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WO2022047243A1 - Méthodes et compositions pour traiter des maladies auto-immunes et un cancer - Google Patents

Méthodes et compositions pour traiter des maladies auto-immunes et un cancer Download PDF

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Publication number
WO2022047243A1
WO2022047243A1 PCT/US2021/048074 US2021048074W WO2022047243A1 WO 2022047243 A1 WO2022047243 A1 WO 2022047243A1 US 2021048074 W US2021048074 W US 2021048074W WO 2022047243 A1 WO2022047243 A1 WO 2022047243A1
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construct
seq
tnfr2
tnfr1
linker
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PCT/US2021/048074
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English (en)
Inventor
Harold Michael Shepard
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Enosi Life Sciences Corp
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Enosi Life Sciences Corp
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Priority to KR1020237009895A priority Critical patent/KR20230078657A/ko
Priority to IL300930A priority patent/IL300930A/en
Priority to EP21786626.8A priority patent/EP4204094A1/fr
Priority to CN202180072889.2A priority patent/CN116847887A/zh
Priority to AU2021332460A priority patent/AU2021332460A1/en
Priority to CA3193273A priority patent/CA3193273A1/fr
Priority to JP2023514071A priority patent/JP2023541816A/ja
Publication of WO2022047243A1 publication Critical patent/WO2022047243A1/fr
Priority to US17/731,595 priority patent/US20220288226A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
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    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C—CHEMISTRY; METALLURGY
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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    • 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
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Definitions

  • This application is directed to nucleic acid constructs and encoded products for use as anti-TNF therapies.
  • the treated diseases are those in which TNF receptors and/or TNF or the TNF/TNF receptor(s) pathways is involved or plays a role in the etiology thereof.
  • Anti-TNF therapies/TNF -blockers typically are prescribed after the failure of conventional DMARDs.
  • These therapies include monoclonal antibodies (mAbs), such as the chimeric mAb infliximab (Remicade®); containing a murine variable region and a human IgGl constant region, and the fully humanized mAbs (IgGls) adalimumab (sold, for example under the trademark Humira®), and golimumab (Simponi® antibody); the PEGylated humanized Fab’ fragment of a mAb targeting TNF, certolizumab pegol (Cimzia® antibody); and TNFR2 fusion proteins, such as the TNFR2-Fc fusion protein etanercept (sold under the trademark Enbrel®), which contains the extracellular receptor region that contains the binding site of human TNFR2 fused to the Fc of human I
  • mAbs monoclonal antibodies
  • mAbs such as
  • the drugs sold under the trademarks Remsima® and Inflectra® are biosimilars of infliximab that are approved for use in the European Union for the treatment of various autoimmune and chronic inflammatory diseases and disorders.
  • These TNF inhibitors which sequester TNF, are used for the treatment of various diseases and conditions, including, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA), and/or inflammatory bowel disease (IBD; such as, Crohn’s disease and ulcerative colitis).
  • Such therapies are associated with severe side effects, including, for example, an increased risk of sepsis and serious infections, such as listeriosis, reactivation of tuberculosis, reactivation of hepatitis B/C, reactivation of herpes zoster, and invasive fungal and other opportunistic infections, including reactivation of M. tuberculosis infection.
  • These therapies have been shown to induce macrophage apoptosis in the rheumatoid synovium.
  • Infliximab is associated with increased apoptosis in the inflammatory cell infiltrate in the guts of patients with Crohn’s disease.
  • anti-rheumatic drugs such as methotrexate and glucocorticoids
  • CNS central nervous system
  • DMARDs Disease Modifying Anti -Rheumatic Drugs
  • DMARDs Disease Modifying Anti -Rheumatic Drugs
  • Other adverse side effects include liver injury, demyelinating disease/CNS disorders, lupus, psoriasis, sarcoidosis, and an increased susceptibility to the development of additional autoimmune diseases, as well as cancers, including lymphomas and solid malignancies (see, e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. U.S.A.
  • TNFR1 tumor necrosis factor receptor 1
  • TNFR2 tumor necrosis factor receptor 2
  • the constructs are for treating diseases, disorders, and conditions in which these receptors and/or TNF are involved in the etiology or in which their inhibition or activation can ameliorate the disease, disorder, and/or condition or a symptom thereof.
  • the constructs provided herein include agonists and antagonists of TNFR1 and TNFR2.
  • TNFR1 antagonist constructs are engineered to inhibit TNFR1 function, and to avoid TNFR1 agonist activity.
  • Agonists of TNFR2 increase regulatory T-cell function to control acute or chronic inflammation.
  • Antagonists of TNFR2 decrease regulatory T- cell function thus increasing immunity, and are for treating cancer and certain immunodeficiency diseases
  • TNF receptors TNFR1 and TNFR2. These pathways balance one another in normal physiology. TNF/TNFR1 drives inflammation, while TNF/TNFR2 is anti-inflammatory. TNFR2 generally is activated later than TNFR1, and so does not immediately impact useful TNF-induced inflammation but activates later to suppress overactivation of inflammatory pathways. Simultaneous inhibition of both pathways removes the inflammation-dampening effect of TNFR2.
  • TNF blockers limit their own efficacy because the Treg generator (TNFR2), which is anti- inflammatory, is turned down/off
  • TNFR2 Treg generator
  • the constructs provided herein among other properties that differ from prior therapeutics that target TNF/TNFRs, inhibit TNFR1 signaling or activity without compromising the ability of a treated subject to fight opportunistic infections.
  • the constructs provided herein is one type that is a modified single chain antibody that specifically targets and inhibits TNFR1, but does not antagonize TNFR2, thereby preventing transient activation of TNFR1 via receptor clustering.
  • Constructs provided herein silence the TNF inflammatory pathway mediated by TNFR1, but retain, and in some embodiments enhance, the healing pathway of TNFR2.
  • constructs can be administered to treat indications where TNF blockers have failed.
  • constructs that specifically inhibit tumor necrosis factor receptor type 1; provided are methods and uses of the constructs for treating diseases, disorders, and conditions in which TNF or receptors therefor play a role in the etiology or in the symptoms.
  • autoimmune diseases including rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, axial spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohn's disease, pediatric Crohn's disease, and ulcerative colitis.
  • the constructs herein can be used to treat the same diseases, but avoid the deleterious or adverse side effects.
  • Constructs provided herein are more effective at suppressing inflammatory cytokines in vivo than prior therapeutics such as s the TNFR2-Fc fusion protein etanercept (sold under the trademark Enbrel®), and preserve regulatory T-cell function.
  • the constructs can include activity modifiers or property modifiers to increase serum half-life, have demonstrated activity in blocking TNFR1 signaling, such as in TNF assays that compare activity with adalimumab and/or etanercept.
  • the constructs preserve macrophage function better than adalimumab, showing they do not lead to opportunistic infections; they also preserve Treg function substantially better than adalimumab or etanercept, and are as therapeutically effective in treating diseases, disorders, and conditions, such as rheumatoid arthritis.
  • the Kd is ⁇ 1 nM
  • the ti/2 in vivo is about 10-12 days.
  • the constructs can be administered by any suitable route for the particular indication. Routes include, but are not limited to, subcutaneously, intravenously, intratumorally, intra-hepatically, topically, mucosally, intradermally, and any other suitable route.
  • constructs that are a tumor necrosis factor receptor 1 (TNFR1) antagonist construct of formula 1 : (TNFR1 inhibitor) n -linker p - (activity modifier) q , wherein: each of n and q is an integer, and each is independently 1, 2, or 3; p is 0, 1, 2 or 3; a TNFR1 inhibitor is a molecule that binds TNFR1 to inhibit (antagonize) activity of TNFR1; an activity modifier is a moiety that modulates or alters the activity or a pharmacological property of the construct compared to the construct in the absence of the activity modifier; and linkers increases flexibility of the construct, and/or moderates or reduces steric effects of the construct or its interaction with a receptor, and/or increases solubility in aqueous media of the construct.
  • Linkers can contain a plurality of components
  • Linkers include chemical linkers, a polypeptide linkers, and combinations thereof.
  • the constructs can be linked via
  • the TNFR1 inhibitor can comprise a domain antibody (dAb) or a single chain antibody.
  • the construct includes those in which the TNFR1 inhibitor is a domain antibody (dAb), the activity modifier is not an unmodified single Fc region or a human serum albumin antibody.
  • the activity modifier or property modifier
  • the TNFR1 inhibitor can be one that inhibits TNFR1 signaling, and/or the activity modifier increases serum half-life of the construct.
  • the constructs include those in which the activity modifier is albumin or an Fc that is modified to have reduced or no ADCC (antibody dependent cellular cytotoxicity) activity and/or reduced or no CDC (complement-dependent cytotoxicity) activity.
  • the TNFR1 inhibitor can be one that inhibits a TNFR1 activity, but does not antagonize tumor necrosis factor receptor 2 (TNFR2) activity.
  • the TNFR1 inhibitor can be one that inhibits TNFR1 signaling.
  • multi-specific constructs comprising a TNFR1 inhibitor and a Treg expander, wherein a bi-specific construct interacts with two different target receptors or antigens or epitopes on a receptor.
  • multi-specific constructs are those that are bi-specific for TFNR1 and a Treg expander.
  • the Treg expander can be a TNFR2 agonist.
  • the constructs can comprise a linker to provide flexibility, increase solubility, and/or to relieve and/or reduce steric hindrance and/or Van der Waals interactions.
  • the constructs optionally, but generally comprise an activity modifier to alter or modulate the activity or a property of the construct.
  • constructs among any of those provided herein, where the TNFR1 inhibitor moiety inhibits binding of TNF- ⁇ binding to TNFR1 and/or inhibits signaling.
  • constructs of formula 3a or 3b (TNFR2 agonist or Treg expander)n- linker p - (activity modifier) q , formula 3a, or (activity modifier) q - linker p -(TNFR2 agonist or Treg expander) n , formula 3b, where: each of n and q is an integer, and each is independently 1, 2, or 3; p is 0, 1, 2 or 3; an activity modifier is a moiety that alters a pharmacological property or an activity of the construct; a TNFR2 agonist interacts with TNRFR2 resulting in TNFR2 activity; a Treg expander, includes TNFR2 agonists, and is molecule that results in increased Treg cells; and a linker increases flexibility and/or moderates or reduces steric effects of the construct or its interaction with a receptor; and/or alters solubility of the construct.
  • the activity modifier is an Fc region or a modified Fc region or a short FcRnBP; and the linker comprise a hinge region, or is a linker comprising G and S residues.
  • linkers are those that increase serum half-life of the construct.
  • the linker can have a sequence set forth in any of SEQ ID NOs: 812-834 or is a PEG moiety linker.
  • the construct comprises an activity modifier that is a modified Fc region or a peptide that increases serum half-life of the construct.
  • the Fc region can be an Fc dimer; the Fc region can be modified to have reduced ADCC and/or CDC activity, such as an Fc modified to have reduced or no ADCC activity.
  • the TNFR1 inhibitor is any as defined in the sequence listing, listed below, or known in the art
  • the Treg expander is any known in the art, is a TNFR2 agonist, or any Treg expander set forth in the sequence listing, or known in the art
  • the linker is any listed in the sequence listing or below or known in the art
  • the activity modifier is any set forth in the sequence listing, known in the art, and/or set forth below.
  • constructs that are TNFR1 antagonist constructs, comprising a TNFR1 inhibitor that is single chain antibody or antigen-binding portion thereof that specifically targets and inhibits TNFR1, but does not antagonize TNFR2, thereby preventing transient activation of TNFR1 via receptor clustering.
  • antibody or antigen-binding portion thereof comprises a modification that improves a pharmacological property and/or structure of the construct.
  • the constructs include component(s) that agonizes TNFR2 signaling to thereby increase expression of regulatory T cells (Tregs), thereby providing TNFR1 antagonism and concomitant (or substantially concomitant) increase in expression of Tregs.
  • the TNFR1 inhibitor can be a single chain antibody that inhibits TNFR1 by inhibiting TNFR1 signaling, such as, for example, where the antibody portion or antigen binding portion of the construct inhibits binding of TNF ⁇ binding to TNFR1.
  • the constructs are those where the TNFR1 inhibitor is an antibody or antigen binding portion that does not inhibit binding of TNF- ⁇ to TNFR1, but does inhibit TNFR1 signaling.
  • the property or activity that can be modulated/altered can be serum half- life.
  • the constructs can comprise an Fc modified to eliminate ADCC and/or CDC activity.
  • the construct can comprise an Fc dimer, such as one in which one Fc monomer comprises holes, and the other comprises knobs, to form heterodimer.
  • the knob mutation(s) is/are selected from among S354C, T366Y, T366W, and T394W by EU numbering
  • the hole mutation(s) is/are selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering, whereby the Fc monomers form the heterodimer.
  • the construct comprises an Fc
  • the Fc is from trastuzumab.
  • the construct can be dimerized by fusion of the N-terminus with the C-terminus of trastuzumab.
  • the linker is or comprises a hinge region from an Fc region.
  • the hinge region is from trastuzumab, and it is linked to the Fc region.
  • the constructs include those that comprise a linker that is linked to the anti-TNFRl antagonist antibody or antigen- binding portion thereof.
  • the linker can be linked to the anti-TNFRl antagonist antibody or antigen-binding portion thereof, and directly or via a hinge region to an Fc region.
  • the Fc region or modified Fc region for example, comprises the sequence of amino acids set forth in any of SEQ ID NOs: 10, 12, 14, 16, 27, 30, 1469, and 1470.
  • constructs that binds to neonatal Fc receptor include TNFR1 constructs that comprise a short FcRn-binding peptide (FcRnBP), where a short FcRn-binding peptide (FcRnBPs) provides for the interaction of the construct with FcRn, and contains 6-25, or 10-20 amino acid residues.
  • FcRnBP contains 12-20 residues or 15 residues or 16 residues.
  • TNFR1 antagonist constructs where the FcRn- binding peptide (FcRnBP) comprises or consists of a peptide of any SEQ ID NOs:48- 51.
  • the constructs include TNFR1 constructs that comprise an Fc heterodimer, where one Fc monomer comprises holes, and the other comprises knobs, whereby the Fc dimer that results is a heterodimer.
  • constructs that are TNFR1 antagonist constructs that comprise: a TNFR1 inhibitor; an Fc dimer; and a Treg expander, where: the Fc dimer comprises two complementary Fc monomers; the TNFR1 inhibitor is linked to one of the Fc monomer, and the Treg expander is linked to the other Fc monomer.
  • the Treg expander can be a TNFR2 agonist.
  • the second Treg expander can be a TNFR2 agonist.
  • the Treg expanders are the same.
  • the TNFR1 inhibitor can be one that inhibits or blocks TNFR1 signaling.
  • the TNFR1 inhibitor binds to TNFR1 and blocks or inhibits
  • the TNFR1 inhibitor binds to TNFR1, does not or interfere with TNF- ⁇ binding, and blocks or inhibits TNFR1 signaling.
  • the Treg expander is a TNFR2 agonist.
  • the TNRF2 agonist can be one that stimulates or induces TNFR2 signaling.
  • Exemplary of the Treg expanders is a TNFR2 agonist that is an scFv, VHH single domain antibody, or Fab of aTNFR2 agonist monoclonal antibody.
  • the Treg expander can be a TNFR2 agonist that is a small molecule, or a nucleic acid aptamer, or a peptide aptamer.
  • the TNFR2 agonist is a construct of formula 3a or 3b, where: formula 3a is (Treg expander)n- linker p - (activity modifier) q , and formula 3b is (activity modifier) q - linker p - (Treg expander) n .
  • each of n and q is an integer, and each is independently 1, 2, or 3; p is 0, 1, 2 or 3; an activity modifier is a moiety that modulates or alters the activity or a pharmacological property of the construct compared to the construct in the absence of the activity modifier; and the linker increases flexibility of the construct, and/or moderates or reduces steric effects of the construct or its interaction with a receptor, and/or increases solubility in aqueous media of the construct.
  • the Treg expander in the construct is a TNFR2 agonist.
  • the TNFR2 agonist stimulates or induces TNFR2 signaling.
  • the Treg expander is a TNFR2 agonist that is an scFv, VHH single domain antibody, or Fab of aTNFR2 agonist monoclonal antibody.
  • the Treg expander can be a TNFR2 agonist that is a small molecule, or a nucleic acid, or peptide aptamer.
  • the construct can be dimerized by N-terminal fusion with the C-terminus of trastuzumab.
  • constructs that comprise a TNFR1 inhibitor moiety linked via a central PEG linker to one more Treg expanders, or that comprise at least two TNFR1 inhibitors that are the same or different, or that comprise two Treg expanders that are the same or different.
  • the constructs that comprise a PEG moiety, such as a central PEG linker can comprise a a branched PEG moiety linking the TNFR1 inhibitor and one or more Treg expanders.
  • n is 1 to 5;
  • R 1 is H or CH 3 , or CH 2 CH 3 or other C1-C5 alkyl is aTNFRl inhibitor (TNFR1 antagonist); is a Treg expander; or
  • Formula 4B is a TNFR1 inhibitor (TNFR1 antagonist) is a Treg expander; n is 1 to 5; or
  • Formula 4C is a TNFR1 inhibitor (TNFR1 antagonist), or a Treg expander; and n is 1 to 5; or
  • Formula 4D each is same or different and each is independently selected from a TNFR1 inhibitor (TNFR1 antagonist), and a TNFR2 agonist; the activity modifier is optional, and can be linked to any suitable locus in the molecule; and n is 1 to 5.
  • the Treg expander can be a TNFR2 agonist.
  • These constructs can include an activity modifier, such as, for example, where the activity modifier is an Fc region, or is an Fc region that includes a hinge region or other linker; and the Fc region or Fc region with hinge region is an Fc that is modified to reduce or eliminate ADCC and/or CDC activity.
  • the Fc or modified Fc is an IgG Fc or is an IgGl or IgG4 Fc, and/or are constructs that bind to neonatal Fc receptor (FcRn).
  • the construct comprises a short FcRn- binding peptide (FcRnBP), where the short FcRn-binding peptide (FcRnBPs) provides for the interaction of the construct with FcRn, and contains 6-25, such as 10-20 amino acid residues; wherein the FcRnBP contains 12-20 residues or 15 residues or 16 residues, such as, for example where the FcRn-binding peptide (FcRnBP) comprises or consists of a peptide of any SEQ ID NOs:48-51.
  • FcRnBP short FcRn-binding peptide
  • TNFR1 antagonist constructs of any of the formulae above and in the application that comprise: a) a TNFR1 inhibitor moiety that is a TNFR1- selective; b) optionally one or more linkers; and c) optionally a half-life extending moiety, where the antagonist construct comprises at least one of b) and c).
  • the TNFR1 -selective antagonist selectively binds and inhibits TNFR1 signaling, but not TNFR2 signaling.
  • the TNFR1 inhibitor, linkers, and other components can be those as described above.
  • the TNFR1 inhibitor that is a selective antagonist comprises an antigen-binding fragment that selectively binds and inhibits TNFR1 signaling but not TNFR2 signaling.
  • the antigen-binding fragment that selectively binds and inhibits TNFR1 signaling but not TNFR2 signaling can comprise a domain antibody (dAb), scFv, or Fab fragment.
  • the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFRl antagonist monoclonal antibody.
  • the human anti- TNFR1 antagonist monoclonal antibody is H398 that comprises SEQ ID NO:678, or ATROSAB, or an antigen binding portion thereof or a sequence having at least 95% sequence identity to SEQ ID NO:31 or 32 or 673 or 678 or an antigen-binding portion thereof that binds to TNFR1.
  • TNFR1 inhibitors are those that comprise a domain antibody (dAb) or antigen binding portion thereof or comprises the sequence of amino acids set forth in any of SEQ ID NOs: 52-672 or a sequence having at least 95% sequence identity thereto that retains TNFR1 inhibitor activity; and/or comprise the scFv set forth in any of SEQ ID NOs:673-678 or variants of these polypeptides having at least 90% or 95% sequence identity thereto that retains TNFR1 inhibitor activity; and/ or comprise the Fab set forth in any of SEQ ID NOs:679-682 or a sequence having at least 90% or 95% sequence identity thereto that retains TNFR1 inhibitor or binding activity; and/or comprises the nanobody whose sequence is set forth in SEQ ID NO: 683 or 684 or a sequence having at least 90% or 95% sequence identity thereto that retains TNFR1 inhibitor or binding activity.
  • dAb domain antibody
  • antigen binding portion thereof comprises the sequence of amino acids set forth in any of SEQ ID NOs: 52
  • TNFR1 inhibitors are those, for example, that comprise a dominant-negative tumor necrosis factor (DN-TNF) or TNF mutein, such as, for example, a DN-TNF or TNF mutein is a soluble TNF molecule, comprising one or more amino acid replacements that confer selective inhibition of TNFR1 and are selected from among:
  • DN-TNF dominant-negative tumor necrosis factor
  • TNF mutein such as, for example, a DN-TNF or TNF mutein is a soluble TNF molecule, comprising one or more amino acid replacements that confer selective inhibition of TNFR1 and are selected from among:
  • the TNFR1 inhibitor is a TNF mutein that comprises the sequence of residues set forth in any one of SEQ ID N0s:701-703, or a sequence with at least or at least about 90% or 95% sequence identity to the sequence of residues set forth in any one of SEQ ID NOs: 701-703 or fragment thereof that retains TNFR1 inhibitor activity.
  • any of the foregoing constructs provided herein can include a linker, where the linker comprises all or a portion of the hinge sequence of trastuzumab, SCDKTH corresponding to residues 222-227 of SEQ ID NO:26 or up to the full sequence of the hinge region of trastuzumab, that contains or has the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO:26), or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof, or residues ESKYGPPCPPCP residues 212-223 of SEQ ID NO:29, or a sequence having at least 98% or 99% sequence identity thereto that is a linker.
  • the linker comprises all or a portion of the hinge sequence of trastuzumab, SCDKTH corresponding to residues 222-227 of SEQ ID NO:26 or up to the full sequence of the hinge region of trastuzumab, that contains or has the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-
  • the construct can comprise a linker, where the linker comprises the sequence SCDKTH, corresponding to residues 222-227 of SEQ ID NO:26.
  • the constructs can comprise in place of or in addition to another of the linkers, a linker that comprises glycine and serine (GS) residues, a GS linker.
  • linkers that comprise a GS linker and all or a portion of the hinge sequence of trastuzumab, corresponding to residues EPKSCDKTHTCPPCP ( residues 219-233 of SEQ ID NO:26), for example, the linker can comprises a GS linker and comprise or contain the only the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID NO:31, from the hinge sequence.
  • Such linkers include, for example, those that comprise a GS linker and all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
  • the constructs herein can contain an activity modifier.
  • the activity modifiers include any described herein, including those described above, and below, and others know to those of skill in the art; the activity modifier alters and activity or property of the construct.
  • the activity modified can be one that is a half-life extending moiety that is an IgG Fc, a polyethylene glycol (PEG) molecule, or human serum albumin (HSA). Examples of IgG Fc is an IgGl or IgG4 Fc.
  • the IgGl Fc can be the Fc of trastuzumab, set forth in SEQ ID NO:27 or a sequence of amino acids having at least 95% sequence identity therewith; the IgG4 Fc can be the Fc of nivolumab, set forth in SEQ ID NO:30 or a sequence of amino acids having at least 95% sequence identity therewith.
  • the IgGl Fc is the Fc of human IgGl, set forth in SEQ ID NO: 10
  • the IgG4 Fc is the Fc of human IgG4, set forth in SEQ ID NO: 16.
  • constructs described herein include those that are TNFR1 inhibitors or comprise a TNFR1 inhibitor(s). These include constructs where the TNFR1 inhibitor is monovalent. These can include linkers, such as where the linker comprises (Gly 4 Ser) 3 , and/or linkers that comprise (Gly 4 Ser) 3 and SCDKTH (residues 217-222 of SEQ ID NO:31); and/or linkers that comprise (Gly 4 Ser) 3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26; and/or those that comprise (Gly 4 Ser) 3 and the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
  • linkers such as where the linker comprises (Gly 4 Ser) 3 , and/or linkers that comprise (Gly 4 Ser) 3 and SCDKTH (residues 217-222 of SEQ ID NO:31); and/or linkers that comprise (G
  • constructs provided herein that inhibit TNFR1 are those that comprise the sequence of residues set forth in any of SEQ ID NOs: 704-764, or a construct that inhibits TNFR1 and has a sequence with at least or at least about 95% sequence identity to the sequence of residues set forth in any one of SEQ ID NOs:704-764.
  • TNFR1 antagonist constructs include those where the TNFR1 construct comprises a short FcRn-binding peptide (FcRnBP); and the short FcRn-binding peptide (FcRnBPs) provides for the interaction of the construct with FcRn, and contains 6-25, such as 10-20 amino acid residues, such as for, example, those where the FcRnBP contains 12-20 residues or 15 residues or 16 residues, such as, for example those where the FcRn-binding peptide (FcRnBP) comprises a peptide of any SEQ ID NOs:48-51 or a peptide having at least about 95% sequence identity therewith, or a FcRn-binding peptide (FcRnBP) that consists of a peptide of any SEQ ID NOs:48-51.
  • FcRnBP short FcRn-binding peptide
  • FcRnBPs short FcRn-binding peptide
  • exemplary TNFR1 -inhibiting constructs include constructs that comprise: a) a domain antibody that inhibits TNFR1; b) a linker that increases flexibility; reduces steric effects, or increases solubility; and c) a half-life extending moiety. Included are such constructs where the half-life extending moiety is not a human serum albumin antibody or an unmodified Fc.
  • the GS linker can be (GGGGS) 3 ; and the IgG Fc can be the Fc of trastuzumab or the Fc of nivolumab.
  • constructs comprising: a) the domain antibody (dAb) of any of SEQ ID NOs:52-672, or the scFv of any of SEQ ID NOs:673-678 or the Fab of any of SEQ ID NOs:679-682, or the nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID NOs:685-703; b) a linker selected from among all or a portion of the hinge sequence of trastuzumab and all or a portion of the hinge sequence of nivolumab; and c) a half-life extending moiety that is an IgG Fc.
  • the linker can comprise all or a portion of the hinge sequence of trastuzumab, where the IgG Fc is the Fc of trastuzumab. In other embodiments, the linker can comprise all or a portion of the hinge sequence of nivolumab, where the IgG Fc is the Fc of nivolumab.
  • these constructs can contain a first linker that is a GS linker is (GGGGS) 3 ; and a second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO:31); and the IgG Fc is the Fc of trastuzumab.
  • the first linker is the GS linker is (GGGGS) 3 ; the second linker comprises all or a portion of the hinge sequence of nivolumab; and the IgG Fc is the Fc of nivolumab.
  • the GS linker can be any described herein or known to those of skill in the art, such as (GGGGS) 3 .
  • the PEG molecule can be one that has a molecular weight of at least 25kDa, generally at least 30 kDa or more, such as at least 40 kDa or 50 kDa, or 60 kDa, or 80 kDa, or more..
  • the primary amino acid sequence of any of the constructs provided herein can be optimized or modified to eliminate immunogenic sequences or immunogenic epitopes.
  • the IgG Fc can be modified to comprise one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) a modification(s) to reduce or eliminate immune effector functions.
  • knob mutation can be selected from among S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering.
  • TNFR1 antagonist constructs can be one where the modification(s) to increase or enhance FcRn recycling is selected from among one or more of: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y
  • the TNFR1 antagonist constructs that can be modified to reduce or eliminate immune effector function(s), such as immune effector function(s) that is/are selected from among one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • the modification(s) to reduce or eliminate immune effector functions are selected from among one or more of: in IgGl: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234 A/L235 A/P329G, L234 A/L235 A/G237 A/P238 S/H268 A/A330 S/P331 S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A, by EU numbering; and in IgG4: L235E, F234A/L235A, S228P/L235E, and
  • the TNFR1 antagonist or multispecific constructs can comprise a central PEG linker moiety; and the construct can comprise a modified Fc region, such as those described above, where Fc region is a modified IgG Fc and the modified IgG Fc comprises one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes, wherein: the knob mutation is selected from among S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modification is selected from among one or more of:
  • K326M/E333S K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y; by EU numbering.
  • the construct can comprise an IgGl Fc that comprises one or more modifications to increase binding to the inhibitory Fey receptor (Fc ⁇ R) Fc ⁇ RIIb.
  • the modification or modifications that increase binding to Fc ⁇ RIIb is/are selected from among one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K, by EU numbering.
  • constructs that are a Treg expander construct. Included among such constructs are those comprising: a) a Treg expander; b) a linker, wherein a linker increases flexibility of the construct, and/or moderates or reduces steric effects of the construct or its interaction with a receptor, and/or increases solubility in aqueous media of the construct; and c) an activity modifier, wherein an activity modifier is a moiety that modulates or alters the activity or the pharmacological property of the construct compared to the construct in the absence of the activity modifier.
  • the Treg expander can be a TNFR2 agonist. These constructs can further comprise a TNFR1 -inhibitor.
  • the TNFR2 agonist is a TNFR2 selective agonist.
  • the constructs described herein that are TNFR2 agonist constructs, comprising: a) a TNFR2 agonist; b) a linker, wherein a linker increases flexibility of the construct, and/or moderates or reduces steric effects of the construct or its interaction with a receptor, and/or increases solubility in aqueous media of the construct; and c) an activity modifier, wherein an activity modifier is a moiety that modulates or alters the activity or the pharmacological property of the construct compared to the construct in the absence of the activity modifier.
  • the TNFR2 agonist can be a TNFR2-selective agonist.
  • the constructs can comprise an activity modifier, such as an activity modifier that is a half-life extending moiety.
  • the constructs can be TNFR2 agonist constructs that selectively activates or antagonizes TNFR2, without activating or antagonizing TNFR1. Included are TNFR2 agonist constructs, where the TNFR2 agonist binds to one or more epitopes within TNFR2. These include human TNFR2.
  • Such epitopes include, for example, epitopes selected from among one or more of the epitopes comprising or consisting of the sequences of amino acids set forth in SEQ ID NOs:839-865, 1202 and 1204.
