and 2.7 ⁇ of ExpiFectamineTM transfection reagent (Gibco®) per milliliter of cell culture. The density of cells at the time of transfection was 2.5 X 10 6 cells/ml. Transfection enhancers 1 and 2 were added to the cells 18 hours post- transfection. Six days post-transfection, cell cultures are centrifuged for 15 min at 5,000 x g to pellet the cells. The supernatant media was decanted from the cells and filtered using 0.2 ⁇ filter in preparation for purification.
  • GloResponseTM NFkB-luc29 Jurkat cells stably transfected with GITRL were thawed and aliquoted in 96-well plate (Corning).
  • GloResponseTM NFkB-luc29 U20S cells stably transfected with GITR (Promega) were plated in 96-well plate and incubated overnight at 37 °C in CO2 incubator prior to day of treatment. Cells were then stimulated with increasing concentrations of Fc-GITRL fusion proteins, recombinant ligands, or agonist antibodies for 6 hours. Following incubation, luciferase reporter signal was measured using Bio-Glo on a SYNERGY HI plate reader (BioTek). Luminescence was normalized to untreated controls and duplicates were averaged and plotted as a function of protein concentration. Non-linear regression was fitted using a 4 parameter least square fit using PRISM software (GraphPad).
  • Fc-GITRL protein was assessed using cell lines stably transfected with the cognate TNFSF receptor and a luciferase reporter under the transcriptional control of NFkB response element.
  • Fig. 4 shows the activity of Fc-GITRL (STIM-GITRL), as compared to the corresponding recombinant ligand and agonistic antibody (TRX518). These results demonstrate that Fc-GITRL is a potent activator of GITR receptor activity.
  • Fc-GITRL The activity of Fc-GITRL is also assessed in vitro using cells isolated from human donors. Briefly, PBMC and isolated cell subsets, such as activated or resting T cells, B cells and NK cells, are treated with the molecules of interest and control molecules before examination for phenotypic, functional, and transcriptional changes using flow cytometry, Luminex, and ELISA. Differences between groups are assessed by single and multi-parameter statistical measures. Fc-scGITRL is expected to stimulate cell proliferation and pro-inflammatory cytokine production in activated CD4+ and CD8+ T cells, and in natural killer (NK) cells.
  • NK natural killer
  • Expi293FTM cells (ThermoFisher) was grown in Expi293 expression medium (Gibco®) as suspension cultures in flasks with rotation (125 rpm). Cells were separately and singly co-transfected with a 1 ⁇ g of plasmid DNA and 2.7 ⁇ of ExpiFectamineTM transfection reagent (Gibco®) per milliliter of cell culture. The density of cells at the time of transfection was 2.5 x 10 6 cells/ml. Transfection enhancers 1 and 2 were added to the cells 18 hours post- transfection. Six days post-transfection, cell cultures are centrifuged for 15 min at 5,000 x g to pellet the cells. The supernatant media was decanted from the cells and filtered using 0.2 ⁇ filter in preparation for purification.
  • GloResponseTM NFkB-luc29 Jurkat cells stably transfected with OX40L is thawed and aliquoted in 96-well plate (Corning).
  • GloResponseTM NFkB-luc29 U20S cells stably transfected with OX40 (Promega) were plated in 96-well plate and incubated overnight at 37 °C in CO2 incubator prior to day of treatment. Cells were then stimulated with increasing concentrations of Fc-OX40L fusion proteins, recombinant ligands, or agonist antibodies for 6 hours.
  • luciferase reporter signal was measured using Bio-Glo on a SYNERGY HI plate reader (BioTek). Luminescence was normalized to untreated controls and duplicates were averaged and plotted as a function of protein concentration. Non-linear regression was fitted using a 4 parameter least square fit using PRISM software (GraphPad).
  • Fc-OX40L protein was assessed using cell lines stably transfected with the cognate TNFSF receptor and a luciferase reporter under the transcriptional control of NFkB response element.
  • Fig. 5 shows the activity of Fc-OX40L (STIM-OX40L), as compared to the corresponding recombinant ligand and agonistic antibody (Medi6469).
  • Fc-OX40L The activity of Fc-OX40L is also assessed in vitro using cells isolated from human donors. Briefly, PBMC and isolated cell subsets, such as activated or resting T cells, B cells and NK cells, are treated with the molecules of interest and control molecules before examination for phenotypic, functional, and transcriptional changes using flow cytometry, Luminex, and ELISA. Differences between groups are assessed by single and multi-parameter statistical measures. Fc-OX40L is expected to stimulate cell proliferation and pro-inflammatory cytokine production in activated CD4+ and CD8+ T cells, and natural killer (NK) cells.
  • NK natural killer
  • T cells were isolated from whole blood drawn from healthy donors and labeled with CellTrace Violet to track proliferation. Labeled T cells are incubated in a 96-well plate (1 x 10 ⁇ 5 per well) pre-coated with 50ng anti-CD3 (OKT3 clone) in the presence of 2.5 nM soluble or plate-bound Fc-scOX40L or control rhlgGl Fc.
  • TNF ligand-Fc fusion molecules The activity of the murine versions of the TNF ligand-Fc fusion molecules is tested in vitro.
  • T cells isolated from spleens of Balb/c mice Pan T Cell isolation kit, Miltenyi are labeled with