  • the TNFR2 agonist constructs where the TNFR2 agonist comprises an antigen-binding fragment of an agonist human anti-TNFR2 antibody or humanized anti-TNFR2 antibody, or antigen-binding portion thereof, or a single chain form thereof.
  • agonist anti-TNFR2 antibody is selected from MR2-1 (also designated ab8161; U.S. Patent No. 9,821,010) or MAB2261 (U.S. Patent No. 9,821,010).
  • the TNFR2 agonist can be an antigen- binding fragment selected from a dAb, scFv, or Fab fragment.
  • the TNFR2 agonist is a TNFR2-selective agonist.
  • the selective agonist can comprise a TNFR2 agonist TNF mutein.
  • TNFR2 selective agonist muteins include, but are not limited to soluble TNF variants comprising one or more TNFR2-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145
  • linkers include any described herein or known to those of skill in the art for use as linkers.
  • Exemplary linkers comprise all or a portion of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26, or comprises all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29, or a sequence having at least 95% sequence identity thereto.
  • Other exemplary linkers comprise or consist of the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID NO:31.
  • GS glycine-serine
  • Linkers can comprise combinations of likers, such as, for example, a linker that comprises a GS linker and all or a portion of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26, or a GS linker and the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID NO:31, or a GS linker and all or a portion of the hinge sequence of nivolumab, corresponding to residues 212- 223 of SEQ ID NO:29.
  • All of the constructs provided herein can include a activity modifier that alters or modulates a property or activity of a construct.
  • a half-life extending moiety is an activity or property modifier.
  • IgG Fc IgG Fc
  • PEG polyethylene glycol
  • HSA human serum albumin
  • IgG Fc is an IgGl or IgG4 Fc.
  • the IgGl Fc is the Fc of trastuzumab, set forth in SEQ ID NO:27; and of the IgG4 Fc is the Fc of nivolumab, set forth in SEQ ID NO:30, human versions, where the IgGl Fc is the Fc of human IgGl, set forth in SEQ ID NO: 10, and the IgG4 Fc is the Fc of human IgG4, set forth in SEQ ID NO:16.
  • the TNFR2 agonist is monovalent; in others it is multivalent, such as bivalent or trivalent.
  • the TNFR2 constructs can contain linkers as described herein.
  • the linker can comprise Gly-Ser, such as (Gly 4 Ser) 3 , or (Gly 4 Ser) 3 and SCDKTH (residues 217-222 of SEQ ID NO:31), or (Gly 4 Ser) 3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26, or (Gly 4 Ser) 3 and the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29, or variants of any of the preceding that have at least 95% sequence identity thereto.
  • These constructs also can include an activity modifier, such as a modifier that is a half-life extending moiety, such as a PEG, or HSA as described above.
  • PEG moieties have a size of at least 20 kDa, typically at least 30 kDa or more as described above and below.
  • TNFR2 agonist constructs comprise: a) a TNFR2 agonist that binds to one or more epitopes within human TNFR2 that is selected from among the epitopes set forth in SEQ ID NOs:839-865, 1202 and 1204; b) a linker selected from among all or a portion of the hinge sequence of trastuzumab and all or a portion of the hinge sequence of nivolumab; and c) an activity modifier that is a half-life extending moiety that is an IgG Fc.
  • Exemplary of the linker and activity modifier is the hinge sequence of trastuzumab; and the IgG Fc is the Fc of trastuzumab, or all or a portion of the hinge sequence of nivolumab; and the IgG Fc is the Fc of nivolumab.
  • first GS linker is (GGGGS) 3
  • second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO:31); and the IgG Fc is the Fc of trastuzumab.
  • the first linker is (GGGGS the second linker comprises all or a portion of the hinge sequence of nivolumab; and the IgG Fc is the Fc of nivolumab.
  • GSSSGSGSSG GSSSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and/or ii) all or a portion of the hinge sequence of trastuzumab or all or a portion of the hinge sequence of nivolumab; and c) an activity modifier that is a half-life extending moiety selected from among an IgGl or IgG4 Fc, a PEG molecule, and human serum albumin (HSA), wherein: the IgGl Fc is the Fc of human IgGl, set forth in SEQ ID NO: 10, or is the Fc of trastuzumab, set forth in SEQ ID NO:27; and the PEG molecule has a molecular weight of at least or at least about
  • that construct is a TNFR2 agonist construct, comprising: a) TNFR2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T
  • GSSSGSGSSG GSSSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and/or ii) all or a portion of the hinge sequence of trastuzumab or all or a portion of the hinge sequence of nivolumab; and c) an activity modifier that is a half-life extending moiety selected from among an IgGl or IgG4 Fc, a PEG molecule, and human serum albumin (HSA), wherein: the IgGl Fc is the Fc of human IgGl, set forth in SEQ ID NO: 10, or is the Fc of trastuzumab, set forth in SEQ ID NO:27; and the PEG molecule has a molecular weight of at least or at least about
  • the construct is a TNFR2 agonist construct, comprising: a) a TNFR2 TNF mutein comprising the mutations D143N/A145R; b) a (GGGGS) 3 linker; and c) an activity modifier that is a half-life extending moiety that is the Fc of trastuzumab or the Fc of nivolumab.
  • the construct is a TNFR2 agonist construct that comprises a) a TNFR2-selective TNF mutein comprising the mutations D143N/A145R; b) a (GGGGS) 3 linker and a second linker that comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and c) an activity modifier that is a half-life extending moiety that is the Fc of trastuzumab.
  • the construct is a TNFR2 agonist construct, comprising: a) a TNFR2-selective TNF mutein comprising the mutations D143N/A145R; b) a (GGGGS) 3 linker and a second linker that comprises all or a portion of the hinge sequence of nivolumab; and c) an activity modifier that is a half-life extending moiety that is the Fc of nivolumab.
  • the construct is a TNFR2 agonist construct that comprises: a) a TNFR2-selective TNF mutein comprising the mutations D143N/A145R; b) a linker comprising all or a portion of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26; and c) a half-life extending moiety that is the Fc of trastuzumab.
  • the construct is a TNFR2 agonist construct that comprises: a) a TNFR2-selective TNF mutein comprising the mutations D143N/A145R; b) a linker comprising all or a portion of the hinge sequence of mvolumab, corresponding to residues 212-223 of SEQ ID NO:29; and c) an activity modifier that is a half-life extending moiety that is the Fc of nivolumab.
  • TNFR1 antagonist constructs TNFR2 agonist constructs, and both, where the IgG Fc is a monomer or a dimer.
  • the constructs provided herein can comprise a dAb (or a Vhh).
  • the constructs can comprise Vhh single chain or double chain containing a dAb.
  • These constructs can contain HSA linked to the dAb directly or via a linker. They HSA and dAb can be linked in any order, such as the C-terminus of the dAb linked directly or via a linker, such as any described above, to the N- terminus of HSA.
  • Exemplary of such constructs are those that comprise: a) residues 20-732, which is the dAb Domlh-131-206 of SEQ ID NO:59, linked via a linker to HSA, as set forth in SEQ ID NO: 1475, or a construct having at least 95%, 96%, 97%, 98%, 99% sequence identity to the construct of SEQ ID NO: 1475 or to residues 20-732 of SEQ ID NO: 1475 and having TNFR1 antagonist activity; or b) a dAb set forth in in any of SEQ ID NOs: 53-83 and 503-671, and variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto, whereby the construct has TNFR1 antagonist activity; or c) a dAb that has the sequence set forth in any of SEQ ID NOs: 57-59 and variants thereof have at least 95% sequence identity thereto, whereby the construct has TNFR1 antagonist activity; or d) the dAb is designated DOMl
  • the constructs provided herein that are TNFR1 constructs can further comprise a TNFR2 agonist or the construct can be a TNFR2 agonist construct.
  • the TNFR2 agonist can be modified to eliminate sequences of amino acids or epitopes that are immunogenic in the subject to be treated, such as for administration to a human subject.
  • it can be a TNFR2-selective agonist.
  • These constructs can comprise a modified IgG Fc.
  • the IgG Fc can comprise one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) a modification(s) to reduce or eliminate immune effector functions, selected from among one or more of complement-dependent cytotoxicity (CDC), antibody- dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • a modification(s) to introduce knobs-into-holes are selected from: one or more knob mutations selected from among S354C, T366Y, T366W, and T394W by EU numbering; and one or more hole mutations selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering, whereby the Fc forms a dimer;
  • the modification(s) to increase or enhance FcRn recycling is selected from among one or more of T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T
  • Constructs provided herein include TNFR2 agonist constructs that contain a modified IgG Fc, where the IgG Fc comprises one or more of the following modifications: a) one or more modification(s) to introduce knobs-into-holes, wherein: the knob mutation is selected from among S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modification is selected from among one or more of:
  • K222W/T223W/H224W D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y; by EU numbering.
  • the constructs provided herein that are TNFR2 agonist construct can comprise a modified IgGl Fc, such as where the Fc is modified to increase binding to the inhibitory Fey receptor (Fc ⁇ R) Fc ⁇ RIIb, which can include modifications that increase binding to Fc ⁇ RIIb.
  • modifications are those selected from among one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K, by EU numbering.
  • constructs that are or comprise a TNFR2 agonist construct hat selectively activates or agonizesTNFR2, without activating or antagonizing TNFR1.
  • constructs include those comprising: a) a TNFR2 agonist; b) one or more linkers; and c) an activity modifier that is a half-life extending moiety, where: the TNFR2 agonist construct is a fusion protein comprising single-chain TNFR2-selective TNF mutein trimers fused with a multimerization domain, and comprises the formula:
  • TNFmut-Ll-TNFmut-L2-TNFmut-L3-MD (Formula III);
  • TNFmut is a TNFR2-selective TNF mutein
  • LI, L2 and L3 are linkers that can be the same or different.
  • the TNF muteins can comprise one or more TNFR2-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S/S
  • the multimerization domain can be selected from EHD2 (SEQ ID NO:808), MHD2 (SEQ ID NO:811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or the trimerization domain of human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO:807), or variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto.
  • EHD2 SEQ ID NO:808
  • MHD2 SEQ ID NO:811
  • TNC trimerization domain of chicken tenascin C
  • human TNC trimerization domain of human TNC
  • the multimerization domain is an IgGl Fc or an IgG4 Fc and the IgGl Fc or IgG4 Fc also is a half-life extending moiety.
  • the half-life extending moiety can be selected from among: an IgGl Fc that is the Fc of human IgGl, set forth in SEQ ID NO: 10, or the Fc of trastuzumab, set forth in SEQ ID NO:27; an IgG4 Fc that is the Fc of human IgG4 set forth in SEQ ID NO: 16, or the Fc of nivolumab, set forth in SEQ ID NO:30; a PEG molecule that is at least or at least about 30 kDa in size; human serum albumin (HSA), and variants of the polypeptide portions having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto.
  • HSA human serum albumin
  • constructs that are or comprise a TNFR2 agonist construct. These constructs include those that comprise the formula:
  • TNFmut-Ll-TNFmut-L2-TNFmut-L3-MD (Formula III), where: a) MD is a multimerization domain; TNFmut is a TNFR2-selective TNF mutein; and LI, L2 and L3 are linkers that can be the same or different, wherein: i) the MD is selected from EHD2 (SEQ ID NO:808), MHD2 (SEQ ID NO:
  • the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO:804; SEQ ID NO:805), or the trimerization domain of human TNC (residues 110-139 of SEQ ID NO:806, SEQ ID NO:807);
  • the TNF muteins comprise the TNFR2-selective mutations D143N/A145R;
  • a half-life extending moiety selected from among: an IgGl Fc that is the Fc of human IgGl, set forth in SEQ ID NO: 10, or the Fc of trastuzumab, set forth in SEQ ID NO:27; an IgG4 Fc that is the F
  • the MD can be selected from: an IgGl Fc that is the Fc of human IgGl, set forth in SEQ ID NO: 10, or the Fc of trastuzumab, set forth in SEQ ID NO:27; or an IgG4 Fc that is the Fc of human IgG4 set forth in SEQ ID NO: 16, or the Fc of nivolumab, set forth in SEQ ID NO: 30; or combination or variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto.
  • Exemplary constructs are those that include an MD that is the IgGl Fc of trastuzumab, and the linker between the MD and the adjacent TNF mutein is all or a portion of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26, or an MD that is the IgGl Fc of trastuzumab, and the linker between the MD and the adjacent TNF mutein comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO:31).
  • An exemplary construct is one that comprises an MD that is the IgGl Fc of trastuzumab, where the linker between the MD and the adjacent TNF mutein comprises (Gly 4 Ser) 3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26.
  • the MD is the IgGl Fc of trastuzumab
  • the linker between the MD and the adjacent TNF mutein comprises (Gly 4 Ser) 3 and SCDKTH (residues 222-227 of SEQ ID NO:31), those wherein the MD is the IgG4 Fc of nivolumab, and the linker between the MD and the adjacent TNF mutein comprises all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29, or those where the MD is the IgG4 Fc of nivolumab, and the linker between the MD and the adjacent TNF mutein comprises (Gly 4 Ser) 3 and all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
  • constructs herein can be modified to eliminate immunogenic sequences, such as those immunogenic to humans.
  • TNFR2 agonist constructs where the TNFR2 agonist is modified to eliminate immunogenic sequences or epitopes that are immunogenic in the subject, such as a human subject.
  • the IgG Fc cam comprise one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes, wherein: the knob mutation is selected from among one or more of S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modification is selected from among one or more of: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M4
  • T256N/A378V/S383N/N434Y, by EU numbering and c) a modification(s) to reduce or eliminate immune effector functions, wherein: the immune effector functions are selected from among one or more of CDC, ADCC and ADCP; and the modification(s) in to reduce or eliminate immune effector functions is selected from among one or more of: in IgGl: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G,
  • L234A/L235 A/G237 A/P238 S/H268 A/A330S/P331 S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A, by EU numbering; and in IgG4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A, by EU numbering.
  • any of the foregoing constructs that are TNFR2 agonist constructs that comprise a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes, wherein: the knob mutation is selected from among one or more of S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modification is selected from among one or more of: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E
  • G236A/S239D/I332E G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; introduction of an afucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W;
  • K222W/T223W/H224W D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y; by EU numbering.
  • any of the foregoing TNFR2 agonist constructs of any of claims that comprise an IgGl Fc that is modified to increase binding to the inhibitory Fey receptor (Fc ⁇ R) Fc ⁇ RIIb are provided.
  • the modifications that increase binding to Fc ⁇ RIIb are selected from among one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K, by EU numbering.
  • constructs provided herein can be multi-specific in that they interact with two or more targets.
  • exemplary of such multi-specific constructs are those are multi- specific TNFR1 inhibitor/TNFR2 agonist constructs and are of any of the following formulae:
  • constructs include those that are multi-specific TNFR1 inhibitor/TNFR2 agonist constructs, where: the TNFR1 inhibitor selectively inhibits or antagonizes TNFR1 signaling without inhibiting or antagonizing TNFR2 signaling; the TNFR1 inhibitor does not interfere with the activation or agonism of TNFR2; the TNFR2 agonist selectively activates or agonizes TNFR2 signaling without activating or agonizing TNFR1 signaling; and the TNFR2 agonist does not interfere with the inhibition or antagonism of TNFR1.
  • TNFR1 inhibitor is selected from among: i) an antigen-binding fragment of a human anti-TNFRl antagonist monoclonal antibody selected from H398 or ATROSAB or a polypeptide with a sequence having at least 95% sequence identity therewith; or ii) the domain antibody (dAb) of any of SEQ ID NOs: 52-672, or the scFv of any of SEQ ID NOs: 673 -678 or the Fab of any of SEQ ID NOs: 679- 682, or the nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID N0s:701-703, or a polypeptide with a sequence that has at least 95% sequence identity with any of the preceding polypeptides, and is a TNFR1 inhibitor; or iii) a dominant-negative tumor necrosis factor (DN-TNF) or TNF
  • DN-TNF dominant-negative tumor necrosis factor
  • the IgGl Fc is selected from the IgGl Fc of human IgGl, set forth in SEQ ID NO: 10, or the IgGl Fc of trastuzumab, set forth in SEQ ID NO:27; the IgG4 Fc is selected from the IgG4 Fc of human IgG4, set forth in SEQ ID NO: 16, or the IgG4 Fc of nivolumab, set forth in SEQ ID NO:30; and optionally, the Fc includes one or more
  • MD-Ll-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-Ll-TNFmut-L2-TNFmut-L3-MD (Formula III); whereby MD is a multimerization domain; TNFmut is a TNFR2- selective TNF mutein; and LI, L2 and L3 are linkers that can be the same or different, and wherein: the MD is selected from EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO:804; SEQ ID NO:805), or the trimerization domain of human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
  • TNFR1 inhibitor comprises a domain antibody (dAb) of any of SEQ ID NOs:52-672, or the scFv of any of SEQ ID NOs:673-678 or the Fab of any of SEQ ID NOs:679-682, or the nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID N0s:701-703, or a sequence with at least or at least about 95% sequence identity thereto; b) the linker comprises (GGGGS) 3 , the polypeptide comprising the sequence SCDKTH (residues 222-227 of SEQ ID NO:26), and the Fc of trastuzumab; and c) the TNFR2 agonist comprises a TNFR2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2-se
  • the TNFR1 inhibitor comprises a domain antibody (dAb) of any of SEQ ID NOs:52-672, or the scFv of any of SEQ ID NOs:673-678 or the Fab of any of SEQ ID NOs:679-682, or the nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID N0s:701-703, or a sequence with at least or at least about 95% sequence identity thereto; b) the linker comprises (GGGGS) 3 , all or a portion of the hinge sequence of nivolumab, and the Fc of nivolumab; and c) the TNFR2 agonist comprises a TNFR2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E,
  • the TNFR1 inhibitor comprises a domain antibody (dAb) of any of SEQ ID NOs:52-672, or the scFv of any of SEQ ID NOs:673-678 or the Fab of any of SEQ ID NOs:679-682, or the nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID N0s:701-703, or a sequence with at least or at least about 95% sequence identity thereto; b) the linker comprises (GGGGS) 3 , and the Fc of trastuzumab; and c) the TNFR2 agonist comprises a TNFR2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K
  • the TNFR1 inhibitor comprises a domain antibody (dAb) of any of SEQ ID NOs:52-672, or the scFv of any of SEQ ID NOs:673-678 or the Fab of any of SEQ ID NOs:679-682, or the nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID N0s:701-703, or a sequence with at least or at least about 95% sequence identity thereto; b) the linker comprises (GGGGS) 3 , and the Fc of nivolumab; and c) the TNFR2 agonist comprises a TNFR2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145
  • These multi-specific constructs can comprise a modified Fc, wherein the IgG Fc comprises one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) a modification(s) to reduce or eliminate immune effector functions.
  • the IgG Fc comprises one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) a modification(s) to reduce or eliminate immune effector functions.
  • the knob mutation is selected from among one or more of S354C, T366Y, T366W, and T394W by EU numbering
  • the hole mutation is selected from among one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering.
  • Fc comprises modifications to increase or enhance FcRn recycling is/are selected from among one or more of T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259l/V308F, V259l/V308F/M428L, E294del/T307
  • the Fc can comprise modifications to immune effector functions that are selected from among one or more of complement-dependent cytotoxicity (CDC), antibody- dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP).
  • the Fc can comprise modification(s) to reduce or eliminate immune effector functions in IgGl and/or IgG4: in IgGl : L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235 A/P329G, L234 A/L235 A/G237 A/P238 S/H268 A/A330 S/P331 S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P3
  • the IgG Fc can comprise one or more of the following modifications: a) a modification(s) to introduce knobs-into-holes, wherein: the knob mutation is selected from among one or more of S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering; b) a modification(s) to increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modification is selected from among one or more of:
  • T250Q T250R, M252F, M252W, M252Y, S254T, T256D, T256E,
  • G236A/S239D/I332E G236A/S239D/A330L/I332E; introduction of a biantennary glycan at residue N297; introduction of an afucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W;
  • K222W/T223W/H224W D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y; by EU numbering.
  • multi-specific constructs are those where: the construct that comprises an IgGl Fc that is modified to increase binding to the inhibitory Fey receptor (Fc ⁇ R) Fc ⁇ RIIb.
  • the modifications that increase binding to Fc ⁇ RIIb are selected from among one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K, by EU numbering.
  • constructs that are a multi-specific TNFR1 antagonist/TNFR2 agonist the TNFR1 antagonist is monovalent; and the TNFR2 agonist is monovalent.
  • multi-specific constructs that are a multi-specific TNFR1 antagonist/TNFR2 agonist constructs, where the TNFR1 antagonist is monovalent; and the TNFR2 agonist is bivalent.
  • the multi-specific constructs are multi-specific TNFR1 antagonist/TNFR2 agonist constructs, where: a) the TNFR1 antagonist is selected from: i) an antigen-binding fragment of a human anti-TNFRl antagonist monoclonal antibody selected from H398 or ATROSAB; or ii) the domain antibody (dAb) of any of SEQ ID NOs: 52-672, or the scFv of any of SEQ ID NOs: 673 -678 or the Fab of any of SEQ ID NOs: 679- 682, or the nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID N0s:701-703, or a sequence with at least or at least about 95% sequence identity thereto; or iii) a dominant-negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule, with one or more amino acid replacements that confer selective inhibition of TN
  • MD-Ll-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-Ll-TNFmut-L2-TNFmut-L3-MD (Formula III); whereby MD is a multimerization domain; TNFmut is a TNFR2- selective TNF mutein; and LI, L2 and L3 are linkers that can be the same or different, and wherein: the MD is selected from EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or the trimerization domain of human TNC (residues 110-139 of SEQ ID NO:806, SEQ ID NO:807);
  • EHD2 SEQ ID NO: 808
  • MHD2 SEQ ID NO: 811
  • TNC trimerization domain of chicken tenas
  • each of the TNFR1 antagonist and TNFR2 agonist is monovalent. Also provided are such constructs where the TNFR1 antagonist is monovalent, and the TNFR2 agonist is bivalent.
  • the constructs provided herein can be used for treatments and uses for treatment of various diseases, disorders, and conditions.
  • the multi- specific constructs that are multi-specific TNFR1 antagonist/TNFR2 agonist, for use for the treatment of a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology.
  • TNFR1 antagonist/TNFR2 agonist constructs for the treatment of a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology are provided.
  • compositions comprising a construct of any of the constructs provided herein in a pharmaceutically acceptable carrier or vehicle.
  • These compositions can be used for or in methods of treatment of diseases, disorders, and conditions, such as, but not limited to, a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, and a disease, condition or disorder characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology.
  • a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder or the disease, condition or disorder is a diseases, disorders, and conditions characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology.
  • RA rheumatoid arthritis
  • JIA psoriasis
  • JIA psoriatic arthritis juvenile idiopathic arthritis
  • IBD inflammatory bowel disease
  • IBD uveitis
  • fibrotic diseases endometriosis
  • MS multiple sclerosis
  • congestive heart failure cardiovascular disease
  • myocardial infarction MI
  • atherosclerosis metabolic diseases
  • cytokine release syndrome septic shock
  • sepsis acute respiratory distress syndrome
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, Alzheimer’s disease, Parkinson’s disease, Behget's disease, Dupuytren’s disease, Tumor Necrosis Factor Receptor- Associated Periodic Syndrome (TRAPS)
  • TRAPS Tumor Necrosis Factor Receptor- Associated Periodic Syndrome
  • constructs that are TNFR2 antagonist constructs that comprises a TNFR2 antagonist, and optionally a linker and optionally an activity modifier.
  • Such constructs for example, have formula 5:
  • TNFR2 antagonist n -linker p - (activity modifier) q , or linker p - (activity modifier) q _(TNFR2 antagonist) n
  • n and q are an integer, and each is independently 1, 2, or 3
  • p is 0, 1, 2 or 3
  • a TNFR2 antagonist is a molecule that interacts with TNFR2 to inhibit (antagonize) its activity TNFR2 to thereby inhibit the proliferation of and/or induce the death of Tregs, and also can inhibit the proliferation of and induce the death of TNFR2-expressing tumor cells
  • an activity modifier is a moiety that modulates or alters the activity or the pharmacological property of the construct compared to the construct in the absence of the activity modifier
  • a linker increases flexibility of the construct, and/or moderates or reduces steric effects of the construct or its interaction with a receptor, and/or increases solubility in aqueous media of the construct.
  • each of the activity modifier and linker is as defined and described for the constructs above and below. They can be used in the methods of treatments and uses, and in pharmaceutical compositions.
  • the TNFR2 antagonist can be used for different diseases, disorders, and conditions, such as to reduce and/or inhibit the proliferation of myeloid-derived suppressor cells (MDSCs); and/or induce apoptosis within MDSCs, by binding TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment; and/or induce the expansion of T effector cells, including cytotoxic CD8 + T cells, via the inhibition of Treg expansion and activity.
  • MDSCs myeloid-derived suppressor cells
  • the TNFR2 antagonists in the constructs include an antibody, antigen-binding fragment thereof, or single chain antibody that bind to epitopes within human TNFR2 that contain one or more of the residues KCRPG (corresponding to residues 142-146 of SEQ ID NO:4), or a larger epitope, containing residues 130-149, 137-144 or 142-149, or at least 5 continuous or discontinuous residues within these epitopes, for example, and do not bind to the epitope containing residues KCSPG (corresponding to residues 56-60 of SEQ ID NO:4); or that binds to the TNFR2 epitope PECLSCGS (corresponding to residues 91-98 of SEQ ID NO:4), RICTCRPG (corresponding to residues 116-123 of SEQ ID NO:4), CAPLRKCR (corresponding to residues 137-144 of SEQ ID NO:4), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO:4), and/or VVCK
  • the antibody, fragment thereof, or single chain form thereof binds to an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO:840), with an affinity that is at least 10-fold greater than the affinity of the same antibody or antigen-binding fragment for a peptide that contains the KCSPG sequence of human TNFR2 (SEQ ID NO:839).
  • the TNFR2 antagonist is an antibody or fragment or single chain form of an antibody selected from among:
  • TNFRAB1 see, SEQ ID NOs:1212 and 1213 for the sequences of the heavy and light chains of TNFRAB1, respectively
  • TNFRAB2 and TNFR2A3 see, e.g., U.S. Patent Publication No.
  • TNFRAB1 QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO:1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223), or a CDR-H3 sequence with at least about 85% sequence identity thereto.
  • TNFRAB1 QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO:1212
  • TNFRAB2 ARDDGSYSPFDYWG; SEQ ID NO: 1217
  • TNFR2A3 ARDDGSYSPFDYFG; SEQ ID NO: 1223
  • TNFRAB1 for example, that specifically binds residues 130-149, containing residues KCRPG of TNFR2, with a 40-fold higher affinity than residues 48-67, containing residues KCSPG of TNFR2.
  • the TNFR2 antagonist binds to one or more epitopes in TNFR2 selected from among: the epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO:851) the epitope that includes one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248), and/or 116- 123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR2; and an epitope to which TNFR2A3 selected from a first epitope includes residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and contains the KCRPG motif, and/or a second epitope that contains residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464).
  • CAPLRKCR epitope containing residues 137-144
  • SEQ ID NO:851
  • the TNFR2 antagonist in the construct is an antibody, fragment thereof, or single chain form thereof that contains on or more of the CDR- H1 amino acids with the sequences set forth in any of SEQ ID NOs: 1214, 1215, and 1231-1233, the CDR-H2 sequences set forth in any of SEQ ID NOs: 1216, 1224, and 1230, the CDR-H3 sequences set forth in any of SEQ ID NOs: 1217, 1223, and 1225- 1229, and/or the CDR-H3 of TNFRAB1, corresponding to residues 99-112 of SEQ ID NO: 1212; the CDR-L1 sequences set forth in any of SEQ ID NOs: 1218 and 1234- 1236, and/or the CDR-L1 sequence of TNFRAB1, corresponding to residues 24-33 of SEQ ID NO: 1213; the CDR-L2 sequences set forth in any of SEQ ID NOs: 1219, 1220, 1237 and 1238, or the CDR
  • the construct comprises a TNFR2 antagonist specifically binds to an epitopes within TNFR2 set forth in any one of SEQ ID NOs: 1247-1464.
  • the TNFR2 antagonist specifically binds to an epitope(s) selected from among:
  • TNFR2 epitopes comprising the sequence of amino acids comprising:
  • PECLSCGS corresponding to residues 91-98 of SEQ ID NO:4, and/or RICTCRPG corresponding to residues 116-123 of SEQ ID NO:4, and/or
  • CAPLRKCR corresponding to residues 137-144 of SEQ ID NO:4), and/or LRKCRPGFGVA corresponding to residues 140-150 of SEQ ID NO:4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO:4), and/or an epitope containing at least 5 continuous or discontinuous residues within residues 75-128, 86-103, 111-128, or 150-190 of SEQ ID NO:4.
  • the TNFR2 antagonist construct comprises a TNFR2 antagonist that is a small molecule.
  • the TNFR2 antagonist is thalidomide or an analog thereof, such as lenalidomide and pomalidomide.
  • the TNFR2 antagonist construct comprises a TNFR2 antagonist that that reduces FoxP3 expression and inhibits the suppressive activity of Tregs.
  • a TNFR2 antagonist that reduces FoxP3 expression and inhibits the suppressive activity of Tregs.
  • exemplary of such antagonists is a histone deacetylase inhibitor that reduces FoxP3 expression and inhibits the suppressive activity of Tregs.
  • exemplary of such inhibitor is panobinostat or cyclophosphamide or Triptolide.
  • the TNFR2 constructs can be used in methods of treatment for and uses for treating infectious diseases, and for treating cancers that express TNFR2.
  • Exemplary of such cancers is a cancer selected from among: T cell lymphoma, such as Hodgkin’s lymphoma and cutaneous non-Hodgkin’s lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, breast cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, and lung cancer.
  • Figure 1 depicts a plasmid map of the pCBL-1 expression plasmid containing the CMV promoter where TE19080L is the inserted fragment.
  • Figure 2 sets forth an exemplary bi-specific construct - with a linker (part of a hinge region) and activity modifier joining two ligands, such as TNFR1 inhibitor (TNFR1 antagonist) and a TNFR2 agonist.
  • a linker part of a hinge region
  • activity modifier joining two ligands, such as TNFR1 inhibitor (TNFR1 antagonist) and a TNFR2 agonist.
  • Figures 3 A-3D depict exemplary PEG-centered multi-specific constructs, which are for presenting/providing two or more moieties that interact with one or more targets, or with one target at a plurality of sites.
  • Figure 3 A depicts an exemplary bivalent construct.
  • One of the circles is, for example, a polypeptide agonist, antagonist or a binding protein, such as an antibody or antigen-binding fragment thereof, or an aptamer (nucleic acid or peptide).
  • the other circle represents polysaccharides or receptor ligands or other moieties that interact with a target of interest.
  • the bivalent nature provides for clustering of targets for receptor activation.
  • the targets include TNFR1 and TNFR2; and as described throughout the disclosure herein, moieties include TNFR1 inhibitors, such as moieties that inhibit TNFR1 signaling, and TNFR2 agonists or other moieties that are Treg expanders.
  • Figure 3B depicts a monovalent single ligand, such as CD3+, to prevent cytokine release syndrome, linked via the PEG moieties to the agonist, antagonist, or binding protein, which is bivalent for receptor clustering.
  • exemplary targets include TNFR1 and/or TNFR2.
  • Figure 3C depicts a heterobifunctional PEG for crosslinking two different cell targeting agents, or two agents, such as trastuzumab and pertuzumab or portions thereof, that bind to different sites on the same receptor.
  • This construct can be used, for example, to cluster a checkpoint control receptor for either stimulation or inhibition of an immune response, or to crosslink two different receptors to achieve suppression of receptor activity (i.e., CD3 vs CD450, or to deliver two different ligands, such as a stimulatory and a co-stimulatory ligand, to two different receptors on the same cells.
  • Figure 3D depicts a homobifunctional PEG for clustering identical receptors on the same or different cells, depending upon chain length, or to trap circulating disease target, such as a soluble receptor or ligand, such as TNF. Additionally in all of these embodiments additional PEG side chain, optionally linked to another reactive group or functional group, such as a serum half-life extending moiety, such as HSA, or an FcRn polypeptide, can be included in these constructs.
  • the PEG moieties can be modified or replaced with moieties with similar properties for presentation of the binding moieties.
  • Figure 4 depicts additional exemplary configurations and structures of PEG- centered constructs for displaying or providing binding moieties or reactive moieties, such as the TNFR1 inhibitors and/or the TNFR2 agonists as described herein.
  • Figure 5 depicts additional exemplary configurations and structures of PEG- centered constructs for displaying or providing binding moieties or reactive moieties, such as the TNFR1 inhibitors and/or the TNFR2 agonists.
  • X and Y can be ligands and reactive moieties.
  • TNF Tumor Necrosis Factor
  • TNFRs Tumor Necrosis Factor Receptors
  • TNFRl-Selective Antagonists a. TNFR1 antagonistic Antibodies b. Monovalent TNFR1 antagonistic Antibodies/Antibody Fragments i. Fab- and scFv-Based TNFR1 antagonists ii. Domain Antibody (dAb)-Based TNFR1 antagonists a) Anti-TNFRl dAb-Anti-Albumin dAb Fusion Constructs b) Domain antibody fragments designated GSK1995057 and GSK2862277 iii. Nanobodies (Nbs) iv. Anti-TNFRl Nanobody-Anti-Albumin Nanobody Fusion Constructs c. Dominant-Negative Inhibitors of TNF (DN-TNFs)/TNF Muteins
  • TNFR2-Selective Agonists a. TNFR2 agonistic Antibodies b. TNFR2-Selective TNF Muteins and Fusions Thereof
  • TNFR1 antagonist constructs TNFR2 agonist constructs; Multi-Specific, Including Bi-Specific, TNFR1 Antagonist and TNFR2 Agonist Constructs
  • TNFR1 antagonist constructs Components of the TNFR1 antagonist constructs, TNFR2 agonist constructs, and Multi-Specific, Including Bi-Specific, TNFR1 Antagonist/TNFR2 agonist constructs
  • TNFR1 inhibitor moiety TNFR1 antagonist
  • TNFR2 Agonist Constructs and TNFR2 Antagonist Constructs c.
  • Activity modifiers i.
  • Fc portions a) Knobs-in-Holes b) Modifications that Enhance Neonatal Fc Receptor (F cRn) Recycling c) Enhancement of or Reduction/Elimination of Fc Immune Effector Functions ii. Other Modifications of Fc portions iii. Human Serum Albumin e. Multi-specific TNFR1 antagonist I TNFR2 agonist Constructs PEGylation for Linking Components of the Multi-Specific Constructs, PEG-centered Multi-Specific Construct, such as Bi- Specific, TNFR1 Antagonist/TNFR2 Agonist Constructs f. Additional Activity modifiers - Fusion proteins that include portions or entire polypeptides that increase serum half-life
  • RTKs Receptor Tyrosine Kinases
  • HER Human Epidermal Growth Factor Receptor
  • HER Human Epidermal Growth Factor Receptor
  • HER Human Epidermal Growth Factor Receptor
  • compositions Therapeutic Uses and Methods of Treatment a. Pharmaceutical Compositions b. Therapeutic Uses and Methods of Treatment
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement- Dependent Cytotoxicity
  • a construct is a product that contains one more components, generally at least two.
  • the components can be polypeptides, small molecules, aptamers, nucleic acids, and/or other such components as described herein or known to those of skill in the art.
  • Various constructs are described and exemplified herein; the components and variety thereof is apparent from the description herein. Those of skill in the art in view of the description can envision other constructs that are within the disclosure and claims herein.
  • the term construct is employed because the products can include a variety of different types of components.
  • a construct that is a TNFR1 construct or a TNFR2 antagonist construct is a construct that comprises a TNFR1 inhibitor moiety, which is a moiety that inhibits or reduces a TNFR1 activity, such as signaling.
  • a construct that is a TNFR2 construct or a TNFR2 agonist construct is a construct that comprises a TNFR2 agonist moiety, which is a moiety that activates or induces an activity of a TNFR2, such as signaling or an activity the results in increased Treg cells.
  • a construct that is a TNFR2 antagonist construct is a construct that comprises a TNFR2 antagonist.
  • a construct that is a multi-specific construct is a construct that comprises more than one antagonist or agonist or both moieties, such as a construct that contains a TNFR1 inhibitor and a TNFR2 agonist, or a construct that contains two TNRF1 antagonists, such as where each interacts with a different epitope on TNFR1 or each has a different TNFR1 antagonist activity, or two TNFR2 agonists, such as where each interacts with a different TNFR2 epitope, or each has a different TNFR2 agonist activity.
  • tumor necrosis factor As used herein, “tumor necrosis factor,” “tumor necrosis factor alpha,” “TNF,” “TNF-alpha,” “TNF- ⁇ ” and “TNF- ⁇ ” are used interchangeably to refer to a pleiotropic proinflammatory cytokine that is a member of the TNF superfamily and is associated with inflammatory and immuno-regulatory activities, including the regulation of tumongenesis/cancer, host defense against pathogenic infections, apoptosis, autoimmunity, and septic shock. When other members of the TNF superfamily are intended, they will be identified by name. TNF participates in coordination of innate and adaptive immune responses, as well as in organogenesis, particularly of the lymphoid organs.
  • TNF is produced as a homotrimeric membrane- bound protein containing 233 amino acids that can be cleaved by the protease TACE (TNF alpha converting enzyme; also known as ADAM17) to release soluble TNF (sol TNF), which contains 157 amino acids; membrane-bound and soluble forms of TNF are biologically active.
  • protease TACE TNF alpha converting enzyme
  • ADAM17 soluble TNF
  • Homotrimers of TNF bind to and signal through two high-affinity, specific receptors, TNFR1 and TNFR2; membrane-bound TNF primarily activates TNFR2, while soluble TNF primarily activates TNFR1.
  • TNF chronic inflammatory and autoimmune diseases and conditions
  • chronic inflammatory and autoimmune diseases and conditions including, but not limited to, for example, septic shock, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and inflammatory bowel disease (IBD), as well as neurodegenerative and demyelinating diseases and conditions, including, but not limited to, for example, Alzheimer’s disease, Parkinson’s disease, stroke and multiple sclerosis.
  • IBD inflammatory bowel disease
  • a “TNF mutein” or “TNF- ⁇ mutein” or “modified TNF polypeptide” refers to a polypeptide that has an amino acid sequence that, for TNF from a particular species, differs from the amino acid sequence of a corresponding wild-type TNF (TNF- ⁇ ) by one or more amino acids.
  • modified TNF polypeptides retain the ability to activate or inhibit TNFR1 and/or TNFR2.
  • Specific mutations in TNF can render the resulting TNF mutein selective for binding to TNFR1 or TNFR2, and can result in TNF muteins with antagonistic or agonistic properties.
  • a “dominant-negative inhibitor of TNF” or “DN-TNF” is a TNF mutein with one or more mutations that abrogate binding to and signaling through TNFR1 and/or TNFR2.
  • DN-TNFs selectively inhibit soluble TNF (sTNF or solTNF) by rapidly exchanging subunits with native TNF homotrimers, forming inactive mixed TNF heterotrimers with disrupted receptor binding surfaces, thus preventing interaction with TNF receptors.
  • DN-TNFs leave transmembrane TNF (tmTNF) unaffected, maintaining the protective roles of TNF signaling through TNFR2.
  • Examples of DN-TNFs are TNF mutants containing one or more of the replacements L133Y, S162Q, Y163H, I173T, Y191Q and A221R, with reference to the sequence of amino acids set forth in SEQ ID NO: 1 (corresponding to residues L57Y, S86Q, Y87H, I97T, Y115Q, and A145R, with reference to the sequence of solTNF, as set forth in SEQ ID NO:2), which impair binding to TNFRs.
  • a “modification” is in reference to the modification of a sequence of amino acids in a polypeptide, or a sequence of nucleotides in a nucleic acid molecule, and includes deletions, insertions, transpositions, replacements and combinations thereof of amino acids or nucleotides, respectively.
  • Methods of modifying a polypeptide or nucleic acid are routine to those of skill in the art, such as by using recombinant DNA methodologies.
  • deletion when referring to a nucleic acid or polypeptide sequence, refers to the deletion of one or more nucleotides or amino acids compared to a sequence, such as a target polynucleotide or polypeptide, or a native or wild-type sequence.
  • insertion when referring to a nucleic acid or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids, within a target, native, wild-type or other related sequence.
  • a nucleic acid molecule that contains one or more insertions compared to a wild-type sequence contains one or more additional nucleotides within the linear length of the sequence.
  • addition when referring to a nucleic acid or amino acid sequence, describes the addition of one or more nucleotides or amino acids onto either termini, compared to another sequence.
  • substitution refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence, with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule.
  • one or more substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule.
  • Amino acid replacements compared to a particular polypeptide can be expressed in terms of the number of the amino acid residue along the length of the polypeptide sequence.
  • a modified polypeptide having a modification in the amino acid at the 100 th position of the amino acid sequence that is a substitution/replacement of tyrosine (Tyr; Y) with glutamic acid (Glu; E), can be expressed as Y100E, TyrlOOGlu, or 100E.
  • Y100 can be used to indicate that the amino acid at the modified 100 th position is a tyrosine.
  • modifications are in a heavy chain (HC) or light chain (LC) of an antibody, modifications also can be denoted by reference to HC- or LC- to indicate the chain of the polypeptide.
  • nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence refers to nucleotides or amino acid positions identified upon alignment with a referenced sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm.
  • aligning the sequences one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
  • alignment of a sequence refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned.
  • An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with a genomic DNA sequence.
  • Related or variant polypeptides or nucleic acid molecules can be aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods, such as using manual alignments and by using the numerous alignment programs available (e.g., BLASTP) and others known to those of skill in the art.
  • one skilled in the art can identify analogous portions or positions, using conserved and identical amino acid residues as guides. Further, one skilled in the art also can employ conserved amino acid or nucleotide residues as guides to find corresponding amino acid or nucleotide residues between and among human and non-human sequences. Corresponding positions also can be based on structural alignments, for example, by using computer simulated alignments of protein structure. In other instances, corresponding regions can be identified. One skilled in the art also can employ conserved amino acid residues as guides to find corresponding amino acid residues between and among human and non-human sequences.
  • proteins are “compared under the same conditions” means that different proteins are treated identically or substantially identically such that any one or more conditions that can influence the activity or properties of a protein or agent are not varied or not substantially varied between the test agents.
  • any one or more conditions such as the amount or concentration of the polypeptide; the presence, including amount, of excipients, carriers or other components in a formulation other than the active agent (e.g., antibody); temperature; pH; time of storage; storage vessel; properties of storage (e.g., agitation); and/or other conditions associated with exposure or use, are identical or substantially identical between and among the compared polypeptides/antibodies.
  • an “adverse effect,” or “side effect,” or “adverse event,” or “adverse side effect,” refers to a harmful, deleterious and/or undesired effect associated with administering a therapeutic agent.
  • side effects associated with the administration of an anti-TNF antibody such as adalimumab (sold, for example, under the trademark Humira®)
  • Such adverse side effects include, for example, serious infections, such as tuberculosis, and other infections caused by viruses, fungi and bacteria, including upper respiratory infections, as well as dermatological and dermal toxicity, such as rash, headaches and nausea.
  • “adverse effect” or “side effect” refers to a harmful, deleterious and/or undesired effect of administering a therapeutic agent.
  • Side effects or adverse effects are graded on toxicity, and various toxicity scales exist, providing definitions for each grade. Examples of such scales are toxicity scales of the National Cancer Institute Common Toxicity Criteria version 2.0, and the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Assigning grades of severity is within the skill of an experienced physician or other health care professional. The severity of symptoms can be quantified using the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system. The CTCAE is a descriptive terminology used for Adverse Event (AE) reporting.
  • CTCAE Adverse Event
  • the grading (severity) scale is provided for each AE term.
  • the CTCAE displays Grades 1 through 5, with clinical descriptions for severity for each adverse event based on the following general guideline: Grade 1 (Mild AE); Grade 2 (Moderate AE); Grade 3 (Severe AE); Grade 4 (Life-threatening or disabling AE); and Grade 5 (Death related to AE/ fatal).
  • a “property” of a polypeptide refers to any property exhibited by a polypeptide, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermal tolerance, and tolerance to pH conditions. Changes in properties can alter an “activity” of the polypeptide. For example, a change in the binding specificity of the antibody polypeptide can alter the ability to bind an antigen, and/or various binding activities, such as affinity or avidity, or in vivo activities of the polypeptide.
  • an “activity” or a “functional activity” of a polypeptide refers to any activity exhibited by the polypeptide. Such activities can be empirically determined. Exemplary activities include, but are not limited to, the ability to interact with a biomolecule, for example, through antigen-binding, DNA binding, ligand binding, or dimerization; and enzymatic activity, for example, kinase activity or proteolytic activity.
  • activities include, but are not limited to, the ability to specifically bind a particular antigen, affinity of antigen-binding (e.g., high or low affinity), avidity of antigen- binding (e.g., high or low avidity), on-rate, off-rate, effector functions, such as the ability to promote antigen neutralization or clearance, virus neutralization, and in vivo activities, such as the ability to prevent infection or invasion of a pathogen, or to promote clearance, or to penetrate a particular tissue or fluid or cell in the body.
  • affinity of antigen-binding e.g., high or low affinity
  • avidity of antigen- binding e.g., high or low avidity
  • effector functions such as the ability to promote antigen neutralization or clearance, virus neutralization
  • in vivo activities such as the ability to prevent infection or invasion of a pathogen, or to promote clearance, or to penetrate a particular tissue or fluid or cell in the body.
  • Activity can be assessed in vitro or in vivo using recognized assays, such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure on- or off-rate, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry, and binding assays (e.g., panning assays).
  • recognized assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure on- or off-rate, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry, and binding assays (e.g., panning assays).
  • activities can be assessed by measuring binding affinities, avidities, and/or binding coefficients (e.g., for on-/off-rates), and other activities in vitro, or by measuring various effects in vivo, such as immune effects, e.g., antigen clearance; penetration or localization of the antibody into tissues; protection from disease, e.g., infection; serum or other fluid antibody titers; or other assays that are well-known in the art.
  • the results of such assays that indicate that a polypeptide exhibits an activity can be correlated to activity of the polypeptide in vivo, in which in vivo activity can be referred to as therapeutic activity, or biological activity.
  • Activity of a modified polypeptide can be any level of percentage of activity of the unmodified polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more, of activity compared to the unmodified polypeptide.
  • Assays to determine functionality or activity of modified (or variant) antibodies are well-known in the art.
  • Binding interactions include, but are not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules, such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as chemical compounds, including drugs.
  • bonds are antibody-antigen interactions and receptor-ligand interactions.
  • binding refers to the specific recognition of the antigen by the antibody, through cognate antibody-antigen interaction, at antibody combining sites. Binding also can include the association of multiple chains of a polypeptide, such as antibody chains, which interact through disulfide bonds.
  • binding activity refers to characteristics of a molecule, e.g., a polypeptide, relating to whether or not, and how, it binds one or more binding partners.
  • Binding activities include the ability to bind the binding partner(s), the affinity with which it binds to the binding partner (e.g., high affinity), the avidity with which it binds to the binding partner, the strength of the bond with the binding partner, and/or the specificity for binding with the binding partner.
  • affinity or “binding affinity” describes the strength of the interaction between two or more molecules, such as binding partners, and typically, the strength of the noncovalent interactions between two binding partners.
  • the affinity of an antibody or antigen-binding fragment thereof for an antigen epitope is the measure of the strength of the total noncovalent interactions between a single antibody combining site and the epitope. Low-affinity antibody-antigen interaction is weak, and the molecules tend to dissociate rapidly, while high affinity antibody- antigen binding is strong and the molecules remain bound for a longer amount of time.
  • Binding affinity can be determined in terms of binding kinetics, such as by measuring rates of association (k a or k on ) and/or dissociation (kd or k off ), half maximal effective concentration (EC 50 ) values, and/or thermodynamic data (e.g., Gibbs free energy (AG), enthalpy (AH), entropy (-TAS), and/or calculating association (K a ) or dissociation ( Kd) constants.
  • EC 50 also called the apparent Kd, is the concentration (e.g., ng/mL) of antibody, where 50% of the maximal binding is observed to a fixed amount of antigen.
  • EC 50 values are determined from sigmoidal dose- response curves, where the EC 50 is the concentration at the inflection point.
  • a high antibody affinity for its substrate correlates with a low EC 50 value, and a low affinity corresponds to a high EC 50 value.
  • Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, such as ELISA, followed by curve-fitting analysis.
  • affinity constant refers to an association constant (K a ) used to measure the affinity of an antibody for an antigen.
  • K a association constant
  • affinity constants are expressed in units of reciprocal molarity (i.e., M' 1 ), and can be calculated from the rate constant for the association-dissociation reaction, as measured by standard kinetic methodology for antibody reactions (e.g., immunoassays, surface plasmon resonance, or other kinetic interaction assays known in the art).
  • the binding affinity of an antibody also can be expressed as a dissociation constant, or Kd.
  • an affinity constant also can be represented by the Kd.
  • Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (see, e.g., Rich and Myszka (2000) Curr. Opin. Biotechnol 11 :54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also, U.S. Patent No. 7,229,619, for a description of exemplary SPR and ITC methods for calculating the binding affinity of antibodies).
  • high binding affinity means that the antibody specifically binds to a target protein with an EC 50 that is less than about 10 ng/mL, 9 ng/mL, 8 ng/mL, 7 ng/mL, 6 ng/mL, 5 ng/mL, 3 ng/mL, 2 ng/mL, 1 ng/mL or less.
  • High binding affinity also can be characterized by an equilibrium dissociation constant (Kd) of 10 -6 M or lower, such as 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10' 12 M, or lower.
  • Kd equilibrium dissociation constant
  • high binding affinity is generally associated with K a values of greater than or equal to about 10 6 M- 1 greater than or equal to about 10 7 M -1 , greater than or equal to about 10 8 M -1 , or greater than or equal to about 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , or 10 12 M -1 .
  • Affinity can be estimated empirically, or affinities can be determined comparatively, e.g., by comparing the affinity of two or more antibodies for a particular antigen, for example, by calculating pairwise ratios of the affinities of the antibodies tested.
  • affinities can be readily determined using conventional techniques, such as by ELISA; equilibrium dialysis; surface plasmon resonance; by radioimmunoassay using a radiolabeled target antigen; or by another method known to the skilled artisan.
  • the affinity data can be analyzed, for example, by the method of Scatchard et al., (1949) Ann N. Y. Acad.
  • antibody avidity refers to the strength of multiple interactions between a multivalent antibody and its cognate antigen, such as with antibodies containing multiple binding sites associated with an antigen with repeating epitopes or an epitope array. A high avidity antibody has a higher strength of such interactions compared to a low avidity antibody.
  • binding for a target refers to a preference, higher binding affinity, for binding to the target compared to a non-target.
  • Selective binding refers to binding to a target with an affinity, generally, of at least about 10 7 - 10 8 M -1 . It also can refer to relative activity in which the affinity of a moiety or molecule for one target molecule is compared to the affinity for another molecule, and if the difference is of a certain magnitude, such as about 10-fold, the moiety or molecule is said to have greater specificity for the first target relative to the second.
  • “specifically binds” or “immunospecifically binds,” with respect to an antibody or antigen-binding fragment thereof, are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of the antibody and the antigen.
  • an antibody that immunospecifically binds (or that specifically binds), for example, to TNFR1 is one that binds to TNFR1 with an affinity constant (K a ) of about or lx 10 7 M -1 or lx 10 8 M -1 or greater (or a dissociation constant (Kd) of lx 10 -7 M or lx 10 -8 M or less).
  • affinity constant K a
  • Antibodies or antigen-binding fragments that immunospecifically bind to a particular antigen can be identified, for example, by immunoassays, such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISAs), surface plasmon resonance (SPR), or other techniques known to those of skill in the art.
  • steric effects refer to the effects of the size of atoms or groups on the molecule. Steric effects include, but are not limited to, steric hindrance and van der Waals repulsion. Steric effects are the effects resulting from the fact that atoms occupy space; when atoms are put close to each other, this costs energy, as the electrons near the atoms repel each other.
  • “exhibits at least one activity” or “retains at least one activity” refers to the activity exhibited by an antibody polypeptide, such as a variant antibody or other therapeutic polypeptide, compared to the target or unmodified polypeptide, that does not contain the modification.
  • a modified, or variant, polypeptide that retains an activity of a target polypeptide can exhibit improved activity, decreased activity, or maintain the activity of the unmodified polypeptide.
  • a modified, or variant, polypeptide can retain an activity that is increased compared to a target or unmodified polypeptide.
  • a modified, or variant, polypeptide can retain an activity that is decreased compared to an unmodified or target polypeptide.
  • Activity of a modified, or variant, polypeptide can be any level of percentage of activity of the unmodified or target polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity, compared to the unmodified or target polypeptide.
  • the change in activity is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times, greater than the unmodified or target polypeptide.
  • Assays for retention of an activity depend on the activity to be retained. Such assays can be performed in vitro or in vivo. Activity can be measured, for example, using assays known in the art and described below for activities, such as, but not limited to, ELISA and panning assays. Activities of a modified, or variant, polypeptide compared to an unmodified or target polypeptide also can be assessed in terms of an in vivo therapeutic or biological activity or result following administration of the polypeptide.
  • the "surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix.
  • Commercial systems are available.
  • the BIAcore system GE Healthcare Life Sciences
  • BIAcore system GE Healthcare Life Sciences
  • antibody refers to immunoglobulins and immunoglobulin fragments, whether natural, or partially or wholly synthetically, such as recombinantly, produced, including any fragment thereof containing at least a portion of the variable heavy chain and/or variable light chain regions of the immunoglobulin molecule that is sufficient to form an antigen-binding site and, when assembled, to specifically bind an antigen.
  • an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site).
  • an antibody refers to an antibody that contains two heavy chains (which can be denoted H and H’) and two light chains (which can be denoted L and L’), where each heavy chain can be a full- length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen- binding site (e.g., heavy chains include, but are not limited to, V H chains, V H -C H 1 chains, and V H -C H 1-C H 2-C H 3 chains), and each light chain can be a full-length light chain or a portion thereof sufficient to form an antigen-binding site (e.g., light chains include, but are not limited to, V L chains and V L -CL chains).
  • antibodies typically include all or at least a portion of the variable heavy (V H ) chain and/or the variable light (V L ) chain.
  • An antibody also can include other regions, such as, for example, all or a portion of the constant region, and/or all or a portion (sufficient to provide flexibility) of the hinge region.
  • antibody includes full-length antibodies and portions thereof, including antibody fragments, such as, for example, anti-TNFRl, antibody fragments.
  • Antibody fragments include, but are not limited to, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFvs), single-chain Fabs (scFab), hsFv (helix-stabilized Fv), single domain antibodies (dAbs, or sdAbs), minibodies, diabodies, anti -idiotypic (anti-Id) antibodies, nanobodies and camelid antibodies, free light chains, V H H antibodies (or nanobodies), or antigen-binding fragments of any of the above.
  • Antibody fragments also can include combinations of any of the above fragments, such as, for example, tandem scFv, Fab-scFv (HC C-term, or LC C-term), Fab-(scFv)2 (C-term), scFv-Fab-scFv, Fab-Cu2-scFv, scFv fusions (C term, or N term), Fab-fusions (HC C-term, or LC C- term), scFv-scFv-dAb, scFv-dAb-scFv, dAb-scFv-scFv, and tribodies.
  • tandem scFv HC C-term, or LC C-term
  • Fab-(scFv)2 C-term
  • scFv-Fab-scFv Fab-Cu2-scFv
  • scFv fusions C term, or N term
  • Fab-fusions HC C-term
  • antibody includes synthetic antibodies, recombinantly produced antibodies, multi- specific and heteroconjugate antibodies (e.g., bi-, tri- and quad-specific antibodies, diabodies, triabodies and tetrabodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies.
  • Antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or sub- subclass (e.g., IgG2a and IgG2b).
  • a “form of an antibody” refers to a particular structure of an antibody.
  • Antibodies herein include full-length antibodies and portions thereof, such as, for example, a Fab fragment or other antibody fragment. Thus, a Fab is a particular form of an antibody.
  • a “corresponding form” of an antibody means that, when comparing a property or activity of two antibodies, the property is compared using the same form of the antibody. For example, if it is stated that an antibody has less activity compared to the activity of the corresponding form of a first antibody, that means that a particular form, such as a Fab of that antibody, has less activity compared to the Fab form of the first antibody.
  • a full-length antibody is an antibody having two full-length heavy chains (e.g., V H -C H 1-C H 2-C H 3, or V H -C H 1-C H 2-C H 3-C H 4), two full-length light chains (V L -CL), and hinge regions, such as human antibodies produced by antibody secreting B cells, and antibodies with the same domains that are produced synthetically.
  • multi-specific construct refers to a construct, such as an antibody or construct comprising portions of an antibody, that exhibits affinity for more than one target antigen so that it can specifically interact with the targets.
  • Multi- specific constructs herein can have structures similar to full immunoglobulin molecules and include Fc regions, for example IgG Fc regions, and antigen-binding regions, such as portions that specifically bind to TNFR1 or TNFR2.
  • a “bispecific construct” refers to a multi-specific construct that has binding specificity for two different antigens.
  • Bispecific constructs include, for example, monoclonal antibodies or antigen-binding fragments thereof linked to a polypeptide region, such as Fc or modified Fc, that modifies the activity of the construct.
  • the constructs are derived from human sources or are derived from a human source or are humanized, and the constructs have binding specificities for at least two different antigens.
  • Bi-specific constructs/molecules provided herein can have binding specificities that are directed to TNFR1, and TNFR2.
  • the bi-specific constructs include a TNFR1 antagonist and a TNFR2 agonist.
  • a bispecific antibody or construct includes antibodies and antigen- binding fragment thereof that includes two separate antigen-binding domains (e.g., two scFvs, or two dAbs, or two Fabs, joined by a linker).
  • the antigen-binding domains can bind to the same antigen or different antigens.
  • antibody fragment refers to any portion of a full-length antibody that is less than full-length, but contains at least a portion of the variable region(s) of the antibody sufficient to form an antigen-binding site (e.g., one or more complementarity-determining region (CDRs)), and thus, retains the binding specificity and/or an activity of the full-length antibody; antibody fragments include antibody derivatives produced by enzymatic treatment of full- length antibodies, as well as synthetically, e.g., recombinantly, produced derivatives.
  • CDRs complementarity-determining region
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab)2, single-chain Fvs (scFvs), Fv, dsFv, diabody, triabody, affibody, nanobody, aptamer, dAb, Fd and Fd fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; pp. 3-25, Kipriyanov).
  • the fragment can include multiple chains linked together, such as by disulfide bridges, and/or by peptide linkers.
  • An antibody fragment generally contains at least about 50 amino acids, such as at about or at least 100 amino acids, and typically, at least about or at least 110, 120, 150, 170, 180, or 200 amino acids.
  • an “Fv antibody fragment” is composed of one variable heavy domain (V H ) and one variable light (V L ) domain, linked by noncovalent interactions.
  • a dsFv disulfide-linked Fv refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the V H -V L pair.
  • an “scFv fragment” refers to an antibody fragment that contains a variable light chain (V L ) and variable heavy chain (V H ), covalently connected by a polypeptide linker in any order.
  • the linker is of a length, such that the two variable domains are bridged without substantial interference.
  • Exemplary linkers are (Gly-Ser) n residues with some Glu or Lys residues dispersed throughout to increase solubility.
  • diabodies are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs, and preferentially dimerize.
  • triabodies are trimeric scFv; they contain three peptide chains, each of which contains one V H domain and one V L domain joined by a short linker (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of V H and V L domains within the same peptide chain; triabodies typically trimerize.
  • a short linker e.g., a linker composed of 1-2 amino acids
  • a “Fab fragment” is an antibody fragment that results from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g., by recombinant methods.
  • a Fab fragment contains a light chain (containing a V L and CL), and another chain containing a variable domain of a heavy chain (V H ) and one constant region domain of the heavy chain (C H 1).