  • CD25+ T cells are increased in the CD8+ subpopulation after treating isolated T cells with 2.5 nM of Fc-scOX40L. Similarly, increased cytokine production is observed, for instance for pro-inflammatory cytokines IL-2 and GM-CSF.
  • Expi293FTM cells (ThermoFisher) were grown in Expi293 expression medium (Gibco®) as suspension cultures in flasks with rotation (125 rpm). Cells were separately and singly co-transfected with a 1 ⁇ g of plasmid DNA and 2.7 ⁇ of ExpiFectamineTM transfection reagent (Gibco®) per milliliter of cell culture. The density of cells at the time of transfection was 2.5 10 6 cells/ml. Transfection enhancers 1 and 2 were added to the cells 18 hours post- transfection. Six days post-transfection, cell cultures are centrifuged for 15 min at 5,000 x g to pellet the cells. The supernatant media was decanted from the cells and filtered using 0.2 ⁇ filter in preparation for purification.
  • GloResponseTM NFkB-luc29 Jurkat cells stably transfected with CD40L were thawed and aliquoted in 96-well plate (Corning).
  • GloResponseTM NFkB-luc29 U20S cells stably transfected with CD40 (Promega) were plated in 96-well plate and incubated overnight at 37 °C in CO2 incubator prior to day of treatment. Cells were then stimulated with increasing concentrations of Fc-CD40L fusion proteins, recombinant ligands, or agonist antibodies for 6 hours. Following incubation, luciferase reporter signal was measured using Bio-Glo on a SYNERGY HI plate reader (BioTek). Luminescence was normalized to untreated controls and duplicates were averaged and plotted as a function of protein concentration. Non-linear regression was fitted using a 4 parameter least square fit using PRISM software (GraphPad).
  • Fc-CD40L The activity of Fc-CD40L protein was assessed using cell lines stably transfected with the cognate TNFSF receptor and a luciferase reporter under the transcriptional control of NFkB response element.
  • Fig. 6 shows the activity of Fc-CD40L (STIM-CD40L), as compared to the corresponding recombinant ligand and agonistic antibody (CP-870893).
  • Fc-CD40L The activity of Fc-CD40L is also assessed in vitro using cells isolated from human donors. Briefly, PBMC and isolated cell subsets, such as activated or resting T cells, B cells and NK cells, are treated with the molecules of interest and control molecules before examination for phenotypic, functional, and transcriptional changes using flow cytometry, Luminex, and ELISA. Differences between groups are assessed by single and multi-parameter statistical measures. Fc-CD40L is expected to activate dendritic cells (DCs) leading to increased priming and proliferation of antigen-specific CD4+ and CD8+ T cells. Fc-CD40L is also expected to induce macrophage differentiation and antibody production in activated B cells.
  • DCs dendritic cells
  • Fc-CD40L is also expected to induce macrophage differentiation and antibody production in activated B cells.
  • Expi293FTM cells (ThermoFisher) are grown in Expi293 expression medium (Gibco®) as suspension cultures in flasks with rotation (125 rpm). Cells are separately and singly co-transfected with a 1 ⁇ g of plasmid DNA and 2.7 ⁇ of ExpiFectamineTM transfection reagent (Gibco®) per milliliter of cell culture. The density of cells at the time of transfection is 2.5 X 10 6 cells/ml. Transfection enhancers 1 and 2 are added to the cells 18 hours post- transfection. Six days post-transfection, cell cultures are centrifuged for 15 min at 5,000 x g to pellet the cells. The supernatant media is decanted from the cells and filtered using 0.2 ⁇ filter in preparation for purification.
  • GloResponseTM NFkB-luc29 Jurkat cells stably transfected with LIGHT are thawed and aliquoted in 96-well plate (Corning).
  • GloResponseTM NFkB-luc29 U20S cells stably transfected with TNFRSF14 (Promega) are plated in 96-well plate and incubated overnight at 37 °C in CO2 incubator prior to day of treatment. Cells are then stimulated with increasing concentrations of Fc-LIGHT fusion proteins, recombinant ligands, or agonist antibodies for 6 hours. Following incubation, luciferase reporter signal are measured using Bio-Glo on a SYNERGY HI plate reader (BioTek). Luminescence was normalized to untreated controls and duplicates are averaged and plotted as a function of protein concentration. Non-linear regression is fitted using a 4 parameter least square fit using PRISM software
  • Fc-LIGHT protein is assessed using cell lines stably transfected with the cognate TNFSF receptor and a luciferase reporter under the transcriptional control of NFkB response element. These results demonstrate that Fc-LIGHT is a potent activator of TNFRSF14 receptor activity.
  • Fc-LIGHT The activity of Fc-LIGHT is also assessed in vitro using cells isolated from human donors. Briefly, PBMC and isolated cell subsets, such as activated or resting T cells, B cells and NK cells, are treated with the molecules of interest and control molecules before examination for phenotypic, functional, and transcriptional changes using flow cytometry, Luminex, and ELISA. Differences between groups are assessed by single and multi-parameter statistical measures. Fc-LIGHT is expected to activate NK cells, increasing their direct tumor cytotoxicity as well as production of IFN -gamma, which enhances DC maturation and thus tumor-specific CD8+ T cell priming.
  • CD70 CSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIAS

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Abstract

L'invention concerne des polypeptides 4-1BBL modifiés et des polypeptides de fusion Fc-4-1BBL, ainsi que des méthodes d'utilisation de ces polypeptides et de compositions pour traiter des troubles liés à 4-1BB (par exemple, le cancer). L'invention concerne de plus des polypeptides CD70 modifiés et des polypeptides de fusion Fc-CD70, ainsi que des méthodes d'utilisation de ces polypeptides et de compositions pour traiter des troubles liés à CD70 (par exemple, le cancer). L'invention concerne en outre des polypeptides GITRL modifiés et des polypeptides de fusion Fc-GITRL, ainsi que des méthodes d'utilisation de ces polypeptides et de compositions pour traiter des troubles liés à GITRL (par exemple, le cancer). L'invention concerne également des polypeptides OX40L modifiés et des polypeptides de fusion Fc-OX40L, ainsi que des méthodes d'utilisation de ces polypeptides et de compositions pour traiter des troubles liés à OX40L (par exemple, le cancer). L'invention concerne par ailleurs des polypeptides CD40L modifiés et des polypeptides de fusion Fc-CD40L, ainsi que des méthodes d'utilisation de ces polypeptides et de compositions pour traiter des troubles liés au CD40L (par exemple, le cancer). L'invention concerne de plus encore des polypeptides LIGHT modifiés et des polypeptides de fusion Fc-LIGHT, ainsi que des méthodes d'utilisation de ces polypeptides et de compositions pour traiter des troubles liés au LIGHT (par exemple, le cancer).
PCT/US2018/016100 2017-02-01 2018-01-31 Polypeptides de fusion de la superfamille du tnf WO2018144514A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3706786A4 (fr) * 2017-11-09 2021-09-01 Medimmune, LLC Polypeptides de fusion bispécifiques et leurs procédés d'utilisation
WO2022192657A3 (fr) * 2021-03-12 2022-10-20 Janssen Biotech, Inc. Compositions de protéines de fusion immunomodulatrices obtenues par bio-ingénierie
EP4041410A4 (fr) * 2019-10-08 2023-12-06 Fred Hutchinson Cancer Center Protéines cd70 trimériques génétiquement modifiées et leurs utilisations

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* Cited by examiner, † Cited by third party
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US8664366B2 (en) * 2009-01-09 2014-03-04 Apogenix Gmbh Fusion proteins forming trimers
CN114751989B (zh) * 2015-03-31 2025-03-14 豪夫迈·罗氏有限公司 包含三聚体tnf家族配体的抗原结合分子

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3706786A4 (fr) * 2017-11-09 2021-09-01 Medimmune, LLC Polypeptides de fusion bispécifiques et leurs procédés d'utilisation
EP4041410A4 (fr) * 2019-10-08 2023-12-06 Fred Hutchinson Cancer Center Protéines cd70 trimériques génétiquement modifiées et leurs utilisations
WO2022192657A3 (fr) * 2021-03-12 2022-10-20 Janssen Biotech, Inc. Compositions de protéines de fusion immunomodulatrices obtenues par bio-ingénierie
EP4304629A4 (fr) * 2021-03-12 2025-02-19 Janssen Biotech Inc Compositions de protéines de fusion immunomodulatrices obtenues par bio-ingénierie

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