  • a “F(ab')2 fragment” is an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure that is produced synthetically, e.g., by recombinant methods.
  • the F(ab')2 fragment essentially contains two Fab fragments, where each heavy chain portion contains an additional few amino acids, such as, for example, all or a portion, sufficient to provide flexibility, of the hinge region, including cysteine residues that form disulfide linkages joining the two fragments.
  • a Fab' fragment is a fragment containing one half (i.e., one heavy chain and one light chain) of the F(ab')2 fragment.
  • an Fd fragment is a fragment of an antibody containing a variable domain (V H ) and one constant region domain (C H 1) of an antibody heavy chain.
  • an Fd’ fragment is a fragment of an antibody containing one heavy chain portion of a F(ab')2 fragment.
  • an Fv’ fragment is a fragment containing only the V H and V L domains of an antibody molecule.
  • hsFv helix-stabilized Fv refers to an antibody fragment in which the constant domains normally present in a Fab fragment have been substituted with a heterodimeric coiled-coil domain (see, e.g., Arndt et al. (2001) J. Mol. Biol. 7:312:221-228).
  • dAbs are the smallest antigen-binding fragments of antibodies; they are about approximately 11-15 kDa in size (about 100-150 amino acids), which is approximately one-tenth the size of a full monoclonal antibody (mAb).
  • Each dAb contains three out of the six CDRs, which are the highly diversified loop regions that bind to the target antigen, from a V H -V L pair in an antibody.
  • a camelid antibody also referred to as a nanobody or VHHs, lacks a light chain and is composed of two identical heavy chains. They occur naturally in camelids, such as camels and alpacas.
  • a polypeptide “domain” is a part of a polypeptide (a sequence of 3 or more, generally 5, 10, or more, amino acids) that is structurally and/or functionally distinguishable or definable.
  • An exemplary polypeptide domain is a part of the polypeptide that can form an independently folded structure within a polypeptide made up of one or more structural motifs (e.g., combinations of alpha helices and/or beta strands connected by loop regions), and/or that is recognized by a particular functional activity, such as enzymatic activity, dimerization or antigen- binding.
  • a polypeptide can have one or more, typically more than one, distinct domains.
  • the polypeptide can have one or more structural domains and one or more functional domains.
  • a single polypeptide domain can be distinguished based on structure and function.
  • a domain can encompass a contiguous linear sequence of amino acids.
  • a domain can encompass a plurality of non- contiguous amino acid portions, which are non-contiguous along the linear sequence of amino acids of the polypeptide.
  • a polypeptide contains a plurality of domains.
  • each heavy chain and each light chain of an antibody molecule contains a plurality of immunoglobulin (Ig) domains, each about 110 amino acids in length.
  • Ig immunoglobulin
  • a “functional region” of a polypeptide is a region of the polypeptide that contains at least one functional domain (which imparts a particular function, such as an ability to interact with a biomolecule, for example, through antigen-binding, DNA binding, ligand binding, or dimerization, or by enzymatic activity, for example, kinase activity or proteolytic activity);
  • exemplary functional regions of polypeptides are antibody domains, such as V H , V L , C H , CL, and portions thereof, such as CDRs, including CDR1, CDR2 and CDR3, or anti gen -binding portions, such as antibody combining sites.
  • a “structural region” of a polypeptide is a region of the polypeptide that contains at least one structural domain.
  • an “Ig domain” is a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond.
  • Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (V L ) and one constant region domain (CL), while a heavy chain contains one variable region domain (V H ) and three or four constant region domains (C H ).
  • V L , CL, V H , and C H domain is an example of an immunoglobulin domain.
  • variable domain is a specific immunoglobulin (Ig) domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies.
  • Ig immunoglobulin
  • Each light chain and each heavy chain has one variable region domain (V L and V H , respectively).
  • the variable domains provide antigen specificity, and thus, are responsible for antigen recognition.
  • Each variable region contains complementarity-determining regions (CDRs) that are part of the antigen-binding site domain and framework regions (FRs).
  • variable region domain contains three CDRs, named CDR1, CDR2, and CDR3.
  • the three CDRs are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide.
  • the CDRs are located within the loops that join the parallel strands of the beta sheets of the variable domain.
  • antigen-binding domain As used herein, “antigen-binding domain,” “antigen-binding site,” “antigen- binding fragment,” “antigen combining site” and “antibody combining site” are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with the cognate antigen.
  • a native conventional full-length antibody molecule has two conventional antigen-binding sites, each containing portions of a heavy chain variable region and portions of a light chain variable region.
  • a conventional antigen-binding site contains the loops that connect the anti-parallel beta strands within the variable region domains.
  • the antigen combining sites can contain other portions of the variable region domains.
  • Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions also are called complementarity-determining regions (CDRs).
  • portion thereof refers to a contiguous portion thereof that is sufficient to form an antigen-binding site such that, when assembled into an antibody containing a heavy and light chain, it contains at least 1 or 2, typically 3, 4, 5 or all 6 CDRs of the variable heavy (V H ) and variable light (V L ) chains sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody containing all 6 CDRs.
  • a sufficient antigen-binding site requires the CDR3 of the heavy chain (CDRH3). It typically further requires the CDR3 of the light chain (CDRL3).
  • frame regions are the domains within the antibody variable region domains that are located within the beta sheets; the FR regions are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
  • Each variable region contains four framework regions that separate the three hypervariable regions.
  • a “constant region” domain is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved among antibodies than the variable region domain.
  • Each light chain has a single light chain constant region (CL) domain, and each heavy chain contains one or more heavy chain constant region (C H ) domains, which include, C H 1, C H 2, C H 3 and C H 4.
  • C H heavy chain constant region
  • Full-length IgA, IgD and IgG isotypes contain C H 1, C H 2 and C H 3 domains and a hinge region, while IgE and IgM contain C H 1, C H 2, C H 3 and C H 4 domains.
  • C H 1 and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with the antigen and rotation of the antibody arms.
  • Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g., through interactions with various cells, biomolecules and tissues.
  • an “antibody hinge region” or “hinge region” refers to a polypeptide region in the heavy chain of the gamma, delta and alpha antibody isotypes, that occurs between the C H 1 and C H 2 domains, joins the Fab and Fc regions, and has no homology with the other antibody domains.
  • This region is rich in proline residues and provides flexibility to IgG, IgD and IgA antibodies, allowing the two “arms” (each containing one antibody combining site) of the Fab portion to be mobile, assuming various angles with respect to one another as they bind an antigen. This flexibility allows the Fab arms to move in order to align the antibody combining sites to interact with epitopes on cell surfaces or other antigens.
  • the synthetically produced antibody fragments contain one or more hinge regions, for example, to promote stability via interactions between two antibody chains. Hinge regions are examples parts of dimerization domains, and, for purposes herein are part of the linkers.
  • a “fragment crystallizable region” or “Fc” or “Fc region” or “Fc domain” refers to a polypeptide containing the constant region of an antibody heavy chain, excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG (C H 2 and C H 3, also referred to as Cy2 and Cy3), or the last three constant region immunoglobulin domains of IgE and IgM (C H 2, C H 3 and C H 4).
  • an Fc domain can include all or part of the flexible hinge region, which is N-terminal to these domains.
  • the Fc can include the J chain.
  • Fc contains immunoglobulin domains C H 2 and C H 3, and optionally, all or part of the hinge between C H 1 and C H 2 (also referred to as Cyl and Cy2).
  • the boundaries of the Fc region can vary, but typically, include at least part of the hinge region.
  • Fc also includes any allelic or species variant, or any variant or modified form, such as any variant or modified form of Fc that has altered binding to an Fc receptor (FcR) or alters an Fc-mediated effector function. Mutations in the Fc region and their effects are well-documented in the art.
  • Fc chimera refers to a chimeric polypeptide in which one or more polypeptides is/are linked, directly or indirectly, to an Fc region or a derivative thereof. Typically, an Fc chimera combines the Fc region of an immunoglobulin with another polypeptide. Derivatives of, or modified Fc polypeptides, are known to those of skill in the art.
  • Kabat numbering refers to the index numbering of the IgGl Kabat antibody (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242); it permits easy comparison among antibodies, similar to way chymotrypsin numbering permits comparison among proteases.
  • Kabat E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242
  • chymotrypsin numbering permits comparison among proteases.
  • One of skill in the art can identify regions of the constant region using Kabat numbering.
  • EU numbering or “EU index” refer to the numbering scheme of the EU antibody described in Edelman et al., (1969) Proc. Natl. Acad. Sci. USA 63:78-85.
  • EU index as in Kabat refers to EU index numbering of the human IgGl Kabat antibody as set forth in Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242.
  • EU numbering, or EU numbering as in Kabat are frequently used by those of skill in the art to number amino acid residues of the Fc regions of the light and heavy antibody chains.
  • the CL domain of the Ig kappa light chain corresponds to residues R108-C214 according to Kabat and EU numbering (see, e.g., Table 2 below).
  • the C H 1 domain of IgGl corresponds to residues 118-215 (EU numbering) or 114-223 (Kabat numbering);
  • C H 2 corresponds to residues 231-340 (EU numbering) or 244-360 (Kabat numbering);
  • C H 3 corresponds to residues 341-447 (EU numbering) or 361-478 (Kabat numbering).
  • Table 1 shows the IgGl heavy chain constant domain by EU, Kabat and sequential numbering, where sequential numbering is with respect to the sequence of amino acids set forth in SEQ ID NO:9, and identifies residues within the C H 1, C H 2 and C H 3 domains, as well as the hinge region.
  • Table 2 shows the immunoglobulin (Ig) kappa light chain constant domain by EU, Kabat and sequential numbering, where sequential numbering is with respect to the sequence of amino acids set forth in SEQ ID NO: 17.
  • the top row sets forth the amino acid residue number by sequential numbering (with reference to SEQ ID NO: 17); the second row (bold) provides the 1 -letter code for the amino acid residue at the position indicated by the number in the top row; the third row (in italics) indicates the corresponding Kabat number according to Kabat numbering; and the fourth row indicates the corresponding EU index number according to EU numbering.
  • Table 3 shows the IgG4 heavy chain constant domain by EU, Kabat and sequential numbering, where sequential numbering is with respect to the sequence of amino acids set forth in SEQ ID NO: 15, and identifies residues within the C H 1, C H 2 and CH3 domains, as well as the hinge region.
  • Such fragments can be derived by a variety of methods known in the art, including, but not limited to, enzymatic cleavage, chemical crosslinking, recombinant means, or combinations thereof.
  • the derived antibody fragment shares the identical, or substantially identical, heavy chain variable region (V H ) and light chain variable region (V L ) of the parent antibody, such that the 0 antibody fragment and the parent antibody bind the same epitope.
  • a “parent antibody” or “source antibody” refers to an antibody from which an antibody fragment (e.g., Fab, F(ab'), F(ab)2, single-chain Fv (scFv), Fv, dsFv, dAb, diabody, Fd and Fd' fragments) is derived.
  • an antibody fragment e.g., Fab, F(ab'), F(ab)2, single-chain Fv (scFv), Fv, dsFv, dAb, diabody, Fd and Fd' fragments
  • epitopic determinants refers to any antigenic determinant on an antigen or protein, to which the paratope of an antibody can bind.
  • Epitopic determinants typically contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • humanized antibodies and human therapeutics refer to antibodies and other protein therapeutics that are modified to include “human” sequences of amino acids, so that administration to a human does not provoke an immune response.
  • a humanized antibody typically contains complementarity determining regions (CDRs or hypervariable loops) derived from a non-human species immunoglobulin, and the remainder of the antibody molecule derived mainly from a human immunoglobulin.
  • CDRs or hypervariable loops complementarity determining regions
  • Methods for humanizing proteins, including antibodies, and producing them are well known and readily available to those of skill in the art. For example, DNA encoding a monoclonal antibody can be altered by recombinant DNA techniques to encode an antibody in which the amino acid composition of the non-variable regions is based on human antibodies.
  • the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (e.g., CDRs) correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions (FRs) are those of a human immunoglobulin sequence.
  • the humanized antibody optionally, also contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • a “multimerization domain” refers to a sequence of amino acids that promotes stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each containing a complementary multimerization domain, which can be the same or a different multimerization domain, to form a stable multimer with the first domain.
  • a polypeptide is joined directly or indirectly to the multimerization domain.
  • Exemplary multimerization domains include the immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains.
  • the multimerization domain can be an immunoglobulin constant region or domain, such as, for example, the Fc domain or portions thereof from IgG, including IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD and IgM, and modified forms thereof.
  • dimerization domains are multimerization domains that facilitate interaction between two polypeptide sequences (such as, but not limited to, antibody chains). Dimerization domains include, but are not limited to, an amino acid sequence containing a cysteine residue that facilitates the formation of a disulfide bond between two polypeptide sequences, such as all or a part of a full-length antibody hinge region, or one or more dimerization sequences, which are sequences of amino acids known to promote interaction between polypeptides (e.g., leucine zippers, GCN4 zippers).
  • a “chimeric polypeptide” refers to a polypeptide that contains portions from at least two different polypeptides or from two non-contiguous portions of a single polypeptide.
  • a chimeric polypeptide generally includes a sequence of amino acid residues from all or a part of one polypeptide, and a sequence of amino acids from all or a part of another different polypeptide.
  • the two portions can be linked directly or indirectly and can be linked via peptide bonds, other covalent bonds, or other non-covalent interactions of sufficient strength to maintain the integrity of a substantial portion of the chimeric polypeptide under equilibrium conditions and physiologic conditions, such as in isotonic pH 7 buffered saline.
  • a “fusion protein” is a polypeptide engineered to contain sequences of amino acids corresponding to two distinct polypeptides, which are joined together, such as by expressing the fusion protein from a vector containing two nucleic acids, encoding the two polypeptides, in close proximity, e.g., adjacent, to one another along the length of the vector. Accordingly, a fusion protein refers to a chimeric protein containing two, or portions from two, or more proteins or peptides that are linked directly or indirectly via peptide bonds. The two molecules can be adjacent in the construct, or can be separated by a linker, or spacer polypeptide.
  • linker refers to a peptide or chemical moiety containing a chain of atoms that covalently attaches an antibody or antigen-binding fragment thereof to another therapeutic moiety or another antibody or fragment thereof.
  • Linkers are included, for example, to increase flexibility, modify steric effects, including steric hindrance, and increase solubility in aqueous medium.
  • linker peptide refers to short sequences of amino acids that join two polypeptide sequences (or nucleic acids encoding such as an amino acid sequence).
  • “Peptide linker” refers to the short sequence of amino acids joining the two polypeptide sequences.
  • Exemplary of polypeptide linkers are linkers joining a peptide transduction domain to an antibody, or linkers joining two antibody chains in a synthetic antibody fragment, such as an scFv fragment. Linkers are well-known, and any known linkers can be used in the provided methods.
  • Exemplary polypeptide linkers include (Gly-Ser) n amino acid sequences, with some Glu or Lys residues dispersed throughout to increase solubility. Other exemplary linkers are described herein; any of these and other known linkers can be used with the polypeptides, antibodies, and other products and methods provided herein.
  • a “tag” or an “epitope tag” refers to a sequence of amino acids, typically added to the N- or C- terminus of a polypeptide, such as an antibody and an antibody fragment/construct, provided herein.
  • the inclusion of tags fused to a polypeptide can facilitate polypeptide purification and/or detection.
  • a tag or tag polypeptide refers to a polypeptide that has enough residues to provide an epitope recognized by an antibody, or that can serve for detection or purification, yet is short enough such that it does not interfere with activity of the polypeptide to which it is linked.
  • the tag polypeptide typically is sufficiently unique so that an antibody that specifically binds thereto does not substantially cross-react with epitopes in the polypeptide to which it is linked. Suitable tag polypeptides generally have at least 5 or 6 amino acid residues, and usually between about 8-50 amino acid residues, typically between 9-30 residues.
  • the tags can be linked to one or more chimeric polypeptides in a multimer and permit detection of the multimer or its recovery from a sample or mixture. Such tags are well-known and can be readily synthesized and designed.
  • Exemplary tag polypeptides include those used for affinity purification and include, for example, FLAG tags; His tags; the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5 (see, e.g., Field et al. (1988) Mol. Cell. Biol. 5:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (see, e.g., Evan et al. (1985) Molecular and Cellular Biology 5:3610-3616); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (see, e.g., Paborsky et al. (1990) Protein Engineering 3:547-553).
  • An antibody used to detect an epitope-tagged antibody is typically referred to herein as a secondary antibody.
  • a “label” or “detectable moiety” is a detectable marker (e.g., a fluorescent molecule, chemiluminescent molecule, bioluminescent molecule, contrast agent (e.g., a metal), radionuclide, chromophore, detectable peptide, or an enzyme that catalyzes the formation of a detectable product) that can be attached or linked directly or indirectly to a molecule (e.g., an antibody or antigen-binding fragment thereof, such as an anti-TNFRl_antibody or antigen-binding fragment thereof provided herein), or associated therewith, and can be detected in vivo and/or in vitro.
  • a detectable marker e.g., a fluorescent molecule, chemiluminescent molecule, bioluminescent molecule, contrast agent (e.g., a metal), radionuclide, chromophore, detectable peptide, or an enzyme that catalyzes the formation of a detectable product
  • the detection method can be any method known in the art, including known in vivo and/or in vitro methods of detection (e.g., imaging by visual inspection, magnetic resonance (MR) spectroscopy, ultrasound signal, X-ray, gamma ray spectroscopy (e.g., positron emission tomography (PET) scanning, single-photon emission computed tomography (SPECT)), fluorescence spectroscopy, or absorption).
  • MR magnetic resonance
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • Indirect detection refers to measurement of a physical phenomenon, such as energy or particle emission or absorption, of an atom, molecule or composition that binds directly or indirectly to the detectable moiety (e.g., detection of a labeled secondary antibody or antigen-binding fragment thereof that binds to a primary antibody (e.g., an anti-TNFR antibody or antigen-binding fragment thereof provided herein)).
  • a physical phenomenon such as energy or particle emission or absorption
  • nucleic acid refers to at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), joined together, typically by phosphodiester linkages. Also included in the term “nucleic acid” are analogs of nucleic acids, such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof.
  • PNA peptide nucleic acid
  • Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (i.e., peptide nucleic acid).
  • the term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double- stranded nucleic acids.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine.
  • the uracil base is uridine.
  • an “isolated nucleic acid molecule” is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • An “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium, when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals, when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding an antibody or antigen-binding fragments provided.
  • operably linked with reference to nucleic acid sequences, regions, elements or domains, means that the nucleic acid regions are functionally related to each other.
  • nucleic acid encoding a leader peptide can be operably linked to nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide.
  • the nucleic acid encoding a first polypeptide is operably linked to nucleic acid encoding a second polypeptide, and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed.
  • an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide.
  • a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.
  • synthetic with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or gene or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.
  • residues of naturally occurring a-amino acids are the residues of those 20 a-amino acids found in nature which are incorporated into a protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.
  • polypeptide refers to two or more amino acids covalently joined.
  • polypeptide and protein are used interchangeably herein.
  • peptide refers to a polypeptide that is from 2 to about or 40 amino acids in length.
  • amino acid is an organic compound containing an amino group and a carboxylic acid group.
  • a polypeptide contains two or more amino acids.
  • amino acids in the polypeptides, such as antibodies, provided include the twenty naturally-occurring amino acids (Table 4), non-natural amino acids, and amino acid analogs (e.g., amino acids wherein the a-carbon has a side chain).
  • amino acids which occur in the various amino acid sequences of polypeptides appearing herein, are identified according to their well- known, three-letter or one-letter abbreviations (see, Table 4).
  • the nucleotides, which occur in the various nucleic acid molecules and fragments are designated with the standard single-letter designations used routinely in the art.
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are generally in the “L” isomeric form. Residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide.
  • amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl- terminus.
  • amino acid residue is defined to include the amino acids listed in the Table of Correspondence (Table 4), modified, non-natural and unusual amino acids.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, or to an amino-terminal group, such as NH2, or to a carboxyl-terminal group, such as COOH.
  • Suitable conservative substitutions of amino acids are known to those of skill in the art and generally can be made without altering a biological activity of a resulting molecule.
  • Those of skill in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224).
  • naturally occurring amino acids refer to the 20 L-amino acids that occur in polypeptides.
  • non-natural amino acid refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid.
  • Non- naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-stereoi somers of amino acids.
  • non-natural amino acids are known to those of skill in the art, and include, but are not limited to, 2- Aminoadipic acid (Aad), 3 -Aminoadipic acid (bAad), P-alanine/p- Amino-propionic acid (Bala), 2-Aminobutyric acid (Abu), 4-Aminobutyric acid/piperidinic acid (4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe), 2- Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm), 2,4-Diaminobutyric acid (Dbu), Desmosine (Des), 2,2'-Diaminopimelic acid (Dpm), 2,3 -Diaminopropionic acid (Dpr), N-Ethylglycine (EtGly), N-Ethylasparagine (EtA
  • DNA construct is a single- or double-stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
  • a “DNA segment” is a portion of a larger DNA molecule having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5’ to 3’ direction, encodes the sequence of amino acids of the specified polypeptide.
  • polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5’ to the 3’ end.
  • Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
  • the length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated “nt”) or base pairs (abbreviated “bp”).
  • nt nucleotides
  • bp base pairs
  • double-stranded molecules When the term is applied to double-stranded molecules, it is used to denote overall length and is understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide can differ slightly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded polynucleotide molecule cannot be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length.
  • production by recombinant means by using recombinant DNA methods refers to the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.
  • expression refers to the process by which polypeptides are produced by transcription and translation of polynucleotides.
  • the level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a “host cell” is a cell that is used to receive, maintain, reproduce and/or amplify a vector.
  • a host cell also can be used to express the polypeptide encoded by the vector.
  • the nucleic acid in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.
  • a “vector” is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation.
  • Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide, such as a modified anti-TNFRl antibody. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid, or for expression/display of the polypeptide encoded by the nucleic acid.
  • the vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome.
  • vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well-known to those of skill in the art.
  • a vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.
  • an “expression vector” includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well- known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells, and those that remain episomal, or those which integrate into the host cell genome.
  • primary sequence refers to the sequence of amino acid residues in a polypeptide or the sequence of nucleotides in a nucleic acid molecule.
  • sequence identity refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference poly- peptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. The alignment can be local or global. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g., terminal gaps are not penalized). Alternatively, sequence identity can be determined without taking into account gaps, as the number of identical positions/length of the total aligned sequence x 100.
  • a “global alignment” is an alignment that aligns two sequences from beginning to end, aligning each letter in each sequence only once. An alignment is produced, regardless of whether or not there is similarity or identity between the sequences. For example, 50% sequence identity based on “global alignment” means that in an alignment of the full sequence of two compared sequences, each of 100 nucleotides in length, 50% of the residues are the same. It is understood that global alignment also can be used in determining sequence identity even when the length of the aligned sequences is not the same. The differences in the terminal ends of the sequences are taken into account in determining sequence identity, unless the “no penalty for end gaps” is selected.
  • a global alignment is used on sequences that share significant similarity over most of their length.
  • Exemplary algorithms for performing global alignment include the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48:443).
  • Exemplary programs for performing global alignment are publicly available and include the Global Sequence Alignment Tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/), and the program available at deepc2.
  • NCBI National Center for Biotechnology Information
  • a “local alignment” is an alignment that aligns two sequences, but only aligns those portions of the sequences that share similarity or identity. Hence, a local alignment determines if sub-segments of one sequence are present in another sequence. If there is no similarity, no alignment will be returned.
  • Local alignment algorithms include BLAST or Smith-Waterman algorithm (Adv. Appl. Math. 2:482 (1981)). For example, 50% sequence identity based on “local alignment” means that in an alignment of the full sequence of two compared sequences of any length, a region of similarity or identity of 100 nucleotides in length has 50% of the residues that are the same in the region of similarity or identity.
  • sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier.
  • Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • nucleic acid molecules have nucleotide sequences, or any two polypeptides have amino acid sequences, that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical,” or other similar variations reciting a percent identity, can be determined using known computer algorithms based on local or global alignment (see, e.g., wikipedia.org/wiki/Sequence_alignment_software, providing links to dozens of known and publicly available alignment databases and programs).
  • the full-length sequence of each of the compared polypeptides or nucleotides is aligned across the full-length of each sequence in a global alignment. Local alignment also can be used when the sequences being compared are substantially the same length.
  • identity represents a comparison or alignment between a test and a reference polypeptide or polynucleotide.
  • “at least 90% identical to” refers to percent identities from 90% to 100%, relative to the reference polypeptide or polynucleotide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes, when a test and reference polypeptide or polynucleotide with a length of 100 amino acids or nucleotides are compared, no more than 10% (i.e.
  • a “disulfide bond” (also called an S-S bond or a disulfide bridge) is a single covalent bond derived from the coupling of thiol groups. Disulfide bonds in proteins are formed between the thiol groups of cysteine residues, and stabilize interactions between polypeptide domains, such as antibody domains.
  • Coupled means attached via a covalent or noncovalent interaction.
  • conjugation means that the moiety is attached to the antibody or antigen- binding fragment thereof by any known means for linking peptides, such as, for example, by production of fusion proteins by recombinant means, or post- translationally by chemical means.
  • Conjugation can employ any of a variety of linking agents to effect conjugation, including, but not limited to, peptide or compound linkers, or chemical cross-linking agents.
  • antibody-dependent cell-mediated cytotoxicity As used herein, “antibody-dependent cell-mediated cytotoxicity,” “antibody- dependent cellular cytotoxicity” and “ADCC” refer, interchangeably, to cell-mediated reactions in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • NK cells e.g., natural killer (NK) cells, neutrophils, and macrophages
  • the primary cells for mediating ADCC NK cells, express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. (1991) Annu. Rev.
  • ADCC activity of a molecule of interest may be assessed in vitro, e.g., in an animal model, such as that disclosed in Clynes et al. (1998) Proc. Natl. Acad. Sci. USA 95:652-656.
  • complement-dependent cytotoxicity is an effector function of IgG and IgM antibodies.
  • target cell such as a bacterial cell or viral-infected cell
  • the classical complement pathway is triggered by bonding protein Clq to these antibodies, resulting in formation of a membrane attack complex (MAC) and subsequent cell lysis.
  • MAC membrane attack complex
  • ADCP antibody-dependent cellular phagocytosis
  • phagocytic potential such as monocytes and macrophages
  • therapeutic activity refers to the in vivo activity of a therapeutic polypeptide.
  • the therapeutic activity is the activity that is associated with treatment of a disease or condition.
  • Therapeutic activity of a modified polypeptide can be any level of percentage of the therapeutic activity of the unmodified polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more, of the therapeutic activity compared to the unmodified polypeptide.
  • assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a protein, such as an antibody, or an antigen-binding fragment thereof, present in the sample, and also, of obtaining an index, ratio, percentage, visual, or other value indicative of the level of the activity. Assessment can be direct or indirect.
  • a “disease or disorder” refers to a pathological condition in an organism, resulting from a cause or condition including, but not limited to, infections, acquired conditions, and genetic conditions, and characterized by identifiable symptoms.
  • treating means that the subject’s symptoms are partially or totally alleviated, or remain static following treatment.
  • treatment encompasses prophylaxis, therapy and/or cure.
  • Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease.
  • Treatment also encompasses any pharmaceutical use of any antibody or antigen-binding fragment thereof, or compositions, provided herein.
  • treatment means amelioration of a symptom or manifestation of a disease, disorder, or condition.
  • prevention refers to methods in which the risk of developing a disease or condition is reduced. To prevent a disease means to reduce the risk of developing the disease.
  • a “pharmaceutically effective agent” includes any therapeutic agent or bioactive agent, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.
  • a “therapeutic effect” means an effect resulting from treatment of a subject that alters, typically improves or ameliorates, the symptoms of a disease or condition, or that cures a disease or condition.
  • a “therapeutically effective amount” or a “therapeutically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.
  • therapeutic efficacy refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, or composition containing a compound has been administered.
  • a “prophylactically effective amount” or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound, that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses.
  • a prophylactically effective amount can be administered in one or more administrations.
  • amelioration of the symptoms of a particular disease or disorder by a treatment refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms, that can be attributed to or associated with administration of the composition or therapeutic.
  • a “prodrug” is a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form (see, e.g., Wilman, 1986, Biochemical Society Transactions, 615th Meeting Harbor, 14:375-382; and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt el al., (ed.), pp. 247-267, Humana Press, 1985).
  • an “anti-cancer agent” refers to any agent that is destructive or toxic to malignant cells and tissues.
  • anti-cancer agents include agents that kill cancer cells or otherwise inhibit or impair the growth of tumors or cancer cells.
  • exemplary anti-cancer agents are chemotherapeutic agents.
  • an “anti -angiogenic agent” or “angiogenesis inhibitor” is a compound that blocks, or interferes with, the development of blood vessels.
  • a TNF-related or TNF-mediated disease refers to a disease, condition, or disorder in which TNFR1 or TNFR1 signaling plays a role in the etiology; included are diseases, disorders, and conditions in which inhibition of TNFR1 signaling can be ameliorative of a symptom of the disease, condition, or disorder.
  • a “TNFR2 agonist,” or an “anti-TNFR2 agonist,” refers to compounds, including small molecules and TNFR2 antibodies or antigen-binding fragments thereof, and other polypeptides that initiate, promote, or increase activation of TNFR2 and/or potentiate one or more signal transduction pathways mediated by TNFR2.
  • TNFR2 agonists can promote or increase the proliferation of a population of Treg cells.
  • TNFR2 agonists can promote or increase TNFR2 activation by binding to TNFR2, e.g., to induce a conformational change that renders the receptor biologically active.
  • TNFR2 agonists can nucleate the trimerization of TNFR2 in a manner similar to or that mimics the interaction between TNFR2 and its cognate ligand, TNF (TNF ⁇ ), thus inducing TNFR2-mediated signaling.
  • TNF TNF
  • TNFR2 agonists also can induce the proliferation of CD4 + , CD25 + , FOXP3 + Treg cells.
  • TNFR2 agonists can also suppress the proliferation of cytotoxic T lymphocytes (e.g., CD8 + T-cells), e.g., through activation of immunomodulatory Treg cells or by directly binding to TNFR2 on the surface of an autoreactive cytotoxic T- cell and inducing apoptosis.
  • a TNFR2 agonist antibody or fragment thereof, for use in the methods herein, can specifically bind to TNFR2, and generally is sufficiently specific so that it does not specifically binding to another receptor of the tumor necrosis factor receptor (TNFR) superfamily member, such as TNFR1.
  • TNFR tumor necrosis factor receptor
  • a TNFR2-selective agonist is a TNFR2 agonist that does not or substantially does not result in TNFR1 signaling activity.
  • a Treg expander is a molecule, including small molecules and polypeptides, that increases regulatory T cells (Treg cells or Tregs), which are an immunosuppressive subpopulation of T cells with immunosuppressive properties via production of cytokines.
  • pan-growth factor trap construct As used herein, the terms “pan-growth factor trap construct,” “pan-EGFR ligand trap construct,” “growth factor trap,” “multi-specific growth factor trap construct,” “bi-specific growth factor trap construct,” “EGFR ligand trap construct,” “pan-HER ligand trap construct,” “pan-HER therapeutic,” “EGFR ligand trap construct,” “HER ligand trap construct” and “growth factor trap construct” are used interchangeably to refer to pan-cell surface receptor molecules, including peptide- based compounds, that modulate the activity of two or more human epidermal growth factor receptors (EGFRs), also referred to as HER or ErbB receptors.
  • EGFRs human epidermal growth factor receptors
  • ErbB receptors human epidermal growth factor receptors
  • a pan-growth factor trap targets at least two different HER receptors, such as via ligand binding and/or interaction with the receptors.
  • an “extracellular domain” or “ECD” is the portion of a cell surface receptor that occurs on the surface of the receptor and includes the ligand- binding site(s).
  • ECD polypeptide includes any ECD-containing molecule, or portion thereof, as long as the ECD polypeptide does not contain any contiguous sequence associated with another domain (e.g., transmembrane domain, protein kinase domain, or others) of a cognate receptor.
  • knobs into holes or “knobs-in-holes” (KIH) refers to multimerization domains, such as immunoglobulin Fc domains, engineered so that steric interactions between and/or among such domains, promote stable interaction, and promote the formation of heterodimers (or heteromultimers) compared to homodimers (or homomultimers) from a mixture of monomers. This can be achieved, for example, by constructing knobs or protuberances and holes or cavities in the complementary multimerizing domains.
  • “Knobs” can be constructed by replacing small amino acid side chains from the interface of the first multimerizing domain polypeptide (e.g., first Fc monomer) with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “holes” of identical or similar size to the knobs optionally are created on the interface of the second complementary multimerizing polypeptide (e.g., second Fc monomer) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • tethering refers to the interaction between two domains of a receptor monomer, whereby the monomer occurs in a conformation that renders it less available for interaction.
  • subdomain II in HER1, HER3 and HER4 can interact with subdomain IV, forming a tethered, inactive structure.
  • a receptor or isoform thereof is less available, or is unavailable, for dimerization and/or ligand binding.
  • the ECDs of the monomeric forms of HER1, HER3 and HER4 occur in a tethered form that exhibits lower ligand affinity than the untethered form.
  • HER2 which lacks certain residues in subdomain IV, occurs in an untethered form and is available for dimerization with HER1, HER3 and HER4.
  • the tethering interaction is released, and the ECD (or receptor) is in a conformation available for dimerization, which involves interactions between domains II of two ECDs.
  • HER-related diseases are any diseases, conditions or disorders in which an epidermal growth factor receptor (HER) and/or ligand is implicated in some aspect of the etiology, pathology development thereof, or symptom thereof. Involvement includes, for example, expression, overexpression, or activity of a HER family member or ligand.
  • Diseases include, but are not limited to, proliferative diseases, including cancers, such as, but not limited to, glioma, and pancreatic, gastric, head and neck, cervical, lung, colorectal, endometrial, prostate, esophageal, ovarian, uterine, bladder or breast cancers.
  • Other conditions include those involving cell proliferation and/or migration, including those involving pathological inflammatory and/or autoimmune responses, such as rheumatoid arthritis (RA), non-malignant hyperproliferative diseases, ocular conditions, skin conditions (e.g., psoriasis), conditions resulting from smooth muscle cell proliferation and/or migration, such as stenosis, including restenosis, atherosclerosis, muscle thickening of the bladder, heart or other muscles, or endometriosis.
  • RA rheumatoid arthritis
  • non-malignant hyperproliferative diseases e.g., non-malignant hyperproliferative diseases
  • ocular conditions e.g., psoriasis
  • smooth muscle cell proliferation and/or migration such as stenosis, including restenosis, atherosclerosis, muscle thickening of the bladder, heart or other muscles, or endometriosis.
  • stenosis including restenosis, atherosclerosis, muscle thick
  • a “patient” refers to a human subject.
  • animal includes any animal, such as, but not limited to, primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, and sheep; pigs; and other animals.
  • Non-human animals exclude humans as the contemplated animal.
  • the polypeptides provided herein are from any source, animal, plant, prokaryotic and fungal. Most polypeptides are of animal origin, including mammalian origin, and generally, for therapeutic use, are human or humanized.
  • composition refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.
  • a “stabilizing agent” refers to compound added to the formulation to protect either the antibody or conjugate, such as under the conditions (e.g., temperature) at which the formulations herein are stored or used.
  • agents that prevent proteins from degradation from other components in the compositions include amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, inhibitors, or substrates and other agents as described herein.
  • a “combination” refers to any association between or among two or more items.
  • the combination can be two or more separate items, such as two compositions or two collections, a mixture thereof, such as a single mixture of the two or more items, or any variation thereof.
  • the elements of a combination are generally functionally associated or related, such as elements used in a method.
  • “combination therapy” refers to the administration of two or more different therapeutics, such as an anti-TNFR construct or such as an antibody or antigen-binding fragment thereof, provided herein, and one or more therapeutics or other treatment(s), such as radiation and surgery.
  • Multiple therapeutic agents can be provided and administered separately, sequentially, intermittently, simultaneously, or in a single composition.
  • a “kit” is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof, for a purpose including, but not limited to, activation, administration, diagnosis, and assessment of a biological activity or property.
  • unit dose form refers to physically discrete units suitable for human and animal subjects, and packaged individually, as is known in the art.
  • single dosage formulation refers to a formulation for direct administration.
  • multi-dose formulation refers to a formulation that contains multiple doses of a therapeutic agent and that can be directly administered to provide several single doses of the therapeutic agent. The doses can be administered over the course of minutes, hours, weeks, days or months. Multi-dose formulations can allow dose adjustment, dose-pooling, and/or dose-splitting. Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.
  • an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass any of the compositions provided herein contained in articles of or for packaging.
  • Fluids refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
  • an isolated or purified polypeptide or protein e.g., an isolated antibody or antigen-binding fragment thereof, or biologically-active portion thereof (e.g., an isolated antigen-binding fragment), is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • a “cellular extract” or “lysate” refers to a preparation or fraction which is made from a lysed or disrupted cell.
  • control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest.
  • a control also can be an internal control.
  • ranges and amounts can be expressed as “about” a particular value or range. “About” also includes the exact amount. Hence “about 5 amino acids” means “about 5 amino acids” and also “5 amino acids.” For particular parameters about is a range within experimental error or a range acceptable to one of skill in the art for a particular parameter.
  • an optionally variant portion means that the portion is variant or non-variant.
  • TNF tumor necrosis factor
  • TNF interacts with immune cells via two receptors, TNFR1, which is overactive in autoimmune disease, and TNFR2 which suppresses autoimmune disease, but is muted when TNRF1 is overactive.
  • TNF blockers such as infliximab (sold as Remicade®), adalimumab (sold as Humira®), and etanercept (sold as Enbrel®) block TNFR1 and TNFR2, resulting in the adverse side effects.
  • Constructs provided herein address this problem. Constructs provided herein shut down only TNFR1, which leads to increased TNFR2 activity, thereby not only treating autoimmune disease symptoms, but providing improved treatment and reduced or no adverse side effects because TNFR2 activity is not blocked.
  • TNF blockers such as infliximab (sold as Remicade®), adalimumab (sold as Humira®), and etanercept (sold as Enbrel®) block TNFR1 and
  • the TNFR1 antagonists can be used for treatment of a variety of disorders, including autoimmune disorders, and also diseases and conditions, such as endometriosis, brain fog, such as from chemotherapy and COVID, Alzheimer’s disease, acute inflammation, such as results from infection by influenza viruses, and SARS-COV2, which results in long-lasting or permanent damage to the lungs, kidneys, and other tissues. Because of the adverse effects and consequent safety concerns with prior TNF blockers, they cannot be used for most of these indications.
  • TNFR1 antagonist constructs provided herein can be used. These constructs as described herein are monovalent in that they only inhibit TNFR1 and do not cause receptor clustering, they are specific, non-immunogenic, and have a half-life of at least about 3-4 weeks, permitting approximately once-a-month dosing.
  • TNFR1 antagonist constructs include at least one moiety that specifically interacts with TNFR1 or TNFR2, and, generally, a further moiety that modulates the interaction directly or indirectly or that provides a pharmacological (pharmacodynamic or pharmacokinetic or both) property to the construct.
  • a construct as provided herein includes at least two moi eties: a binding moiety that interacts with TNFR1 or TNFR2, and a second moiety that modulates or alters pharmacological properties or activities of the construct or the binding moiety.
  • the constructs provided herein are those that are antagonists of TNFR1 activity.
  • the TNFR1 antagonist constructs contain a portion that binds to or interacts with TNFR1 and inhibits TNFR1 -mediated signaling, and a second portion that confers additional properties, such as extended serum half-life, elimination of ADCC and/or CDC activity, and modulation of interaction with particular receptors.
  • the TNFR1 antagonists and constructs also include modification(s) so that they have none or reduced immunogenicity, particularly in a human, and also can include modifications to eliminate or reduce binding to pre-existing antibodies.
  • the TNFR1 antagonist constructs are selected to specifically bind to TNFR1, and to have minimal or no binding to TNFR2 or no TNFR2 antagonist activity. Thus, the constructs only modulate TNFR1. In some embodiments, the TNFR1 antagonist constructs are selected to also have or to be linked to a second domain or moiety that has TNFR2 agonist activity.
  • the TNFR1 constructs include those that are designed or selected to interact with TNFR1 with affinity, such as Kd ⁇ 50 nM or ⁇ 10 nM or ⁇ 5 nM, and particularly with higher affinity (as Kd ⁇ 1 nM or ⁇ 0.1 nM or higher affinity) and/or potent inhibition of TNFR1 signaling (e.g., IC50 50 nM or ⁇ 10 nM or ⁇ 5 nM or ⁇ 3 nM or, 1 nM or ⁇ 0.5 nM).
  • affinity such as Kd ⁇ 50 nM or ⁇ 10 nM or ⁇ 5 nM, and particularly with higher affinity (as Kd ⁇ 1 nM or ⁇ 0.1 nM or higher affinity) and/or potent inhibition of TNFR1 signaling (e.g., IC50 50 nM or ⁇ 10 nM or ⁇ 5 nM or ⁇ 3 nM or, 1 nM or ⁇ 0.5
  • the linker provides advantageous properties to the molecules, such as, for example, increased serum half-life, increased stability, proper three-dimensional structure and flexibility, and improved pharmacological properties.
  • TNF Blockers examples include Etanercept, adalimumab (Humira®), Infliximab
  • TNFR1 inflammatory blockade increases the specificity of TNFR1 inflammatory blockade and result in conservation or amplification of TNFR2 function, which is a natural immunosuppressor, at least in part by up-regulation of immunosuppressive Tregs, and the induction of protective and anti-inflammatory signaling pathways.
  • TNF Blockade resulting in inhibition of TNFR2 function also reduces the T cell-induced monocyte activation leading to increased possibility of opportunistic infections (see e.g., Rossel et al. (2007) J. Immunol. 179:4239-48).
  • TNF Blockers such as etanercept, adalimumab, infliximab
  • TNF Blockers such as etanercept, adalimumab, infliximab
  • Other blockers such as like IL6, IL17, IL23 only block their own part of the cytokine cascade, not the whole thing.
  • Existing TNF blockers have the same mechanism of action for TNFR1 and TNFR2, thereby blocking the activity of both.
  • JAK inhibitors pose similar problems; they have inflammatory and anti-inflammatory activities.
  • the inflammatory cytokine Ill is not blocked by JAK inhibitors
  • the inflammatory cytokine IL6 is blocked by JAK inhibitors (a second line use for rheumatoid arthritis treatment)
  • IL10 which is anti-inflammatory, is not blocked by JAK inhibitors.
  • Constructs provided herein combine the effectiveness of TNFR1 and TNF inhibitor therapies with the benefits of TNFR2 agonists that eliminate or reduce the adverse effects of anti-TNFRl/anti-TNF therapies, and also contribute additional therapeutic modalities advantages, including the up-regulation of immunosuppressive Tregs, and the induction of protective and anti-inflammatory signaling pathways.
  • the TNFR1 antagonist constructs contain one or more TNFR1 inhibitors, one or more linkers, and one or more activity modifiers.
  • the structure of the TNFR1 antagonist constructs provided herein can be represented by the formulae 1 : (TNFR1 inhibitor) n -linker p - (activity modifier) q , Formula la, or (activity modifier)q-linker p - (TNFR1 inhibitor) n Formula lb, where: each of n and q is an integer, and each is independently 1, 2, or 3; p is 0, 1, 2 or 3; and an activity modifier is a moiety, such as a polypeptide, such as albumin, or an Fc that is modified to have reduced or no ADCC activity, that increases serum half-life of the TNFR1 inhibitor; and the TNFR1 inhibitor is a molecule, such as a polypeptide or small drug molecule that binds to TNFR1 and inhibits its activity, such as signaling activity.
  • the activity modifier is not a human serum albumin antibody or an unmodified single Fc.
  • Activity modifiers include modified Fc regions, such as Fc modified to eliminate ADCC and/or CDC activity, Fc dimers, and other antibody domains.
  • the linkers include chemical linkers, and polypeptides, such as GS linkers, and hinge regions, such as from antibodies, so that the constructs include chemical conjugates, fusion proteins, and combinations of both.
  • linker can include additional linkers as required for conferring properties such as flexibility.
  • Each linker can contain a plurality of components.
  • Formula 2 also can include an activity modifier in place of or in addition to a linker.
  • Activity modifiers and linkers include, an Fc or and Fc with a hinge region, or an Fc with a GS linker, or other combinations of components.
  • the Fc in these constructs include unmodified Fc regions; the linkers are as described above, and detailed below.
  • TNFR2 agonist constructs that have formulae 3:
  • Linkers a. Glycine-Serine Linkers b. Hinge Regions c. chemical linkers
  • Activity modifiers a. Modified Fes b. Polypeptides and other moi eties that confer improved or altered pharmacological properties, such as increased serum half-life, resistance to degradation by endogenous proteases, and other such properties.
  • the constructs are used in methods of treatment of diseases, disorders, and conditions in which TNF in a pathologic modifier of the disease, condition, or disorder, such that inhibition TNFR1 signaling is reduced or inhibited, and/or in which inhibition of TNF or TNFR1 signaling can suppress or cause regression of the disease, disorder, or condition, and/or in which inhibition ameliorates a symptom of the disease, disorder, and/or condition.
  • TNF in a pathologic modifier of the disease, condition, or disorder
  • inhibition TNFR1 signaling can suppress or cause regression of the disease, disorder, or condition, and/or in which inhibition ameliorates a symptom of the disease, disorder, and/or condition.
  • compositions for use in the methods and uses and nucleic acids and vectors for producing constructs that include polypeptides and those that are fusion proteins.
  • the following sections describe diseases, disorders, and conditions, TNFR1/TNFR2 activities and their roles in the diseases, disorders, and conditions, existing treatments for the diseases, disorders, and conditions, constructs and components thereof that are provided herein, methods of producing the constructs, pharmaceutical compositions containing the constructs and/or encoding nucleic acids, and methods of treatment.
  • TNF tumor necrosis factor
  • TNF Tumor Necrosis Factor
  • Tumor necrosis factor (TNF; see e.g., SEQ ID NO:1; also referred to as TNF alpha, TNF- ⁇ , or TNF- ⁇ ) is a pleiotropic, proinflammatory cytokine that is associated with inflammatory and immuno-regulatory activities, including the regulation of tumorigenesis/cancer, host defense against pathogenic infections, apoptosis, autoimmunity, and septic shock, and that plays an important role in the coordination of innate and adaptive immune responses, as well as organogenesis, particularly of the lymphoid organs.
  • TNF Tumor necrosis factor
  • TNF is produced primarily by macrophages, and also can be produced by monocytes, dendritic cells (DCs), B cells, T cells, fibroblasts and other cell types. It is produced as a homotrimeric membrane-bound protein containing 233 amino acids (26 kDa) that can be cleaved by the protease TACE (TNF alpha converting enzyme; also known as ADA17) to release soluble TNF, which contains 157 amino acids (17 kDa); membrane-bound and soluble forms of TNF are biologically active.
  • TACE TNF alpha converting enzyme
  • Transmembrane human TNF contains 233 amino acids, and contains a cytoplasmic domain, corresponding to residues 1-35, a transmembrane domain, corresponding to residues 36-56, and an extracellular domain, corresponding to residues 57-233, with reference to SEQ ID NO: 1.
  • the soluble form of TNF corresponds to amino acid residues 77-233, as set forth in SEQ ID NO: 1 (see, SEQ ID NO:2 for the sequence of amino acid residues of soluble TNF).
  • TNF Uncontrolled production of TNF is associated with several inflammatory and autoimmune diseases and conditions, including, for example, septic shock, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the overexpression of TNF also has been associated with neurodegenerative diseases and conditions, such as, for example, Alzheimer’s disease, Parkinson’s disease, stroke and multiple sclerosis. Additionally, TNF promotes osteoclastogenesis, and overproduction of TNF is associated with bone loss.
  • TNF TNF receptors
  • TNFRs TNF receptors
  • mice overexpression of human TNF in mice results in the development of spontaneous RA-like lesions in the joints with the formation of hyperplastic synovial membranes and the destruction of cartilage and bone (see, e.g., Blüml et al. (2010) Arthritis & Rheumatism 62(6): 1608- 1619; Keffer et al. (1991) EMBO J 10(13):4025-4031 ; Esperito Santo et al. (2015) Biochem. Biophys. Res. Commun. 464: 1145-1150; Blüml et al. (2012) International Immunology 24(5):275-281; Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43): 12304-12309).
  • TNF signals through two high-affinity, specific receptors, TNFR1 and TNFR2; TNFR1 is associated with detrimental inflammatory processes, while TNFR2 is associated with beneficial immuno-regulatory processes. It has been shown that membrane-bound TNF primarily activates TNFR2, while soluble TNF primarily activates TNFR1 (Blüml et al. (2010) Arthritis & Rheumatism 62(6): 1608-1619).
  • Soluble TNF (solTNF; corresponding to residues 77-233 of SEQ ID NO: 1; see, also, the sequence set forth in SEQ ID NO:2), which is involved in paracrine signaling (primarily via TNFR1), is associated with chronic inflammation, whereas transmembrane TNF (tmTNF), which acts via cell-to-cell contact to induce juxtacrine signaling (primarily via TNFR2), is associated with the resolution of inflammation and with the induction of immunity against pathogens, such as Listeria monocytogenes and Mycobacterium tuberculosis (Zalevsky et al. (2007) J. Immunol. 179: 1872-1883).
  • TNF signaling through TNFR1 and TNFR2 effects different outcomes, depending on the receptor type.
  • TNF blockade of TNF Due to the association between TNF overexpression and the development of inflammatory and autoimmune diseases and conditions, the blockade of TNF has been used in the treatment of various such diseases and conditions, including, but not limited to, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA), and inflammatory bowel disease (IBD; e.g., Crohn’s disease, ulcerative colitis).
  • RA rheumatoid arthritis
  • JIA juvenile idiopathic arthritis
  • IBD inflammatory bowel disease
  • TNF blockers not only block detrimental inflammatory signaling via TNFR1, but also block beneficial, immune-regulatory signaling via TNFR2.
  • TNF blockers particularly in the case of chronic diseases/conditions that require long-term administration, such as arthritis or IBD, can be limited.
  • Approximately one-third of RA patients are non -responsive, or therapeutic benefits are not sustained, with the use of anti-TNF therapies.
  • therapies with improved therapeutic efficacy and safety, particularly therapies that block the inflammatory effects of TNFR1 signaling, but maintain, or boost, the beneficial anti-inflammatory effects of TNFR2 signaling.
  • Such therapies are provided herein.
  • TFRs Tumor Necrosis Factor Receptors
  • TNFR1 TNF receptor type 1; also known to as TNFRI, p55, p60, CD 120a, TNF receptor superfamily member 1A, and TNFRSF1A
  • TNFR2 TNF receptor type 2; also known as TNFRII, p75, p80, CD120b, TNF receptor superfamily member IB, and TNFRSF1B).
  • TNFRI is expressed by all nucleated cells types; TNFR2 expression is restricted to immune cells (e.g., monocytes, macrophages, activated T cells, regulatory T cells (Tregs), B cells and natural killer (NK) cells), endothelial cells, particular central nervous system (CNS) cells, and particular cardiac cells.
  • TNFR2 expression on Tregs is induced upon T-cell receptor activation.
  • TNFR1 and TNFR2 exist as membrane-bound receptors, and as soluble, “decoy” (i.e., non-signaling) receptors, following shedding from cell surfaces.
  • Soluble TNF preferentially/selectively binds to TNFR1; binding of the membrane-bound and soluble forms of TNF, however, activates TNFR1.
  • the primary ligand for TNFR2 is membrane-bound TNF. Soluble TNF does not fully activate TNFR2, but the soluble form of TNFR2 (following TNFR2 shedding) has a high binding affinity for TNF, allowing it to scavenge and inhibit TNF from binding membrane-bound, signaling receptors, which contributes to the anti-inflammatory effects of TNFR2.
  • TNFR1 and TNFR2 binds TNF with rapid on and off kinetics, allowing TNFR2 to concentrate TNF on cell surfaces and pass the ligand to TNFR1, which mediates TNFR1 signaling.
  • Each of TNFR1 and TNFR2 contains extracellular, transmembrane and cytoplasmic domains.
  • the extracellular domains of TNFR1 and TNFR2 contain four cysteine-rich domains (CRDs) that are required for ligand binding.
  • CCDs cysteine-rich domains
  • the intracellular domains of TNFR1 and TNFR2 initiate different signaling cascades, and mediate different effector functions, in response to TNF ligand binding.
  • TNFR signaling abnormalities are associated with several autoimmune diseases, and the administration of TNF can be used as a treatment strategy for such diseases.
  • low dose TNF selectively destroys autoreactive T cells in blood samples from type I diabetes and scleroderma patients, and in an animal model of Sjogren’s syndrome.
  • the administration of TNF can result in systemic toxicity, for example, in cancer patients with high TNF levels.
  • the toxicity results from the ubiquitous cellular expression of TNFR1; as described herein, agonizing TNFR2 is a safer therapeutic option than administration of TNF, due to its more restricted cellular expression.
  • TNFR1 TNFR1
  • Human TNFR1 (see, SEQ ID NO:3), is the major inflammatory receptor, and accounts for the majority of the proinflammatory, cytotoxic and apoptotic effects attributed to TNF. Human TNFR1 is a homotrimeric receptor, and its binding by TNF induces a pro-inflammatory response (see, e.g., Morton et aL (2019) Sci Signal. 12(592):eaaw2418, for a description of TNFR1 signaling). TNFR1 contains 455 amino acid residues; residues 1-29 correspond to the signal peptide, residues 30-211 correspond to the extracellular domain, residues 212-232 correspond to the transmembrane domain, and residues 233-455 correspond to the cytoplasmic domain.
  • TNFR1 contains cysteine-rich domains (CRDs) 1-4, corresponding to amino acid residues 43-82, 83-125, 126-166 and 167-196 of SEQ ID NO:3, respectively.
  • CRDs 2 and 3 contact bound TNF, and CRD1, particularly amino acid residues 30-82 with reference to SEQ ID NO:3, forms the pre-ligand binding assembly domain (PLAD), a hemophilic interaction motif that is necessary for ligand binding and receptor function.
  • PAD pre-ligand binding assembly domain
  • the cytoplasmic domain contains a death domain (corresponding to residues 356-441 of SEQ ID NO:3) that binds to the TNFR1- associated death domain (TRADD) and the Fas-associated death domain (FADD) following the binding of TNF to TNFR1, resulting in signaling pathways that activate caspases and induce apoptosis.
  • TRADD TNFR1-associated death domain
  • FADD Fas-associated death domain
  • the binding of TNF to TNFR1 also initiates proinflammatory cascades through MAPK (mitogen-activated protein kinase; e.g., p38, JNK, ERK) and NF-excellentB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathways.
  • MAPK mitogen-activated protein kinase
  • NF-excellentB nuclear factor kappa-light-chain-enhancer of activated B cells
  • TNFR1 plays a role in lymphatic organogenesis and in the immune response to pathogens, and is the primary receptor associated with host anti- viral defense mechanisms. It has been shown that mycobacterial containment depends on TNF-derived signals, and that patients treated with TNF-blockers can suffer from endogenous reactivation of latent tuberculosis.
  • TNFR1 which primarily is involved in pro-inflammatory signaling, is the driving force in the development of arthritis.
  • knockout of TNFR1 in mice, as well as silencing of TNFR1 expression by RNA interference results in the attenuation of collagen-induced arthritis (CIA), an animal model of arthritis.
  • CIA collagen-induced arthritis
  • TNFR1 deficient mice that overexpress TNF are protected from the development of arthritis, and the reintroduction of TNFR1 on mesenchymal cells results in the development of TNF-dependent arthritis.
  • TNFR1 enhances the generation of osteoclasts, resulting in local bone destruction, and it has been shown that the lack of TNFR1 on hematopoietic cells attenuates bone destruction in a model of erosive arthritis.
  • TNFR1 also has been associated with cardiotoxic effects in TNF-induced models of heart failure and myocardial infarction, and has been shown to promote neurodegeneration in an animal model of retinal ischemia (see, e.g. , Schmidt et al. (2013) Arthritis & Rheumatism 65(9 ⁇ .2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065; McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Ruspi et al. (2014) Cellular Signaling 26:683-690; Faustman and Davis (2013) Front. Immunol. 4:478; Blüml et al. (2012) International Immunology 24(5):275-281 ; Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43): 12304-12309).
  • b. TNFR2
  • TNFR2 Human TNFR2 (see, SEQ ID NO:4) contains 461 amino acid residues; residues 1-22 correspond to the signal peptide, residues 23-257 correspond to the extracellular domain, residues 258-287 correspond to the transmembrane domain, and residues 288-461 correspond to the cytoplasmic domain.
  • TNFR2 which, unlike TNFR1, lacks a death domain, has a TNF receptor-associated factor 2 (TRAF2) binding site.
  • TRAF2 signaling via TRAF2 promotes cell survival and proliferation through NF-excellentB and activator protein 1 (API) activation, and has been associated with PI3K-PKB/Akt-mediated repair and migration.
  • API3K-PKB/Akt-mediated repair and migration TNF receptor-associated factor 2
  • TNF signaling via TNFR2 also promotes the expansion and activation of regulatory T cells (Tregs), which play an important role in the suppression of inflammatory and autoimmune diseases and disorders.
  • Tregs regulatory T cells
  • TNFR2 signaling has been implicated in repair and regeneration in models of wound healing and myocardial infarction, while knockout of TNFR2 in a mouse model of erosive arthritis results in joint inflammation and bone destruction.
  • TNFR2 which primarily is involved in anti-inflammatory signaling, has been associated with neuro-, cardio-, gut- and osteo-protective effects.
  • TNFR2 exhibits anti-inflammatory and protective effects; these effects have been demonstrated, for example, in experimental autoimmune encephalomyelitis (EAE), experimental colitis, heart failure/heart disease, myocardial infarction, inflammatory arthritis, demyelinating and neurodegenerative disorders, and infectious disease.
  • EAE experimental autoimmune encephalomyelitis
  • TNF activation of TNFR2 by TNF inhibits seizures, attenuates cognitive dysfunction following brain injury, promotes survival following myocardial infarction in mice, protects against myocardial ischemia/reperfusion injury, and reduces remodeling and hypertrophy following heart failure.
  • TNFR2 agonism also is associated with pancreatic regeneration, remyelination, survival of neuron subtypes, and stem cell proliferation.
  • TNFR2 agomsm selectively destroys autoreactive T cells, but not healthy cells, in blood samples from patients with type I diabetes, multiple sclerosis, Graves’ disease and Sjogren’s syndrome.
  • TNF signaling through TNFR2 has been shown to induce regeneration of oligodendrocyte precursors in myelin, and thus, can be of use for the treatment of demyelinating disorders, such as multiple sclerosis (MS).
  • TNFR2 also has been shown to promote neuroprotection in an animal model of retinal ischemia.
  • TNFR2 also regulates osteoclastogenesis.
  • Osteoclasts are a type of bone cells that break down bone tissue; the regulation of osteoclastogenesis is important for maintaining bone mass, and protecting against joint inflammation and erosive destruction.
  • Mice lacking TNFR2 display enhanced osteoclastogenesis, worsening TNF-driven arthritis, and local bone destruction.
  • the lack of TNFR2 in an animal model of erosive arthritis results in disease progression, and TNFR2-deficient mice overexpressing TNF develop aggravated arthritis and joint destruction compared with control mice.
  • Expression of TNFR2 on hematopoietic cells attenuates TNF-driven arthritis, while the loss of TNFR2 on hematopoietic cells increases the recruitment of inflammatory cells to the synovial membrane.
  • Polymorphisms in the TNFR2 gene are correlated with a variety of autoimmune diseases, including, for example, RA, Crohn’s disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis and scleroderma; the polymorphisms hinder the binding of TNF to TNFR2, which limits activation of NF-excellentB and hampers TNFR2 signaling pathways in Tregs (see, e.g., Yang et al.
  • TNFR1 contains an intracellular death domain and can activate apoptotic and/or inflammatory pathways, while TNFR2 binds TRAFs and can activate the canonical and non-canonical NF-excellentB pathways to control cell survival and proliferation.
  • TNFR2 binds TRAFs and can activate the canonical and non-canonical NF-excellentB pathways to control cell survival and proliferation.
  • cells that express TNFR2 also express TNFR1, at varying ratios, depending on the cell type and function. Since TNFR1 signaling generally induces cell death, whereas TNFR2 signaling generally induces cell survival, the ratio of their co-expression on cells shifts the balance towards apoptosis or cell survival.
  • TNFR1 is the primary TNF receptor involved in the pathogenesis of RA, while TNFR2 plays an immunoregulatory role. Both receptors, however, are involved in mediating the antiviral activity of TNF.
  • Animal disease models show that TNFR1 is associated with inflammatory neurodegeneration, while TNFR2 is associated with neuroprotection.
  • TNFR1 TNFR1 -selective antagonistic IgGl antibody
  • EHD2-SCTNF R2 an agonistic TNFR2-selective TNF mutein (i.e. , mutated protein ).
  • EHD2-SCTNF R2 contains a covalently stabilized human TNFR2-selective single-chain TNF trimer with the mutations D143N/A145R (residue numbering with respect to soluble TNF as set forth in SEQ ID NO:2, and corresponding to D219N and A221R, respectively, with respect to SEQ ID NO: 1; these mutations abrogate affinity for TNFR1), fused to the dimerization domain EHD2, which is derived from the heavy chain C H 2 domain of IgE and creates a disulfide bonded dimer that contains hexameric TNF domains.
  • NMD A and ATROSAB Simultaneous injection of NMD A and ATROSAB, or NMD A and EHD2-SCTNF R2 , into the nucleus basalis magnocellularis results in significant but incomplete neuroprotective effects compared with controls, in an in vivo mouse model.
  • the incomplete nature of these responses was due to the agonistic activity of ATROSAB, a byproduct of the bivalent antibody inducing aberrant receptor clustering and activation (Richter et al. (2013) PLoS One 8(8):e72156.
  • the EHD2- scTNFR2 is immunogenic in humans because of its multiple fusion partners, and an immune response to the IgE fragments result in an autoimmune reaction in toxicology studies (see, e.g., Weeratna et al. (2016) Immun. Inflamm. Dis. 4(2)'.135-147).
  • Treg cells are an immunosuppressive subpopulation of T cells with immunosuppressive properties via production of cytokines. These include transforming growth factor beta, interleukin 35, and interleukin 10. Induction of Treg function can inhibit several pathologies. Induction can enhance success of transplantation, suppress allergy, control responses, such as severe acute respiratory syndrome, to infectious disease and autoimmunity. Tregs suppress and/or downregulate the induction and proliferation of effector T cells (Teffs), modulate the immune system, maintain immune homeostasis and tolerance to self-antigens, and can prevent the development of autoimmune disease and tissue destruction.
  • Tregs effector T cells
  • Tregs express, among other markers, CD4, CTLA-4, CD25 (also known as IL-2 receptor alpha chain or IL2RA), and FOXP3 (transcription factor forkhead box P3), and express TNFR2 at a tenfold higher density than they express TNFR1.
  • TNFR2 is expressed by only a subpopulation of Tregs, which is the maximally suppressive subset; this subset contains TNFR2-expressing CD4 + FoxP3 + Tregs.
  • TNF via TNFR2 signaling, promotes Treg cell proliferation, up-regulation of FoxP3 expression, and Treg cell suppressive activity/function.
  • the autoimmune microenvironment contains more autoreactive CD8 + effector T cells than immunosuppressive CD4 + Tregs, resulting in tissue destruction.
  • TNFR2 function As a result, preservation of TNFR2 function, or enhanced TNFR2 function, which expands Tregs and eliminates autoreactive T cells, restores the immune balance (see, Sharma et al. (2016) Front Immunol. 9:883).
  • pharmacological retention of Treg function by selective inhibition of TNFR1, possibly together with TNFR2 stimulation (agonism) would improve outcomes in many acute and chronic inflammatory conditions (severe acute respiratory syndrome, autoimmune diseases).
  • TNF In addition to up-regulating the expression of TNFR2 on Tregs, TNF also up- regulates the Treg surface expression of other co-stimulatory members of the TNF receptor superfamily (TNFRSF), such as 4- IBB and 0X40, result in the optimal activation and proliferation of Tregs, and in the attenuation of excessive inflammatory responses.
  • TNFRSF TNF receptor superfamily
  • Neutralization of TNF blocks in vivo expansion of Tregs (e.g., Hamano et al. (2011) Eur. J. Immunol. 41 :2010-2020).
  • CD4 + FoxP3 + Tregs constitutively express TNFR2, promoting Treg cell activation, expansion and survival.
  • TNF signaling through TNFR2 i.e., TNFR2 agonism
  • TNFR2 agonism promotes the activation and expansion of Tregs
  • TNFR2 antagonism results in Treg contraction.
  • TNFR2 agonism selectively kills autoreactive T cells and expands suppressive Tregs in humans with autoimmune disease, and in animal models of autoimmunity.
  • TNFR2 signaling promotes Treg cell expansion and suppressive activity in experimental autoimmune encephalomyelitis (EAE; an animal model of inflammatory CNS demyelinating disease, e.g., multiple sclerosis), and in a murine model of diabetes, and induces human antigen-specific Treg cells by tolerogenic dendritic cells.
  • EAE experimental autoimmune encephalomyelitis
  • TNFR2-deficient Tregs are reduced in their ability to prevent experimental colitis in vivo, and TNFR2 is required for sustained FoxP3 expression on Tregs, and as a result, for maintaining the phenotypic and functional stability of Tregs, indicating that TNFR2 is required for the in vivo immunosuppressive function of Tregs (see, e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728- 2738; Faustman and Davis (2013) Front. Immunol. 4:478; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Vanamee et al.
  • Tregs were shown to suppress disease and reduce joint inflammation and bone destruction in a well-established antigen- induced arthritis (AIA) model, in which mice are immunized with methylated bovine serum albumin (mBSA) to induce T cell-mediated tissue damage (see, e.g., Wright et al. (2009) Proc. Natl. Acad. Sci. USA 106(45): 19078-19083).
  • AIA antigen-induced arthritis
  • mBSA methylated bovine serum albumin
  • TNFR2 As described and provided herein, TNFR2, and its expression by Tregs, is required for the suppression of inflammatory and autoimmune diseases and conditions.
  • the mycobacterium bovis bacillus Calmetter-Guenn (BCG) induces transient expansion of Tregs.
  • BCG mycobacterium bovis bacillus Calmetter-Guenn
  • Tregs and/or modulators that enhance Treg function in the treatment of type I diabetes (see, e.g., Spence et al. (2016) Curr Diab Rep 16(11): 110. dor. 10.1007/s 11892-016-0807-6).
  • Treg function presents a therapeutic approach for the prevention or treatment of inflammatory and autoimmune diseases and conditions.
  • Tregs only constitute -1-5% of total CD4+ T cells in the blood. Their low numbers hinder their clinical use.
  • Ex vivo generation of Tregs, and/or stimulation of their production in vivo is factor that limits their therapeutic use. For example, in vivo stimulation with IL-2, anti-CD3, or anti- CD28 is too toxic, while ex vivo stimulation using these agents generates heterogeneous CD4 + populations that can release proinflammatory cytokines and have antagonistic properties.
  • TL1 A-Ig a naturally occurring TNF receptor superfamily agonist, or TNFR2 monoclonal antibody agonists
  • a TNFR2 agonist construct, and the multi-specific constructs, provided herein can preserve and/or expand the Treg population in vivo without interfering with the therapeutic activity of anti-TNFRl activity.
  • selective inhibition of inflammatory TNFR1 activity, while maintaining or increasing TNFR2-associated Treg suppressive activity is beneficial in the treatment of inflammatory and autoimmune diseases and conditions. These diseases and conditions include, but are not limited to, RA, type I diabetes, heart failure and multiple sclerosis (see, e.g., Goodall et al. (2015) PLoS ONE 10(9):e0137065).
  • TME tumor microenvironment
  • TNFR2 + Tregs In a tumor microenvironment (TME), in contrast to an autoimmune microenvironment in which the expansion of TNFR2 + Tregs prevents tissue destruction, tumors are infiltrated by large numbers of immunosuppressive TNFR2 + Tregs, which prevent the proliferation of tumor-killing CD8 + cytotoxic T lymphocytes (CTLs), also known as effector T cells (Teffs), allowing for tumor growth.
  • CTLs cytotoxic T lymphocytes
  • Teffs effector T cells
  • Antagonism of TNFR2 on lymphocytes in the TME restores the balance between the two types of T cells, by inhibiting or eliminating Tregs and allowing for the activation and expansion of effector T cells, a condition where tumor growth can be controlled or reversed.
  • the TNFR2 inhibitor must not have the ability to aggregate immune cells via ADCC for two reasons: 1) aggregation transiently leads to ‘super-induction’ of TNFR2 mediated immunosuppression; and 2) eventually leads to systemic depletion of Tregs, which will be detrimental to the patient because it is essential to retain a basal level of Treg activity to maintain immune homeostasis.
  • Tumor cells and myeloid-derived suppressor cells also express TNFR2, and inhibition of TNFR2 in MDSCs control metastasis, as shown in a murine liver cancer model.
  • blockade of TNFR2 presents a useful treatment for certain types of cancers via the inhibition of immunosuppressive Tregs.
  • TNFR2 antagonists only should be administered to patients whose tumors show overexpression of TNFR2 compared to adjacent normal tissue as judged from immunohistochemistry.
  • diagnostics See e.g., Zhang et al. (2019) Thorac Cancer 10(3) A37-444. doi: 10.1111/1759-7714.12948; Yang et al. (2017) Oncol Lett.14 (2) .2393 -2398. doi:10.3892/ol.2017.6410; and Yang et al. (2016) Oncol Lett. 16(3): 2971-2978. doi: 10.3892/ol.2018.8998, for exemplary assays).
  • TNF- ⁇ is involved in numerous diseases, disorders, and conditions. Constructs provided herein can be used for treatment of such diseases, disorders, and conditions. The following discussion describes some exemplary diseases, disorders, and conditions in which blocking TNF can have a therapeutic effect.
  • TNF blockers such as etanercept, infliximab, adalimumab, certolizumab and Golimumab, have adverse side effects that can limit their use for treatment of such diseases, disorders, and conditions.
  • constructs provided herein which avoid some or all of these adverse effects, can be used to treat these diseases, disorders, and conditions (see, e.g., Lis et al. (2014) Arch Med Sci.10(6):1175-1185 for a review of the role of TNF in disease and the use of TNF blockers for treatment thereof).
  • Inflammatory diseases include an array of disorders and conditions that are characterized by inflammation, and include autoimmune diseases.
  • the immune system protects the body by producing antibodies and/or activating lymphocytes in response to invading microorganisms, such as viruses and bacteria. In healthy individuals, the immune system does not trigger a response against the body’s own (i.e., “self’) cells; autoimmune diseases occur when the immune system attacks healthy, non-invading, self, cells and tissues.
  • Autoimmune/inflammatory diseases and disorders associated with elevated TNF levels include, for example, arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, spondyloarthritis), inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis), uveitis, fibrotic diseases, endometriosis, lupus, ankylosing spondylitis, psoriasis, multiple sclerosis (MS), Parkinson’s disease, and Alzheimer’s disease, among others.
  • arthritis e.g., rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, spondyloarthritis
  • inflammatory bowel disease e.g., Crohn’s disease and ulcerative colitis
  • uveitis e.g., Crohn’s disease and ulcerative colitis
  • fibrotic diseases e.g., endometriosis,
  • Rheumatoid arthritis is a chronic autoimmune inflammatory disease.
  • the inflammation associated with rheumatoid arthritis affects the linings of the joints (i.e., the synovial lining), and also the membranes lining the blood vessels, heart and also can become inflamed.
  • RA is characterized by the infiltration of immune cells (e.g., activated B cells) into the synovial membrane and synovial cell proliferation, which results in the thickening of the synovial lining.
  • the proliferative mass known as the pannus, invades and destroys cartilage and bone, irreversibly destroying joint structure and function. This is mediated by the induction of proinflammatory cytokines, such as TNF, IL-1 and IL-6.
  • Tumor necrosis factor a is a key modulator of the induction and perpetuation of the proinflammatory activities that are associated with RA.
  • TNF is over-expressed in synovial fluids and in the synovial membrane, and expression of TNFRs is up-regulated in the synovial membrane (see, e.g., Blüml et al. (2012) International Immunology 24(5):275-281; Schmidt et al. (2013) Arthritis & Rheumatism 65(9y.2262-2223,' Keffer et al. (199V) EMBO J. 10(13):4025-4031).
  • Other types of arthritis that can be treated with constructs herein, include, for example, psoriatic arthritis, juvenile idiopathic arthritis, and spondyloarthritis b. Inflammatory Bowel Disease (IBD) and Uveitis
  • IBD Inflammatory bowel disease
  • Crohn’s disease includes Crohn’s disease and ulcerative colitis, which are inflammatory diseases of the intestine and colon.
  • Mice overexpressing TNF develop intestinal inflammation that resembles Crohn’s disease, while TNFR1 deficiency protects against Crohn’s disease, (see, e.g., Fischer et al. (2015) Antibodies 4:48-70).
  • Uveitis is a form of eye inflammation that affects the eye wall (uvea), the middle layer of the eye between the retina and the sclera (white of the eye), and can lead to vision loss.
  • TNF-alpha is involved in its pathophysiology, and TNF blockers have been used for treatment.
  • Dupuytren’s disease is exemplary of such diseases.
  • Dupuytren’s disease (DD) is a common fibrotic condition of the hands that is characterized by irreversible flexion contractures of the fingers; the condition is limited to the palm of the hand and causes irreversible curling in of the fingers, severely compromising hand function.
  • DD Dupuytren’s disease
  • Treatment involves surgical excision (fasciotomy) of the diseased tissue or cords, or disruption of the cords using collagenase or needle fasciotomy.
  • Myofibroblasts which express the contractile protein a-smooth muscle actin (a-SMA) and aggregate in nodules, deposit excessive collagenous extracellular matrix and are responsible for its remodeling and contraction in all fibrotic conditions, including DD.
  • TNF converts palmar fibroblasts into myofibroblasts in patients with DD, via the Wnt signaling pathway, and DD myofibroblasts exhibit a dose-dependent reduction in contractility and reduction in the expression of a-SMA and pro-collagen, following treatment with anti-TNF therapies.
  • Treatment with the fully humanized IgG mAbs adalimumab and golimumab have been the most effective.
  • anti-TNF therapies such as adalimumab
  • adalimumab is associated with an increased risk of infection, and in a phase 2a trial evaluating the therapeutic efficacy of adalimumab in DD, 1 patient (out of 21 receiving adalimumab) developed a wound infection requiring hospitalization (see, e.g., Nanchahal et al. (2016) EBioMedicine 33:282- 288).
  • TRAPS Tumor Necrosis Factor Receptor-Associated Periodic Syndrome
  • TRAPS Tumor necrosis factor receptor-associated periodic syndrome
  • TRAPS Tumor necrosis factor receptor-associated periodic syndrome
  • TRAPS is the second most common inherited autosomal dominant auto-inflammatory disease, and is caused by mutations in the TNFRSF1A gene, encoding TNFR1.
  • TRAPS is characterized by unprovoked, periodic long-lasting fever, systemic inflammation, abdominal pain, skin lesions, conjunctivitis, myalgia and pericarditis, with inflammatory attacks lasting up to several weeks.
  • a complication associated with more severe clinical phenotypes of TRAPS is AA-type serum amyloidosis, which can result in renal impairment and failure. Disease onset typically occurs in early childhood, but TRAPS can present in adults as well.
  • TRAPS-associated mutations occur in the extracellular domain of TNFR1, which is involved in ligand binding.
  • High-penetrance mutations which are associated with the most severe clinical phenotype, occur in the extracellular cysteine-rich domains (CRDs).
  • CCDs cysteine-rich domains
  • the mutations affect the folding and secondary structure of TNFR1, which can result in defective TNFR1 trafficking, altered ligand binding affinity, reduced activation- induced shedding and impaired cell signaling.
  • ligand-independent gain- of-function of TNFR1 induces TRAPS pathophysiology, and certain mutations result in the constitutive activity of TNFR1, NF-excellentB and caspase 1.
  • TNF central nervous system
  • CNS central nervous system
  • TNF is implicated in initiating and maintaining neuroinflammation, and in modulating other neurological processes, such as synaptic function and plasticity.
  • the levels of TNFR1 in the hippocampus of aged rats is approximately 3-fold higher compared to the levels of TNFR2.
  • TNF is implicated in chronic glial activation and impaired neuronal viability through its actions on TNFR1.
  • neurologic changes include synaptic dysfunction and Ca 2+ dysregulation, which can be replicated in healthy young animals and in neuronal cultures using artificial elevations in TNF.
  • TNF also potentiates the activity of L-type voltage sensitive Ca 2+ channels (L- VSCCs); a similar effect is observed in hippocampal neurons of memory impaired aged rats.
  • L- VSCCs L-type voltage sensitive Ca 2+ channels
  • XProl595 a soluble dominant negative TNF (DN-TNF) that preferentially inhibits TNFR1 signaling, resulted in improved behavioral performance on a Morris swim task, reduced microglial activation, prevention of hippocampal long-term depression (LTD), and reduced the activity of L-VSCCs in CAI neurons.
  • DN-TNF soluble dominant negative TNF
  • TNF is a central player in inflammatory responses; TNF protein levels are low in healthy brain but chronically elevated in many neuroinflammatory diseases, including Alzheimer’s disease (AD).
  • AD Alzheimer’s disease
  • TNF promotes microglial activation, synaptic dysfunction, neuronal cell death, accumulation of plaques and tangles, and cognitive decline.
  • APP amyloid precursor protein
  • tau TNF levels were elevated in entorhinal cortex, coincident with the earliest appearance of pathology (see, e.g., McCoy et al. (2006) J. Neurosci. 26(37):9365-9375).
  • TNF-driven processes are implicated in AD pathology and contribute to cognitive dysfunction and accelerated progression of AD.
  • the bacterial endotoxin lipopolysaccharide (LPS) which induces inflammation and the production of TNF, accelerates the appearance and severity of AD pathology in several animal models of AD.
  • LPS bacterial endotoxin lipopolysaccharide
  • the overproduction of proinflammatory mediators, including TNF, occurs in the brain when microglia, which are often in close physical association with amyloid plaques in AD brains, become chronically activated. Elevated levels of TNF inhibit phagocytosis of amyloid beta (A ⁇ ) in the brains of AD patients, which hinders efficient plaque removal by microglia.
  • a ⁇ amyloid beta
  • the chronic inhibition of solTNF by administering a DN-TNF, such as XENP345, or a lentivirus encoding the DN-TNF prevented the acceleration of AD-like pathology induced by chronic systemic inflammation in an animal model of AD (3xTgAD mice), and decreased the LPS- induced intraneuronal accumulation of 6E10-immunoreactive protein, particularly C- terminal amyloid precursor protein (APP) fragments (P-CTF), in the hippocampus, cortex and amygdala.
  • APP C- terminal amyloid precursor protein
  • Parkinson’s disease is the second most prevalent neurodegenerative disease in the United States, with an incidence of 5% in individuals over 65 years of age.
  • the clinical manifestations of Parkinson’s disease result from the selective loss of dopaminergic neurons in the ventral mesencephalon substantia nigra pars compacta (SNpc), which results in a decrease in striatal dopamine.
  • the cerebrospinal fluid (CSF) and postmortem brains of patients with PD and animal models of PD show elevated levels of TNF.
  • a cohort of early-onset PD patients in Japan showed an increased frequency of a polymorphic allele (-1031C) in the TNF gene promoter that results in higher transcriptional activity and elevated TNF levels.
  • TNFR1 is highly expressed in mgrostnatal dopaminergic neurons, which increases vulnerability to TNF -induced neuroinflammation and dopaminergic neuron toxicity.
  • the in vivo neutralization of soluble TNF (solTNF) by a dominant-negative TNF mutein (XENP345) was neuroprotective, and reduced the retrograde nigral degeneration induced by a striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6- OHDA) by 50% and attenuated amphetamine-induced rotational behavior in rats, indicating preservation of striatal dopamine levels.
  • XENP345 Delayed administration of XENP345 in embryonic rat midbrain neuron/glia cell cultures exposed to lipopolysaccharide (LPS) prevented the degeneration of dopaminergic neurons, despite sustained microglia activation and secretion of solTNF.
  • LPS lipopolysaccharide
  • XENP345 also attenuated 6-OHDA-induced dopaminergic neuron toxicity in vitro.
  • TNF thus, is implicated in the development of Parkinson’s disease, and it may be possible to delay the progressive degeneration of the nigrostriatal pathway in humans by using TNF- blocking therapeutics, particularly in the early stages of Parkinson’s disease (see, e.g., McCoy et al. (2006) J Neurosci. 26(37):9365-9375).
  • MS Multiple Sclerosis
  • TNFR1 multiple sclerosis
  • MS multiple sclerosis
  • EAE experimental autoimmune encephalomyelitis
  • TNFR2 deficiency worsens the disease.
  • Mice expressing non-cleavable membrane-bound TNF are protected against EAE, indicating that the interaction of soluble TNF with TNFR1 is associated with disease pathology (see, e.g., Fischer et al. (2015) Antibodies 4:48-70).
  • TNF- ⁇ has been implicated in the pathophysiology of endometriosis. TNF- ⁇ levels are increased in peritoneal fluid of women with endometriosis, and the levels correlate with severity of disease (see, e.g., Koninckx (2008) Hum Reprod. 23: 2017- 2023). Peritoneal fluid TNF- ⁇ is produced locally by activated peritoneal macrophages, and TNF- ⁇ induces IL-8 secretion by peritoneal mesothelial cells. The peritoneal fluid concentrations of TNF- ⁇ and IL-8 correlate with the size and the number of active peritoneal lesions (Bullimore, (2003) Med Hypotheses. 60:84-88).
  • Serum TNF-a levels are increased, and monocytes from patients with endometriosis release more TNF-a in vitro compared with monocytes from controls.
  • Peritoneal fluid levels of MCP-1 are increased in patients with endometriosis.
  • TNF-a, IL-8 and MCP-1 drive an inflammatory Th- 1 type response in the peritoneal fluid of patients with endometriosis.
  • TNF-a mediated inflammation may be a causal factor in the pain associated with endometriosis.
  • Blocking TNF-a appears to inhibit the development of the disease in animal models, and may be effective for humans. Because of the adverse side effects of existing TNF blockers, treatment of endometriosis with such blockers has not been recommended (see, Koninckx (2008) Hum Reprod. 23: 2017-2023). Constructs provided herein, however, are designed to avoid the deleterious effects, and can be considered for treating TNF-a mediated inflammation in endometriosis.
  • TNF- ⁇ was the first cytokine to be identified in human atherosclerotic plaque
  • TNF- ⁇ is involved in the activation of the endothelium and upregulation of adhesion molecules, which occur early in the development of atherosclerotic disease. TNF also is implicated in the pathogenesis of atherosclerosis by affecting lipid metabolism and inducing vascular inflammation.
  • the plaque area in apoE' 7 ' and TNF’ 7 ' mice on a high fat diet is half the size of the plaque area in mice that are apoE' 7 '.
  • anti-TNF therapies such as infliximab and etanercept
  • infliximab and etanercept have thus not been tested for the treatment of cardiovascular disease (see, e.g., Udalova et al. (2016) Microbial Spectrum 4(4):MCHD-0022-2015; Kalliolias and Ivashkiv (2016) Nat. Rev. Rheumatol. 12(l):49-62).
  • alternative therapies are required.
  • ARDS Acute Respiratory Distress Syndrome
  • ARDS Acute respiratory distress syndrome
  • ARDS is characterized by immune cell-mediated lung injury, which is associated with the release of inflammatory cytokines and proteases.
  • the uncontrolled local inflammatory response in ARDS results in damage to the alveolar-capillary barrier, and non-cardiogenic pulmonary edema.
  • Pulmonary neutrophil recruitment which is central to the pathogenesis of ARDS, is mediated by the interaction of primed and activated neutrophils with the lung microvascular endothelium, and is increased by damage to the alveolar-capillary barrier caused by the action of proinflammatory mediators.
  • TNF- ⁇ has been identified as a key effector molecule in ARDS, as well as in sepsis, which is a common cause of ARDS. For example, TNF- ⁇ contributes to increased endothelial permeability.
  • Clinical trials involving the administration of non-selective anti-TNF antibodies for the treatment of sepsis have failed to demonstrate any survival benefit, and one trial indicated that higher doses were harmful.
  • TNFR1 -deficient mice are protected from lung injury, sepsis and other acute organ injuries, while TNFR2-deficient mice are more susceptible to injury in these models, indicating that selective antagonism of TNFR1 can be therapeutically effective.
  • GSK1995057 a short-acting, fully human domain antibody (dAb) fragment that selectively antagonizes TNFR1, but not TNFR2, attenuated disease severity in a murine model of acute respiratory distress syndrome, and attenuated inflammation and signs of lung injury in non-human primates.
  • SARS- CoV severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 shares several similarities with SARS-CoV, the strain of coronavirus responsible for the SARS pandemic of 2002.
  • SARS-CoV and SARS-CoV-2 use the spike (S)-proteins to engage their cellular receptor, ACE2 (angiotensin-converting enzyme 2), for invading cells.
  • ACE2 angiotensin-converting enzyme 2
  • the expression of the ACE2 receptor is upregulated by SARS-CoV-2 infection and by inflammatory cytokine stimulation.
  • S-proteins induce the TNF- ⁇ -converting enzyme (TACE)-dependent shedding of the ACE2 ectodomain, which is a process that is strictly coupled to TNF- ⁇ production.
  • TACE TNF- ⁇ -converting enzyme
  • ACE2 knockout mice are susceptible to severe respiratory failure following chemical challenge, and ACE2 has been shown to moderate ACE-induced intracellular inflammation. ACE2 downregulation is linked to the severe respiratory distress associated with SARS-CoV infection. Increased TNF- ⁇ production can thus facilitate viral infection and result in organ damage, such as lung injury.
  • Tregs are a type of immunosuppressive cell that display diverse clinical applications in transplantation, allergy, infectious disease, GVHD, autoimmunity, and cancer.
  • Tregs co-express CD4+ and the interleukin-2 receptor alpha chain CD25 hl and feature inducible levels of intracellular transcription factor forkhead box P3 (FOXP3).
  • FoxP3 intracellular transcription factor forkhead box P3
  • Naturally-occurring Tregs express TNFR2 at a higher density than TNFR1.
  • Treg activity TNF-mediated TNFR2 activates and induces proliferation of Tregs (100) and TNFR2 expression indicates maximally suppressive Tregs
  • Treg can prevent overreaction to inflammatory stimuli.
  • Anti-TNF can be a treatment for SARS and COVID- 19.
  • Adalimumab is being used for treatment of COVID-19 (clinical trial China in February, 2020 (ChiCTR2000030089); see, e.g., Lucchino et al. (2020) Rheumatology (Oxford) 59(6) : 1200- 1203; Haga et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 705:7809-7814).
  • TNFR2 Knockout of TNFR2 in mice infected with SARS-CoV does not provide any protective effects; the double knockout of TNFR1 and TNFR2 protected infected mice from weight loss associated with infection (see, e.g., McDermott et al. (2016) BMC Systems Biology 10:93). These results indicate that TNF signaling through TNFR1 primarily contributes to the pathogenesis of SARS-CoV infection, by increasing proinflammatory processes, and that selective inhibition of TNFR1, rather than inhibition of TNF, is a better therapeutic approach.
  • the constructs provided herein can be used to treat the acute inflammatory aspects of SARS and COVID-19. The constructs are used in combination with anti-infective agents; the constructs are used to suppress or ameliorate the acute effects of cytokine storm.
  • TNF- ⁇ inhibition reduces the severity of virally-induced lung diseases, such as those caused by respiratory syncytial virus (RSV) or influenza virus, in mice.
  • RSV respiratory syncytial virus
  • the depletion of TNF using anti-TNF antibody in these mouse models reduced the pulmonary recruitment of inflammatory cells, reduced the production of proinflammatory cytokines (e.g., IFN- ⁇ , IL-4, IL-5, TNF) by T-cells, and reduced the severity of illness without interfering with viral clearance (see, e.g., Hussell et al. (2001) Eur. J. Immunol. 31 :2566-2573).
  • TNF inhibitors and TNF receptor antagonists can be beneficial in the treatment of human viral lung diseases, such as those caused by SARS-CoV and SARS-CoV2, by preventing or reducing TNF-induced immune activation and pulmonary injury.
  • Allogeneic hematopoietic stem cell transplantation is complicated by the development of non-infectious idiopathic pneumonia syndrome (IPS), an acute pulmonary dysfunction that resembles SARS pneumonia. Elevated levels of TNF- ⁇ have been found in the sera of patients who developed lung injury after allogeneic stem cell transplantation (SCT), and it has been shown that donor-derived alloreactive T-cells are associated with this process. In humans, anti-TNF therapy with etanercept is beneficial in the treatment of IPS after allogeneic stem cell transplantation.
  • SCT allogeneic stem cell transplantation
  • Recipients of allogeneic stem cell transplants are at high risk of developing bacterial and fungal infections, due to the immunoablative effects of SCT conditioning regimens, the requirement for long term use of immunosuppressive drugs to prevent or treat graft-vs-host disease (GvHD), and other SCT complications (including acute GvHD) that can impair host defenses (see, e.g., Yanik et al. (2002) Biol. Blood Marrow Transplant. 8:395-400).
  • Other indications that can be treated by constructs provided herein include chemo brain, a condition experienced during and following chemotherapy, particularly women treated for breast cancer. Also, the treatments and constructs herein can be used to treat long COVID.
  • the use of selective TNFR1 antagonists which preserves protective TNF signaling via TNFR2, and, unlike anti-TNF therapies, does not increase the risk of serious infections, provides a safer and more effective therapeutic option for the treatment, prevention or amelioration of virally- and non-virally- induced lung injury.
  • RA rheumatoid arthritis
  • treatments can improve symptoms and slow disease progression, for example, by minimizing pain and swelling, preventing bone deformity, and maintaining day-to-day functioning.
  • the primary treatments for RA are disease-modifying anti-rheumatic drugs (DMARDs), which also are used for the treatment of other chronic inflammatory and autoimmune diseases and disorders, such as, for example, psoriasis, plaque psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, Behçet's disease, inflammatory bowel disease (IBD; e.g., Crohn’s disease and ulcerative colitis), multiple sclerosis, and lupus, as well as for the treatment of some cancers.
  • IBD inflammatory bowel disease
  • DMARDs are immunosuppressive and immunomodulatory agents that are classified as either conventional synthetic DMARDs (csDMARDs), or biological DMARDs (bDMARDs; e.g., antibodies and fusion proteins).
  • Conventional synthetic DMARDs include, for example, methotrexate (MTX), a chemotherapy agent and immunosuppressant; hydroxychloroquine (HCQ; Plaquenil®), an anti-malarial agent; sulfasalazine (Azulfidine®), an anti-inflammatory drug; and leflunomide (Arava®), an immunosuppressant that inhibits dihydroorotate dehydrogenase.
  • Biologic DMARDs include, for example, abatacept (Orencia®), a fusion protein that prevents T cell activation and contains the Fc region of IgGl fused to the extracellular domain of CTLA-4; anakinra (sold, for example, under the trademark Kineret®), a recombinant human IL-1 receptor antagonist; rituximab (sold under trademarks, including) Rituxan®, Truxima®, Mab Thera®), a chimeric monoclonal antibody against CD20, which induces apoptosis in CD20 + cells, such as B cells; tocilizumab (atlizumab, Actemra®, RoActemra®), a humanized monoclonal antibody against the IL-6 receptor (IL-6R); corticosteroids; tofacitinib (Xeljanz®), a small molecule inhibitor of Janus kinase (JAK), a protein tyrosine
  • Combination therapy particularly of methotrexate with a biological DMARD, is more effective than either therapy alone.
  • Combination therapies also can include multiple csDMARDs and multiple csDMARDs with one biological DMARD. Due to the risk of serious side effects, including serious infections, multiple biological DMARDs, particularly anti-TNF DMARDs, typically are not used for combination therapy methods.
  • csDMARDs Conventional synthetic Disease Modifying Anti-Rheumatic Drugs
  • csDMARDS include drugs such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine
  • Methotrexate is the most commonly used agent for initial treatment, and its mechanism of action involves stimulating the release of adenosine from fibroblasts, reducing neutrophil adhesion, inhibiting leukotriene B4 synthesis by neutrophils, inhibiting local IL-1 production, reducing levels of IL-6 and IL-8, suppressing cell-mediated immunity, and inhibiting synovial collagenase gene expression.
  • DMARDs act by inhibiting the proliferation of lymphocytes or causing lymphocyte dysfunction.
  • leflunomide inhibits dihydroorotate dehydrogenase, resulting in inhibition of pyrimidine synthesis, and blocking lymphocyte proliferation.
  • Sulfasalazine mediates its anti-inflammatory effects by preventing oxidative, nitrative and nitrosative damage, and hydroxychloroquine is a mild immunomodulatory agent that inhibits intracellular toll-like receptor 9 (TLR9).
  • TLR9 toll-like receptor 9
  • Hydroxychloroquine which has the best safety profile of conventional DMARDs, does not increase the risk of infections, and does not cause hepatotoxicity or renal dysfunction; common side effects of hydroxychloroquine include rash and diarrhea.
  • Retinopathy/maculopathy is a rare but serious side effect of hydroxychloroquine therapy which is associated with doses of more than 5 mg/kg/day, long-term use (more than 5 years of therapy), older age and chronic kidney disease.
  • Other rare adverse effects of hydroxychloroquine include anemia, leukopenia, myopathy, and cardiomyopathy.
  • methotrexate is associated with nausea, abdominal pain, diarrhea, rash/allergic reaction, bone marrow suppression, hepatotoxicity and higher incidence of common and sometimes serious infections.
  • Methotrexate and leflunomide also cause alopecia.
  • Other side effects associated with methotrexate therapy include interstitial lung disease, folic acid deficiency, and liver cirrhosis.
  • Leflunomide also is associated with hypertension, peripheral neuropathy, and weight loss.
  • Sulfasalazine has a very high risk of gastrointestinal distress and can rarely cause DRESS syndrome (drug reaction with eosinophilia and systemic symptoms) (see, e.g., Benjamin et al. Disease Modifying Anti -Rheumatic Drugs (DMARD) [Updated 2020 Feb 27], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2020 Jan. Available from: URL:ncbi.nlm.nih.gov/books/NBK507863/). These drugs are effective because they are immunosuppressive.
  • the constructs provided herein that are selective anti-TNFRl antagonists that preserve TNFR2 immunosuppressive activity advantageously can avoid the need for these immunosuppressive drugs.
  • Anti-TNF therapies/TNF -blockers typically are prescribed after the failure of conventional DMARDs, and include monoclonal antibodies (mAbs), such as the chimeric mAb infliximab (Remicade®); containing a murine variable region and a human IgGl constant region), and the fully humanized mAbs (IgGls) adalimumab (Humira®) and golimumab (Simponi®); the PEGylated humanized Fab’ fragment of a mAb targeting TNF, certolizumab pegol (Cimzia®); and TNFR2 fusion proteins, such as the TNFR2-Fc fusion protein etanercept (Enbrel®), which contains the extracellular receptor region that contains the binding site of human TNFR2 fused to the Fc of human IgGl.
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • Remsima® and Inflectra® are biosimilars of infliximab that are approved for use in the European Union for the treatment of various autoimmune and chronic inflammatory diseases and disorders.
  • TNF inhibitors which sequester TNF, are used for the treatment of various diseases and conditions, including, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA) and/or inflammatory bowel disease (IBD; e.g., Crohn’s disease and ulcerative colitis).
  • TNF is a key cytokine in the inflammatory and immune responses to infections, and the use of drugs that remove TNF impairs host immunity against microorganisms, increasing the risk of infection.
  • TNF blocking agents are associated with the reactivation of M. tuberculosis infection.
  • TNF plays an important role in the resistance against Mycobacterium tuberculosis, and adalimumab therapy in RA patients significantly reduces reactivity against M. tuberculosis.
  • the reduced immune reactivity can be related to the activation of Tregs and the induction of apoptosis in effector lymphocytes.
  • Anti-TNF therapy has been shown to induce macrophage apoptosis in the rheumatoid synovium.
  • Infliximab is associated with increased apoptosis in the inflammatory cell infiltrate in the guts of patients with Crohn’s disease.
  • anti -rheumatic drugs such as methotrexate and glucocorticoids
  • Other anti -rheumatic drugs also can induce apoptosis in immune cells (see, e.g., Vigna-Perez et al. (2005) Clin. Exp. Immunol. 141 (2):372-380).
  • Adalimumab and infliximab, but not etanercept, a TNFR2-Fc fusion protein induce caspase-dependent apoptosis in cultured monocytes, and downregulate the production of IL- 10 and IL- 12 by monocytes (see, e.g., Shen et al. (2005) Ailment Pharmacol. Ther. 21 :251-258).
  • TNF blockers The most prevalent fungal infections associated with TNF blockers are histoplasmosis, candidiasis, and aspergillosis.
  • Anti-TNF agents also can cause worsening of severe congestive heart failure, drug-induced lupus, and demyelinating central nervous system (CNS) diseases, as well as lymphomas and non-melanoma skin cancers (see, e.g., Benjamin et al. Disease Modifying Anti -Rheumatic Drugs (DMARD) [Updated 2020 Feb 27], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2020 Jan. Available from: ncbi.nlm.nih.gov/books/NBK507863/).
  • DMARD Anti -Rheumatic Drugs
  • Infliximab also has been associated with the development of leukopenia, neutropenia, thrombocytopenia, and pancytopenia (some fatal).
  • Etanercept has been associated with an increased incidence of opportunistic bacterial and viral infections in patients with RA.
  • Etanercept also is used to treat severe refractory graft-versus-host disease (GvHD). Subjects with severe GvHD who are treated with etanercept have a very high risk (100% in one study, see, Zoran et al. (2019) Sci. Rep. 9: 17231) of developing invasive aspergillosis (IA), a life-threatening mold (re., fungal) infection caused by Aspergillus fumigatus.
  • IA invasive aspergillosis
  • IA life-threatening mold
  • Treatment with etanercept results in the downregulation of genes involved in immune responses and TNF signaling, including genes involved in NF-xB signaling, anti-microbial humoral responses and apoptotic processes, as well as a decrease in the secretion of chemokines, such as CXCL10, from immune cells (see, e.g., Zoran et al. (2019) Sci. Rep. 9:17231).
  • chemokines such as CXCL10
  • TNF blocking therapies include congestive heart failure, liver injury, demyelinating disease/CNS disorders, lupus, psoriasis, sarcoidosis, and an increased susceptibility to the development of additional autoimmune diseases, as well as cancers, including lymphomas and solid malignancies (see, e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43): 12304- 12309; Zalevsky et al. (2007) J. Immunol. 179: 1872-1883; Zoran et al. (2019) Sci. Rep. 9: 17231).
  • the abrogation of all TNF-mediated signaling, by sequestering TNF is not an ideal therapeutic strategy, as it results in severe immunosuppression that can lead to serious, sometimes fatal, infections, and other dangerous side effects.
  • Anti-TNF therapies ameliorate RA but are not curative, and require years of continuous and costly therapy.
  • the inhibition/blockade of TNF in RA reduces inflammation and joint destruction, but, as discussed above, is associated with an increased risk of serious infections, such as tuberculosis and listeriosis, due to immunosuppression.
  • the use of TNF blockers particularly in the case of chronic diseases/conditions that require long-term administration, such as arthritis and IBD, is limited.
  • Approximately 30% of RA patients are non-responsive, or therapeutic benefits are not sustained, with the use of anti-TNF therapies (see, e.g., McCann et aL (2014) Arthritis & Rheumatology 66(10):2728-2738).
  • Non-responsiveness also occurs in non-RA patients receiving anti-TNF therapeutics.
  • 13-33% of treated patients do not respond to treatment, and up to 46% stop responding, resulting in discontinuation or dose increase (see, e.g., Richter et aL (2019) MABS 11(4):653-665).
  • Anti-TNF therapeutics block/sequester TNF and inhibit soluble TNF (solTNF) and transmembrane TNF (tmTNF) signaling via TNFR1 and TNFR2, respectively; solTNF signaling has been associated with chronic inflammation, while tmTNF signaling has been associated with the resolution of inflammation and with the induction of immunity against pathogens such as Listeria monocytogenes and Mycobacterium tuberculosis.
  • the primary anti-inflammatory effects of anti-TNF therapies are achieved by blocking TNFR1, while blocking TNFR2 inhibits Treg cell activity.
  • TNFR1 signaling is primarily inflammatory and is involved in the pathogenesis of inflammatory and autoimmune diseases and conditions, such as RA, psoriasis, IBD, and neurodegenerative disorders, such as MS; whereas, TNFR2 signaling has anti-inflammatory and protective effects in various cell and organ types, including neural, cardiac, gut and bone tissues, and also is involved in host defense mechanisms against infection by pathogens.
  • selective blockade of TNFR1 improves the therapeutic efficacy in comparison to anti-TNF therapies, by eliminating undesirable, proinflammatory signaling associated with RA and other autoimmune and inflammatory diseases and conditions, while preserving the beneficial effects of TNFR2 signaling (see, e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Blüml et al. (2012) International Immunology 24(5) :275-281; Zalevsky et al. (2007) J. Immunol. 179: 1872-1883).
  • Anti-TNF therapies have failed in the treatment of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, stroke and multiple sclerosis (MS), which have been associated with the overexpression of TNF.
  • a TNFR1 receptor-Fc IgGl fusion protein anti-TNF therapeutic, lenercept Ros 45-2081
  • failed and symptoms were increased/worsened compared to patients receiving a placebo, with neurologic deficits being more severe in lenercept-treated patients.
  • TNFR1 has been shown to mediate inflammatory neurodegeneration
  • TNFR2 induces neuroprotection, thus, blockade of signaling through both receptors by anti-TNF therapies abrogates the neuroprotective effects of TNFR2 signaling.
  • ATROSAB a humanized monoclonal antibody that blocks TNFR1
  • EHD2-SCTNF R2 an agonistic TNFR2-selective TNF mutein
  • ATROSAB is a partial TNFR1 agonist; those of skill in the art would not administer a TNFR1 agonist.
  • the blockade of TNFR1 and TNFR2 abrogates the therapeutic effect, indicating that TNFR2 plays an essential role in neuroprotection, and that selective blockade of TNFR1 can be used for the treatment of neurodegenerative diseases where anti-TNF therapies have failed (see, e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43): 12304-12309).
  • TNF blockers such as etanercept, infliximab, adalimumab, and others
  • TNF blockers such as etanercept, infliximab, adalimumab, and others
  • TNFR1 signaling results in inflammation, cytotoxicity and apoptosis
  • TNFR2 signaling is protective and anti-inflammatory, partly due to its expansion and activation of immunosuppressive Tregs, which destroy effector T cells in the autoimmune environment, preventing tissue destruction and disease progression.
  • TNF blockers through its inhibition of TNFR2 signaling, and the consequential depletion of immunosuppressive Tregs, which results in a pro- inflammatory microenvironment, can fail in the treatment of, and/or can exacerbate, autoimmune and inflammatory diseases and disorders.
  • the dual blockade of TNFR1 and TNFR2 also can lead to opportunistic infections and cancer.
  • TNFR1 antagonistic Antibodies As provided herein, the specific inhibition of TNFR1 signaling maintains normal TNFR2 function, which is necessary for maintaining the equilibrium between pro-inflammatory and anti-inflammatory activity, via the production of subsets of both regulatory and cytotoxic T cells. Selective TNFR1 inhibition retains the potent anti-inflammatory activity of TNFR2 signaling, results in fewer opportunistic infections and cancer, and preserves TNF-induced Treg functions.
  • ATROSAB Antagonistic TNF Receptor One-Specific Antibody
  • ATROSAB the first TNFR1 blocking antibody, is a full-length IgGl that is a humanized version of the neutralizing mouse anti -human TNFR1 monoclonal antibody H398. It was abandoned as a therapeutic because it has partial agonist activity, which activates TNFR1, thereby mimicking TNF activity, a toxic pathway.
  • ATROSAB maintains the conformation of TNFR1 in an inactive state and obstructs the binding of TNF.
  • the Fc region in ATROSAB is mutated to eliminate FC ⁇ R receptor binding and complement fixation, thereby avoiding unwanted immune system activation (see, e.g., Kalliolias and Ivashkiv (2016) Nat. Rev. Rheumatol. 12(l):49-62).
  • Full-length antibodies have the advantage of increased in vivo half-life, but, as discussed elsewhere herein, are not feasible for the development of TNFR1 antagonists due to receptor cross-linking, which tends to agonize TNFR1 instead of antagonizing it. This did not result from Fc cross-linking because the Fc-interacting part of the antibody was removed by mutation.
  • the IgG ATROSAB exhibited some TNFR1 agonistic activity in the absence of TNF, which was observed to a limited extent at a narrow concentration range, due to its bivalent molecular structure.
  • TNFR1 cross-linking of TNFR1 also can occur due to secondary events, such as interactions with FC ⁇ Rs or anti-drug antibodies (AD As), which must be avoided to maintain the antagonistic nature of TNFR1 inhibitors.
  • AD anti-drug antibodies
  • Small antibody fragments such as domain antibodies and derivatives and modified forms thereof have been developed, and exemplary antibody fragments and modified forms are discussed in the following sections.
  • the small antibody fragments have not been successfully developed into pharmaceuticals. They are limited in their use as therapeutics; they have short serum half-lives and fast peripheral clearance, which are a result of their small size.
  • molecules that are 50-60 kDa in size or smaller are subject to renal filtration; dAbs and other antibody fragments, which are less than 50-60 kDa in size, are rapidly cleared by the kidneys.
  • the dAb designated DMS5541, and similar molecules demonstrate selectivity for TNFR1, and potentially can inhibit the deleterious effects of TNFR1 signaling.
  • DMS5541 which is formed from two dAbs (antiTNFRl and anti-human serum albumin), is only approximately 25 kDa in size, and is too small to have desirable pharmacokinetics for therapeutic purposes. Its association with HSA, which is meant to stabilize its half-life, is only 34nM, which means it is often in a dissociated state with respect to HSA.
  • Single-domain antibodies (sdAbs) that have been tested so far are expressed in E.coli, and are prone to aggregation (unfolding) during manufacturing. Additionally, sdAbs prepared cytoplasmically (from direct expression in E. coli ) often lack the conserved disulfide bond found in variable heavy domains, which both decreases their melting point and can decrease their ability to refold.
  • constructs such as the TNFR1 antagonist constructs, provided herein address this problem as well as other problems, such as the immunogenicity, and reactions with pre-existing antibodies.
  • Therapeutic antibodies of a variety of structures can be potent and well- tolerated therapeutics.
  • Antibodies are used for the treatment of a variety of diseases and conditions, including, for example, rheumatoid arthritis (e.g., adalimumab, sold under the trademark Humira®); cancers, such as non-Hodgkin’s lymphoma (e.g., rituximab and ibritumomab tiuxetan, sold under the trademarks Rituxan® and Zevalin®, respectively) and breast and gastric cancers (e.g., trastuzumab, sold under the trademark Herceptin®); and respiratory syncytial virus infection (e.g., palivizumab, sold under the trademark Synagis®).
  • Antigen-binding fragments of antibodies such as Fabs ( ⁇ 57 kDa) and single chain Fv fragments (scFvs, ⁇ 27 kDa), and other structures, which can be selected in vitro, such as with phage display (circumventing animal immunization), and which can be manufactured in large quantities using bacterial or yeast cell cultures, have been developed.
  • a Fab fragment contains a V H -C H 1 polypeptide, linked to a V L -CL polypeptide via a disulfide bond; an scFv is a fusion protein containing a V H domain and V L domain linked by a short polypeptide linker.
  • Another class of therapeutic, small fragments of antibodies are domain antibodies (dAbs; also known as single domain antibodies, or sdAbs), which are monomeric and contain a variable domain of the heavy chain (V H ) or of the light chain (V L ) of an antibody.
  • dAbs are the smallest antigen-binding fragments of antibodies; they are approximately 11-15 kDa in size, which is about one-tenth the size of a full monoclonal antibody (mAb) (see, e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490). Similar to dAbs, nanobodies (Nbs) are small antigen-binding fragments derived from camelid heavy- chain antibodies that are devoid of light chains.
  • Nanobodies are small ( ⁇ 15 kDa), have low immunogenicity and high affinity, are soluble and stable, and are encoded by a single gene/exon (VHH), so that they are modular, which allows for high yield production in bacteria and yeasts (see, e.g., Steeland et al. (2015) J. Biol. Chem. 290(7):4022-4037; Steeland et al. (2017) Sci. Reports 7:13646).
  • VHH single gene/exon
  • ATROSAB humanized semi-agonistic/antagonistic TNFR1- specific antibody
  • ATROSAB inhibits TNFR1 -mediated cellular responses.
  • ATROSAB exhibits some TNFR1 agonistic activity, likely due to its bivalent molecular structure or by virtue of its binding to TNFR1, in the absence of INF.
  • the parental mouse antibody, H398, possesses stronger inhibitory potential, which is due to the faster dissociation of ATROSAB (z. e. , a higher k O ff value) compared to H398.
  • the single- chain variable fragment (scFv) of ATROSAB was subjected to a first affinity maturation by site-directed mutagenesis of exposed residues within individual CDRs, or combinations of CDRs, and selection by phage display against human TNFRl-Fc.
  • the scFv of ATROSAB contains the V H domain, corresponding to residues 1-115 of the ATROSAB heavy chain (see, SEQ ID NO:31), linked by a short peptide linker to the V L domain, corresponding to residues 1-113 of the ATROSAB light chain (see, SEQ ID NO:32).
  • a clone, scFv IG11 (see, SEQ ID NO:674), with 6 mutations within CDR-H2 of the ATROSAB heavy chain, Y52V, Y54T, S55Q, H57E, Y59K, and E62D, with reference to SEQ ID NO:31, exhibited slower receptor dissociation and improved equilibrium binding to human TNFRl-Fc, and improved inhibition of TNF- induced TNFR1 activation.
  • scFv T12B This clone was further subjected to random mutagenesis, generating the clone scFv T12B (see, SEQ ID NO:675), containing the mutations Q1H, Y52V, Y54S, S55Q, H57E, Y59K, and E62D in the V H domain (with reference to SEQ ID NO:31), and S96G in the V L domain (with reference to SEQ ID NO:32).
  • scFv T12B had reduced dissociation from immobilized TNFRl-Fc compared to the scFv of ATROSAB and to scFv IG11, and increased TNFR1 inhibitory activity (see, e.g., Richter et al.
  • H398 The humanization of H398 was re-engineered by an exchange of VH and VL framework regions of H398 with alternative germline genes to optimize CDR arrangement.
  • scFv 13.7 containing the VH domain of scFV T12B, linked by a short peptide linker to a newly humanized VL domain of H398, had similar binding to human TNFRl-Fc in ELISA and QCM, improved inhibition of TNF-induced TNFR1 activity, and improved thermal stability, with a 10 degree Celsius higher melting temperature compared to scFv T12B.
  • IgG 13.7 and Fab 13.7 were generated (IgG 13.7 and Fab 13.7, respectively), which had increased binding to TNFR1 compared to ATROSAB (1.4- fold) and the Fab of ATROSAB (Fab ATR; 8.7-fold), respectively.
  • Fab 13.7 also had reduced dissociation from immobilized TNFRl-Fc, compared to Fab ATR, with an 18.8-fold improved monovalent affinity.
  • affinity maturation and framework replacement resulted in improved binding to TNFR1 for Fab 13.7.
  • Fab 13.7 and IgG 13.7 displayed selectivity towards TNFRl-Fc and did not bind to a TNFR2-Fc fusion protein; Fab 13.7 bound to human and rhesus TNFRl-Fc, but not to mouse and rat TNFRl-Fc, showing a similar binding patern to ATROSAB.
  • monovalent Fab ATR and Fab 13.7 did not activate TNFR1, while ATROSAB displayed marginal activation of TNFR1 activity and IgG 13.7 strongly activated TNFR1.
  • the agonistic activity of IgG 13.7 can be due to the improved affinity and slower dissociation from TNFR1, resulting in the formation of stable signaling competent receptor-antibody complexes.
  • Fab 13.7 displayed improved inhibition of TNFR1 activity compared to Fab ATR and to ATROSAB, and lacked any agonistic activity. Incubation of Fab 13.7 or ATROSAB with cross-linking anti-human Fab serum, revealed that Fab 13.7 does not activate TNFR-1, while ATROSAB does (see, e g., Richter et al. (2019) mAbs 11(1):166-177).
  • Fab 13.7 (with a molecular mass of ⁇ 47 kDa). Fab 13.7 displayed an initial half-life of 0.08 h, a terminal half-life of 1.4 h, and an AUC of 4.2 pg/ml x h, which were similar to the values obtained for Fab ATR.
  • Fab fragment
  • Fab 13.7 was generated by introducing a free cysteine residue at the C-terminus of the CHI domain, which was chemically coupled to a branched PEG40kDa moiety, generating Fab 13.7PEG.
  • Fab 13.7 also was fused through its Fd and a short flexible linker to the N-terminus of mouse serum albumin (MSA), generating Fabl3.7-MSA.
  • MSA mouse serum albumin
  • a monovalent Fab-Fc fusion protein was generated by fusing Fab 13.7 to a modified Fc, lacking the cysteine residues in the hinge region and the ability to dimerize via the CH3 domain, generating a one-armed half-IgG molecule (IgG 1 half 13.7).
  • a monovalent Fv-Fc molecule also was generated by fusing the VH and VL domains to a hetero-dimerizing knob-into-hole (kih) Fc chain lacking the cysteine residues in the hinge region (Fvl3.7-Fckih). None of the derivatives showed any agonistic TNFR1 activity, and, compared to Fab 13.7, a slightly reduced binding to human TNFRl-Fc was observed for Fabl3.7PEG, Fabl3.7-MSA and IgG half 13.7 ; binding of Fvl3.7- Fckih was not affected.
  • Inhibition of TNF -mediated TNFR1 activity was reduced by 1.5-3.3 fold compared to Fab 13.7; Fabl3.7PEG showed the strongest impairment in function, and Fvl3.7-Fckih showed the lowest change in bioactivity.
  • IgG half 13.7 showed a similar half-life to Fab 13.7, and an AUC value that was increased by 7.1- fold.
  • Fabl3.7PEG, Fabl3.7-MSA and Fvl3.7-Fckih had extended terminal half-lives, with values of 14.4 h, 9.7 h, and 10.5 h, respectively, and increased AUC values.
  • the fusion protein Fvl3.7-Fckih which was engineered for heterodimeric assembly of two peptide chains by using knobs-into-holes technology, displayed the best combination of improved pharmacokinetic properties and TNFR1 antagonistic activity (see, e.g., Richter et al. (2019) mAbs 11(1): 166-177; see, also, Richter, F. Thesis, entitled “Evolution of the Antagonistic Tumor Necrosis Factor Receptor One- Specific Antibody ATROSAB,” Universitat Stuttgart, 2015; available from pdfs.semanticscholar.org/d8e7/8b87d76dce36225cld497939ef37445cfa8a.pdf).
  • the Fc heterodimerization approach is based on interspersed Ig domains, derived from the heterodimerizing IgGl constant heavy chain domain, CHI, and the kappa light chain constant domain, CLK, and containing sections of the IgGl CH3 sequence to mediate FcRn binding and enable FcRn-mediated drug recycling in vivo.
  • the interspersed Ig domains include “CH31,” which contains amino acid sequence fragments of CHI and CH3, and “CH3kappa” (CH3K), which contains amino acid sequence fragments of CLK and CH3.
  • IgGl CH2 domains also were fused to the N- termini of the CH31 and CH3K domains, to include the entire FcRn binding region of the IgG molecule.
  • FCIK covalently linked heterodimerizing Fc moiety
  • Asymmetric SCFV-FCIK fusion proteins were prepared and compared to scFv fusions with Fes containing knobs-into-holes, and heterodimer formation was similar or improved, compared to the fusions containing knobs-into-holes technology (see, e.g., Richter et al. (2019) mAbs 11(4):653-665).
  • the vanable domains of the TNFR1 -specific Fab 13.7 molecule were fused to the CH2 domain N-termini of the CH31- or CH3K-containing Fc chains with a short peptide linker, by fusing the VH to the CH2-CH3K chain and the VL to the CH2- CH31 chain (VL13.7-CH2-CH31/VH13.7-CH2-CH3K; l/LIC/VHKC), generating the monovalent TNFR1 -specific antagonistic antibody-derived molecule (FV-FC1K fusion protein), known as Atrosimab (72 kDa in size).
  • Atrosimab lacks the ability to mediate Fc effector functions, due to mutations that were introduced into FCIK; the lack of binding to effector molecules of the immune system prevents the activation of TNFR1 due to secondary crosslinking of Atrosimab bound to cells expressing Fc ⁇ Rs.
  • TNFR1 binding and inhibition was slightly reduced, which can be attributed to alterations in the VH and VL pairing after fusion to the CH2 domain.
  • the initial and terminal half-lives of Atrosimab were determined to be 2.2 +/- 1.2 h and 41.7 +/- 18.1 h, respectively, and the AUC was 5856 +/- 1369.9 pg/ml x h.
  • dAbs domain antibodies
  • sdAbs single domain antibodies
  • V H variable domain of the heavy chain
  • V L variable domain of the light chain
  • dAbs are the smallest antigen-binding fragments of antibodies; they are approximately 11-15 kDa in size, which is about one-tenth the size of a full monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • each dAb contains three out of the six CDRs from a V H -V L pair in an antibody, which are the highly diversified loop regions that bind to the target antigen.
  • dAbs Due to their smaller size, dAbs are produced at higher yields from bacterial cultures, and are more amenable to phage display, since only a single polypeptide chain is produced. Specific dAbs with high affinities and potencies rapidly can be produced by protein engineering.
  • the small size of dAbs also allows for increased tissue penetration, stability, and choice of delivery formulations. Due to their small size, it is possible to create molecules containing linked dAbs that are specific for different antigens/targets, which is not possible with conventional antibodies, and is difficult to achieve for other antibody fragments, such as Fabs and scFvs. Due to the monomeric and monovalent binding modality of dAbs, they suitable for use where the targets are not amenable to intervention with monoclonal antibodies.
  • TNFR1 is one such target; TNFR1 is activated/agonized by antibody-induced receptor cross-linking (see, e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065).
  • Small size antibody fragments such as dAbs, scFvs, Fvs, disulfide-bonded Fvs and Fabs, are easier to produce and handle, and are distributed rapidly throughout the body, in comparison to larger molecules; however, their short in vivo half-life limits their therapeutic efficacy.
  • increasing the serum half-life of dAbs increases the therapeutic efficacy and decreases the frequency of dosing, particularly in applications that require binding antigens in the bloodstream, such as in the treatment of rheumatoid arthritis or cancer. This can be achieved by PEGylation, conjugation to serum albumin, fusion with a second dAb with specific binding to serum albumin, or fusion to an Fc fragment or complete antibody constant regions.
  • Fusion with an Fc region also allows for the recruitment of Fc effector functions, including complement activation, antibody-dependent cellular cytotoxicity, or Fc-mediated clearance of immune complexes (see, e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490; Goodall et al. (2015) PLoS ONE 10(9):e0137065).
  • Fc effector functions including complement activation, antibody-dependent cellular cytotoxicity, or Fc-mediated clearance of immune complexes (see, e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490; Goodall et al. (2015) PLoS ONE 10(9):e0137065).
  • DMS5540 is a 25 kDa mouse TNFR1 antagonist, that is a bispecific single variable domain antibody, containing a noncompetitive (does not interfere with TNF binding) anti-TNFRl dAb, fused with an albumin-binding dAb (AlbudAb; to extend serum half-life). DMS5540, which does not bind human TNFR1, was found to inhibit
  • TNF- ⁇ -mediated cytotoxicity in the mouse fibroblast cell line L929 (which is highly sensitive to TNF- ⁇ -mediated cytotoxicity).
  • DMS5540 was administered to mice intravenously, followed four hours later with an intravenous bolus injection of TNF ⁇ , and serum IL-6 levels were assessed.
  • DMS5540 demonstrated a dose-dependent inhibition of TNF- ⁇ -mediated signaling effects in vivo, as determined by a decreased IL-6 response, when compared to mice administered a control dAb lacking specific antigen binding but fused to AlbudAb (DMS5538), or no dAb (see, e.g., Goodall et al. (2015) PLoS ONE 10(9):e0137065).
  • mice with collagen-induced arthritis were treated, beginning on the day of arthritis onset, for 10 days with DMS5540, an isotype (negative) control dAb (DMS5538), or murine TNFR2 genetically fused with mouse IgGl Fc domain (mTNFRII-Fc; mTNFR2.Fc), which blocks both receptors (TNFR1 and TNFR2) and inhibits mouse TNF, and disease progression was monitored.
  • DMS5538 an isotype (negative) control dAb
  • mTNFRII-Fc murine TNFR2 genetically fused with mouse IgGl Fc domain
  • TNFR1 and TNFR2 mouse IgGl Fc domain
  • the concentrations of systemic cytokines were measured, the numbers of T cell subsets in lymph nodes and spleens were assessed, and intrinsic Treg cell function was evaluated.
  • TNFR1 disease progression was suppressed similarly by blockade of TNFR1 with DMS5540 and blockade of TNFR1/2 with mTNFRII-Fc, compared to the negative control, indicating that blockade of TNFR1 or TNF protects joints from inflammatory mediators that result in joint damage in arthritis.
  • Effector T cell activity measured in terms of the expression levels of proinflammatory cytokines (e.g., IFN ⁇ , IL- 10 and RANTES), was increased following blockade of TNFR1/2 with mTNFRII-Fc, but not following the selective blockade of TNFR1 with DMS5540, indicating an immunoregulatory role (e.g., T cell effector function suppression) for TNFR2 signaling.
  • proinflammatory cytokines e.g., IFN ⁇ , IL- 10 and RANTES
  • TNFR1 blockade of TNFR1, but not of TNFR1/2, resulted in the expansion and activation of Treg cells, while an increase in the expression of FoxP3 and TNFR2, both of which are expressed by Tregs, was observed in joints undergoing remission, indicating their role in the resolution of inflammation.
  • signaling inhibits inflammation and promotes Treg cell suppressor activity, resulting in enhanced therapeutic efficacy compared to traditional methods of TNF inhibition (see, e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738).
  • DMS5540 also more effectively prevents inflammation-induced osteoclast formation and bone loss, than mTNFR2.Fc (anti-TNF), in an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis.
  • LPS lipopolysaccharide
  • TNFR1 selective inhibition of TNFR1 also can be used for therapeutic intervention in inflammatory bone loss disorders, such as osteomyelitis and periprosthetic osteolysis and aseptic loosening (see, e.g., Esperito Santo et al. Biochem. Biophys. Res. Commun. 464: 1145-1150).
  • DMS5541 also known as TNFRI-AlbudAb
  • TNFRI-AlbudAb which contains a noncompetitive human TNFR1 -specific dAb fused to AlbudAb
  • RA rheumatoid arthritis
  • MNCs synovial membrane mononuclear cells
  • DMS5541 inhibited the production of the proinfl ammatory cytokines GM-CSF, IL-10, IL-1 ⁇ and IL-6, and the chemokines IL-8, RANTES (CCL5) and MCP-1 (CCL2), at similar levels to TNF ligand blockade with etanercept. This inhibition was not due to cellular toxicity, as DMS5541 inhibited TNF- ⁇ -induced cytotoxicity in human rhabdomyosarcoma KYM- 1D4 cells in a dose-dependent manner, similar to TNF blockade with etanercept.
  • the domain antibody fragment designated GSK1995057 is a short-acting, fully human domain antibody (dAb) fragment (containing a V H chain) that selectively antagonizes TNF signaling through TNFR1, but not TNFR2. Due to its small size, GSK1995057 can be nebulized directly to the lungs, and has been investigated in the treatment of animal and human models of acute respiratory distress syndrome (ARDS) via inhalation. GSK1995057 reduces pulmonary inflammation in non-human primate (cynomolgus monkey) and human models of ARDS.
  • ARDS acute respiratory distress syndrome
  • Pulmonary neutrophil infiltration is central to the pathogenesis of ARDS, and is increased by damage to the alveolar-capillary barrier caused by the action of proinflammatory mediators.
  • TNF- ⁇ contributes to increased endothelial permeability, and GSK1995057 prevents this increase, indicating that TNFR1 signaling mediates TNF-induced endothelial permeability (see, e.g., Proudfoot et al. (2016) Thorax 73:723-730). Because of its inherent short half-life and neutralization by auto- antibody, the trial failed.
  • the immunogenicity of GSK1995057 may be due more to improper folding of the protein made in E coll than as a failure to properly humanize the dAb; it was derived from a human antibody fragment, and only the hypervariable sequences were altered to adapt specificity for TNFR1 (see, e.g., International PCT Publication No. WO2008/149148A2).
  • LPS lipopolysaccharide
  • pretreatment with GSK1995057 reduces pulmonary neutrophil infiltration, levels of proinflammatory chemokines, markers of endothelial injury and alveolar-capillary leak, in a dose-dependent manner.
  • the results indicate that inhaled GSK1995057 can effect the same results as higher doses of parenterally administered antibodies.
  • ADAs anti-drug antibodies
  • cytokine release infusion reactions at doses of 2-10 pig/kg, were observed due to high levels of pre-existing, naturally occurring anti-immunoglobulin autoantibodies, (i.e., ADAs) present in approximately 50% of drug naive, healthy subjects.
  • the ADAs were human anti-V H (HAVH) autoantibodies, and the complex of HAVH autoantibodies with framework sequences of GSK1995057 resulted in the activation of TNFR1 signaling, and the occurrence of mild to moderate infusion reactions in subjects with high HAVH autoantibody titers (see, e.g. , Cordy et al. (2015) Clin. Exp. Immunol. 182:139-148).
  • HAVH autoantibodies The binding of HAVH autoantibodies to a framework region of the dAb GSK1995057 induces cytokine release in vitro.
  • the epitope on GSK1995057 for the autoantibodies was characterized.
  • Pre-existing anti-drug antibodies (ADAs) bind to an epitope close to the C-terminal regional of V H dAbs, including the dAb GSK1995057.
  • a modified dAb designated GSK2862277 (see, SEQ ID NO:56) was generated by adding a single alanine residue at the C-terminus of the modified dAb. This modification reduced binding to HAVH autoantibodies.
  • nanobodies are small antigen-binding fragments derived from camelid heavy-chain antibodies that are devoid of light chains. They are small (15 kDa), have low immunogenicity and high affinity, are soluble and stable, and are encoded by a single gene/exon (VHH), making them modular and allowing for high yield production in bacteria or yeasts.
  • VHH single gene/exon
  • TROS TNF Receptor One-Silencer
  • Nb Alb-70-96 NNF Receptor One-Silencer
  • TROS is a trivalent high-affinity nanobody-based selective inhibitor of human TNFR1 that competes with TNF for binding to TNFR1.
  • two anti-human TNFR1 nanobodies Nb 70 and Nb 96; see, SEQ ID NOs: 683 and 684, respectively
  • SEQ ID NOs: 683 and 684 two anti-human TNFR1 nanobodies
  • TROS a model of MS
  • TROS also inhibits inflammation in ex vivo cultured colon biopsies of Crohn’s disease patients, and antagonizes inflammation in a model of acute TNF -induced liver inflammation in liver chimeric humanized mice (see, e.g., Steeland et al. (2015) ./. Biol. Chem. 290(7): 4022-4037; Steeland et al. (2017) Sci. Reports 7:13646).
  • TNF inhibitors are the signaling-incompetent dominant- negative inhibitors of TNF (DN-TNFs), also known as TNF muteins.
  • the DN-TNFs are engineered variants of TNF with mutations that abrogate binding to and signaling through TNFR1 and TNFR2.
  • DN-TNFs selectively inhibit soluble TNF (sTNF or solTNF) by rapidly exchanging subunits with native TNF homotrimers, forming inactive mixed TNF heterotrimers with disrupted receptor binding surfaces, thus preventing interaction with TNF receptors.
  • DN-TNFs leave transmembrane TNF (tmTNF) unaffected, maintaining the protective roles of TNF signaling through TNFR2.
  • DN-TNFs inhibit TNF -induced NF-excellentB activity and caspase-mediated apoptosis, and reduce disease severity in animal models of arthritis and Parkinson’s disease. These molecules because of their structure likely are immunogenic.
  • DN-TNFs unlike anti-TNF therapies that bind to solTNF and to tmTNF, do not inhibit tmTNF signaling, and do not suppress the resistance of mice to infection by L. monocytogenes .
  • Examples of DN- TNFs are TNF mutants containing one or more of the replacements L133Y, S162Q, Y163H, I173T, Y191Q and A221R, with reference to the sequence of amino acids set forth in SEQ ID NO: 1 (corresponding to residues L57Y, S86Q, Y87H, I97T, Y115Q, and A145R, with reference to the sequence of solTNF, as set forth in SEQ ID NO:2), which impair binding to TNFRs. Additional modifications, for example, to improve expression, allow site-specific PEGylation, also can be included (see, e.g., Zalevsky et al. (2007) J. Immunol. 179: 1872-1883).
  • the TNF mutations R32W and S86T result in a several hundred-fold loss in affinity towards TNFR2, but do not affect binding to TNFR1.
  • the R32W/S86T double mutant abrogates all binding to TNFR2, with no loss in binding to TNFR1.
  • the mutations D143N, D143Y, A145R and D143N/A145R, with reference to SEQ ID NO:2, render the TNF variants selective for TNFR2 (see, e.g., Loetscher et al. (1993) J. Biol. Chem. 268(35):26350-26357; U.S. Patent No. 5,422,104).
  • a modified TNF designated XProl595 (INmuneBio; see, SEQ ID NO:701), is a PEGylated, soluble DN-TNF mutein that preferentially inhibits TNFR1 signaling, and contains the mutations VIM, R31C, C69V, Y87H, C101A and A1456R, with reference to SEQ ID NO:2 (see, e.g., U.S. Publication No. 2015/0239951).
  • XProl595 decreases neuroinflammation and is being investigated in the treatment of Alzheimer’s disease (see, e.g, clinical trial identifier No. NCT03943264).
  • XProl595 blocks the development of amyloid pathology in a mouse model of Alzheimer’s Disease (3xTgAD), prevents the loss of neuron communication and cognitive impairment in a different (tgCRND8) mouse model of Alzheimer’s Disease, attenuates the dysfunction in neuronal communication and cognitive deficit in normal aged rats, and prevents young mice from developing amyloid pathology, cognitive impairment, and dysfunction in neuronal communication, in a third model (5xFAD) of Alzheimer’s disease. In older mice that have Alzheimer’ s-like pathology, XProl595 reduced amyloid, improved cognition, rescued neuron communication, and also, normalized innate and adaptive immune responses.
  • TNFR1 The levels of TNFR1 are higher in the hippocampus, in comparison to TNFR2, in aged (22 months) but not young adult (6 months) Fischer 344 rats.
  • aged rats When treated with XProl595, aged rats exhibit improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased levels of the GluRl type glutamate receptors, and lower L-type voltage sensitive Ca 2+ channel (L-VSCC) activity in hippocampal CAI neurons, indicating that functional changes associated with brain aging can occur from selective alterations in TNF signaling.
  • L-VSCC L-type voltage sensitive Ca 2+ channel
  • XProl595 ameliorates disease, improves remyelination and reduces CNS lesions and neuroinflammation. XProl595 also ameliorates inflammatory arthritis, and decreases susceptibility to infection in treated animals. In comparison to etanercept, which had no therapeutic effect, treatment with XProl595 delayed the onset of EAE and ameliorated symptoms more efficiently. XProl595 administration increases the level of TNR2 expression in the lesion area in EAE, indicating that tmTNF signaling via TNFR2 is implicated in neural regeneration (see, e.g., Yang et al. (2016) Front. Immunol. 9:784; Sama et al.
  • XProl595 does not inhibit the activity of transmembrane TNF (which activates TNFR1 and TNFR2), it cannot block the inflammatory effects of TNFR1. This also applies to other dominant negative TNF reagents, described below.
  • XENP345 is a PEGylated DN-TNF mutein, containing the mutations I97T/A145R, with reference to SEQ ID NO:2.
  • the in vivo neutralization of soluble TNF (solTNF) by XENP345 in animal models of Parkinson’s disease and Alzheimer’s disease is neuroprotective, reduces neuronal degeneration and cognitive dysfunction, and slows down neurodegenerative disease progression (see, e.g., McCoy et al. (2006) J. Neurosci. 26(37):9365-9375; McAlpine et al. (2009) Neurobiol. Dis. 34(1): 163-177).
  • RlantTNF (see, SEQ ID NO: 703) is a TNFR1 -selective antagonistic mutant TNF, identified from a phage library displaying structural human TNF variants in which each of the six amino acid residues at the receptor-binding site, corresponding to residues 84-89 of SEQ ID NO:2, were mutated.
  • RlantTNF which contains the mutations A84S, V85T, S86T, Y87H, Q88N and T89Q, has similar affinity to TNFR1 as wild-type human TNF, and does not interfere with TNFR2 activity.
  • RlantTNF ameliorated liver injury, as evidenced by reductions in the serum levels of alanine aminotransferase and the pro-inflammatory cytokines IL-2 and IL-6, in two models of acute hepatitis.
  • the plasma half-life of RlantTNF like wild-type TNF, however, is very short (12 min).
  • PEG-RlantTNF a PEGylated version, in which PEG is bound to the N-terminal site of RlantTNF, was produced.
  • PEG-RlantTNF decreases morbidity, ameliorates disease symptoms, improves demyelination in an EAE mouse model, and suppresses Thl and Th 17 cell activation and inflammatory T-cell infiltration in the spinal cord.
  • PEG-RlantTNF also inhibits NF-excellentB, suppresses smooth muscle cell proliferation, and decreases chemokine and adhesion molecule expression, thus decreasing intimal hyperplasia and arterial inflammation in IL-1 receptor antagonist-deficient mice after inducing femoral artery injury in an external vascular cuff model.
  • PEG-RlantTNF did not reactivate viral infection and did not affect the clearance of injected adenovirus, while viral load increased after treatment with etanercept.
  • PEG-RlantTNF treatment also delayed and ameliorated CIA symptoms in prophylactic and therapeutic settings, and was more effective than etanercept when used for the treatment of established CIA (see, e.g., Yang et al. (2016) Front. Immunol. 9:784; Shibata et a/. (2008) 7. Biol. Chem. 283(2):998-1007; Kitagaki et al.
  • Soluble TNFR1 also has been associated with an increased risk of developing MS; thus, neutralization of soluble TNFR1, which cannot be achieved with DN- TNFs/TNF muteins, can be beneficial.
  • TNFR1 antagonists can block the binding of lymphotoxin-a (LT-a), another member of the TNF superfamily, to TNFR1.
  • LT-a lymphotoxin-a
  • LT-a can have a proinflammatory role in RA and in animal disease models, such as CIA and EAE; thus, simultaneous blocking of TNF and LT-a binding to TNFR1 by TNFR1 antagonists can have additional benefits, in comparison to solTNF inhibition, in acute and chronic inflammatory diseases and disorders (see, e.g., Fischer et al. (2015) Antibodies 4:48-70).
  • Tregs CD4 + FoxP3 + regulatory T cells
  • Tregs maintain immunological homeostasis and inhibit autoimmune responses; Tregs also modulate the antitumor immune response, allowing for tumor immune evasion.
  • Tregs thus, are a therapeutic target in the treatment of, for example, autoimmune and chronic inflammatory diseases and conditions, graft-versus-host disease (GvHD), transplantation rejection, and cancer.
  • TNF signaling via TNFR2 regulates the function and activity of Tregs.
  • TNFR2 agonists upregulate Treg activity, while TNFR2 antagonists downregulate Treg activity.
  • TNFR2 agonists include antibodies, such as monoclonal TNFR2 agonist antibodies, and antigen-binding fragments thereof, peptides and proteins, such as TNFR2-selective TNF muteins, fusion proteins, and small molecules.
  • TNFR2 agonistic Antibodies As provided herein, specific agonism of TNFR2 induces the expansion and activation of Tregs, which modulate the immune system, reduces the activity of autoreactive CD8 + T cells that damage tissues, and induces signaling pathways with anti-inflammatory, as well as cell survival, regeneration and protective effects, including neuro-protective, cardio-protective, gut-protective and osteo-protective effects.
  • the enhancement of TNFR2 signaling with TNFR2-selective agonists can be used to enhance the therapeutic effects of TNFR1 -specific antagonism, particularly in the treatment of autoimmune and chronic inflammatory diseases and disorders, including neurodegenerative diseases in which anti-TNF therapies/TNF-blockers have failed.
  • Human TNFR2-selective agonist antibodies include the commercially available MR2-1 (a monoclonal mouse IgGl that binds human, cynomolgus monkey and rhesus monkey TNFR2; Hycult Biotech), and clone MAB2261 (a monoclonal mouse IgG2A that binds human TNFR2; R&D Systems).
  • TNFR2 agonists, such as antibodies can potently stimulate the expansion of homogeneous populations of FoxP3 + Tregs in CD4 cell cultures, and upregulate the expression of TNF, TRAF2, TRAF3, BIRC3 (cIAP2) and FoxP3 mRNA.
  • Magnetic-activated cell sorting (MACS)- purified CD4 + CD25 + cells cultured using standard in vitro human Treg expansion protocols (i.e. , with anti-CD3 antibodies, anti-CD28 antibodies, IL-2 and rapamycin), yield expanded Tregs with higher levels of FoxP3 (and other characteristic Treg markers), and more potent suppressive capacities, when expanded in the presence of a TNFR2 agonist antibody, compared to in the absence of the TNFR2 agonist.
  • Treg expansion protocols i.e. , with anti-CD3 antibodies, anti-CD28 antibodies, IL-2 and rapamycin
  • Tregs isolated from a patient with type 1 diabetes, that exhibit a resting phenotype are activated and expanded upon in vitro treatment with a TNFR2 agonist antibody; such Tregs are more potent in the inhibition of autologous CD8 + T cells (see, e.g., Zou et al. (2016) Front. Immunol. 9:594).
  • Tregs Treatment of isolated Tregs, expanded using the standard in vitro protocol, with MR2-1, a commercially available agonistic human TNFR2 monoclonal antibody (mAb) containing a mouse IgGl, generates homogenous populations of FoxP3 + Helios + CD127 low Tregs; these Tregs maintain their phenotype and highly suppressive activity in a humanized mouse model.
  • TNFR2 agonists thus, can enhance the ex vivo expansion of Treg cells from impure cell populations, for use in Treg- based immunotherapy (see, e.g., Zou et al. (2016) Front. Immunol. 9:594).
  • TNF can be engineered to selectively bind TNFR1 or TNFR2; for example, a TNFR2 selective TNF mutein is a variant of TNF that contains one or more mutations that increase binding to TNFR2 and/or reduce or eliminate binding to TNFR1.
  • TNFR2-selective mutations include non-conservative substitutions of the Asp residue at position 143 of soluble TNF (see, SEQ ID NO:2), such as, for example, D143Y, D143F or D143N, or non-conservative substitutions of the Ala residue at position 145 of soluble TNF, such as, for example, A145R (see, e.g., U.S. Patent No. 9,081,017).
  • TNF07 is a soluble TNF (sTNF or solTNF) mutein, containing the mutations S95C/G148C (with respect to the sequence of residues set forth in SEQ ID NO:2), that forms a stable TNF trimer and functions as a TNFR2 agonist.
  • TNF07 acts as a TNFR2 agonist despite lacking TNFR2-selective mutations. TNF07 induces potent TNFR2 signaling, expands FoxP3 Treg cells, and selectively induces the death of autoreactive CD8 + T cells isolated from patients with type 1 diabetes (see, e.g., Ban et al. (2015) Molecular and Cellular Therapies 3:7; Zou et al. (2016) Front. Immunol. 9:594).
  • TNFR2 agonists containing fusions of single-chain TNFR2-selective TNF mutein trimers, with multimerization domains, have been generated.
  • the primary ligand for TNFR2 is membrane-bound TNF (mem TNF; also referred to herein as transmembrane TNF or tmTNF).
  • multimerization domains such as dimerization or trimerization domains, generates hexameric or nonameric molecules, respectively, with respect to the TNF subunits; these hexamers and nonamers of TNF mimic membrane-bound TNF trimers and thus, are capable of effectively activating TNFR2 signaling.
  • dimerization domains include EHD2, which is derived from the heavy chain C H 2 domain of IgE and MHD2, which is derived from the heavy chain C H 2 domain of IgM. Dimerization domains also can include Fc domains, such as those derived from IgGl and IgG4, optionally including modifications that alter immune effector functions. Commonly used trimerization domains include chicken tenascin C (TNC) and human TNC. Dimerization and trimerization enhances TNFR2 signaling, and improves the half-life of the fusion protein, for example, by increasing the molecular weight of the molecule, and/or by introducing FcRn recycling, for example, when the dimerization domain is an Fc.
  • TNC chicken tenascin C
  • TNC-sc-mTNF(221N/223R) is a nonameric agonistic TNFR2-specific mouse TNF variant that does not bind TNFR1, and is a single-chain mouse TNF timer, where each TNF subunit is residues 91-235 of SEQ ID NO:5, fused to the trimerization domain of chicken tenascin C (cTNC), corresponding to residues 110-139 of SEQ ID NO:804 (see, also, SEQ ID NO:805).
  • the three single- chain mouse TNF subunits are linked by two (GGGS)4 peptide linkers (see, e.g., SEQ ID NO:707.
  • TNC trimerization domain is linked to the N- terminus of the first TNF subunit in the single-chain trimer.
  • the specificity of STAR2 for TNFR2 results from the mutations D221N and A223R (with reference to the sequence of mouse TNF, set forth in SEQ ID NO: 5) within the individual TNF subunits, which creates a steric clash between STAR2 and mouse TNFR1. Fusion to the TNC tnmenzation domain causes spontaneous oligomer formation, creating three covalently linked TNF trimers, and mimicking membrane-bound TNF.
  • STAR2 stimulates the proliferation of Tregs in vitro and in vivo in a TNFR2-dependent, IL-2- independent mechanism.
  • TNC-scTNF(143N/145R) A human equivalent of the TNFR2-specific STAR2 agonist, TNC-scTNF(143N/145R), made of residues 9- 157 of soluble TNF (see, SEQ ID NO:2), containing the mutations D143N/A145R with reference to SEQ ID NO:2 (solTNF), potently stimulated CD4 + FoxP3 + Treg expansion in vitro from CD4 + T cells isolated from healthy donors (see, e.g., Chopra et al. (2016) J. Exp. Med. 213(9): 1881-1900; Zou et al. (2016) Front. Immunol. 9:594).
  • TNC-SCTNF R2 is a soluble human TNFR2 agonist that is a fusion of the trimerization domain of human tenascin C (hTNC), containing residues 110-139 of SEQ ID NO:806 (see, also, SEQ ID NO:807), to the N-terminus of a TNFR2-selective single-chain TNF variant (SCTNF R2 ; SEQ ID NO:803), contains three TNF domains connected by two short peptide linkers (GGGGS).
  • the TNFR2-selective TNF molecule, SCTNF R2 resembles soluble trimeric TNF, and each TNF subunit includes amino acids 80-233 of the full length TNF set forth in SEQ ID NO: 1 (corresponding to residues 4-157 of SEQ ID NO:2), with the mutations D143N/A145R, with reference to SEQ ID NO:2, which eliminate binding to TNFR1. Because TNFR2 is only fully activated by membrane-bound TNF, but not soluble TNF trimers, the trimerization domain of TNC is fused to the N-terminus of SCTNF R2 , generating TNC- SCTNF R2 .
  • TNC-SCTNF R2 exists in a trimeric assembly of the single stranded fusion protein and resembles a nonameric TNF molecule; this oligomeric TNF mutein, due to its increased avidity, mimics membrane-bound TNF (mem TNF) activity, induces the clustering of TNFR2 and the formation of TNFR2 signaling complexes, efficiently activating TNFR2.
  • TNC-SCTNF R2 exhibits neuroprotective properties; it preserves neurons from superoxide-induced cell death and rescues neurons from catecholaminergic cell death. In an in vitro model of Parkinson’s disease, TNC- SCTNF R2 rescued neurons after induction of cell death by 6-OHDA.
  • EHD2-SCTNF R2 is an agonistic TNFR2-selective TNF mutein fusion protein that contains a covalently stabilized human TNFR2-selective single-chain TNF trimer (SCTNF R2 ; SEQ ID NO:803) with the mutations D143N/A145R (residue numbering with respect to soluble TNF, as set forth in SEQ ID NO:2), which abrogate binding to TNFR1, fused to the dimerization domain EHD2 (SEQ ID NO:808), which is derived from the heavy chain C H 2 domain of IgE, and creates a disulfide bonded dimer that contains hexameric TNF domains.
  • Each TNF subunit within SCTNF R2 contains residues 4-157 of SEQ ID NO:2.
  • EHD2 is fused to the N-terminal end of the trivalent human single-chain SCTNF R2 via a peptide linker (GGGSGGGSGGGSGGGSGGGSGGSEFLA; SEQ ID NO: 809), and the three TNF domains of SCTNF R2 are connected via two GGGGS peptide linkers.
  • EHD2- SCTNF R2 exhibits neuroprotective properties in a mouse model of NMDA-induced acute neurodegeneration (see, e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113(43): 12304-12309; and U.S. Patent Publication No. 2020/0102362).
  • TNFR2 agonist fusion proteins also include single chain TNFR2 agonists (SCTNF R2 ) containing three TNF muteins with the mutations D143N/A145R (with reference to SEQ ID NO:2), which abrogate binding to TNFR1, fused with a dimerization domain that is an Fc, resulting in a protein that is hexameric with respect to the TNF domains (SCTNF R2 -FC).
  • the Fc can be an IgG4 or IgGl Fc, optionally containing mutations that eliminate Fc effector functions, such as ADCC and CDC.
  • the three TNF muteins which contain residues 12-157 of SEQ ID NO:2, are linked together by two short peptide linkers, and the dimerization domain is linked to the N- terminus or C-terminus of the single chain trimeric TNF molecule (SCTNF R2 ) by a third short peptide linker.
  • EHD2 IgE heavy chain domain 2
  • MHD2 IgM heavy chain domain 2
  • SEQ ID NO:811 see, e.g., International Application Publication No. WO 2019/226750.
  • TNFR2 antagonists inhibit the proliferation of and induce the death of Tregs, and also can inhibit the proliferation of and induce the death of TNFR2-expressing tumor cells.
  • TNFR2 antagonists can reduce or inhibit the proliferation of myeloid- derived suppressor cells (MDSCs), and/or induce apoptosis within MDSCs, by binding TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment.
  • MDSCs myeloid- derived suppressor cells
  • TNFR2 antagonists also induce the expansion of T effector cells, including cytotoxic CD8 + T cells, via the inhibition of Treg expansion and activity.
  • TNFR2 antagonists can be useful in the treatment of infectious diseases, and certain cancers that express TNFR2, such as, for example, T cell lymphomas (e.g., Hodgkin’s lymphoma and cutaneous non-Hodgkin’s lymphoma), ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, breast cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, and lung cancer (see, e.g., U.S. Patent Publication No. 2019/0144556; Torrey et al. (2017) Sci. Signal. 10:eaaf8608).
  • T cell lymphomas e.g., Hodgkin’s lymphoma and cutaneous non-Hodgkin’s lymphoma
  • ovarian cancer colon cancer
  • multiple myeloma renal cell carcinoma
  • breast cancer cervical cancer
  • endometrial cancer glioma
  • head and neck cancer glioma
  • TNFR2 is restricted to particular immune cells, including Tregs and MDSCs, endothelial cells, and particular neurons and cardiac cells.
  • the restricted expression of TNFR2 makes it an ideal drug target, as systemic toxicity from anti-TNFR2 therapeutics is less likely to occur.
  • TNFR2 antagonist antibodies and antigen-binding fragments thereof bind epitopes within human TNFR2 that contain one or more of the residues KCRPG (corresponding to residues 142-146 of SEQ ID NO:4), or a larger epitope, containing residues 130-149, 137-144 or 142-149, or at least 5 continuous or discontinuous residues within these epitopes, for example, and do not bind to the epitope containing residues KCSPG (corresponding to residues 56-60 of SEQ ID NO:4).
  • TNFR2 antagonists also can bind the TNFR2 epitopes PECLSCGS (corresponding to residues 91-98 of SEQ ID NO:4), RICTCRPG (corresponding to residues 116-123 of SEQ ID NO:4), CAPLRKCR (corresponding to residues 137-144 of SEQ ID NO:4), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO:4), and VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO:4), and/or an epitope containing at least 5 continuous or discontinuous residues within residues 75- 128, 86-103, 111-128, or 150-190 of SEQ ID N0:4 (see, e.g., U.S. Patent Publication No. 2019/0144556).
  • PECLSCGS corresponding to residues 91-98 of SEQ ID NO:4
  • RICTCRPG corresponding to residues 116-123 of SEQ ID NO:4
  • antagonistic TNFR2 antibodies or antigen-binding fragments thereof bind to an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO:840), with an affinity that is at least 10-fold greater, for example, than the affinity of the same antibody or antigen-binding fragment for a peptide that contains the KCSPG sequence of human TNFR2 (SEQ ID NO:839).
  • Antibodies or antibody fragments that bind epitopes containing one or more residues of the KCRPG sequence, and epitopes containing the KCSPG motif with similar affinity are not antagonistic TNFR2 antibodies.
  • Antagonistic TNFR2 antibodies include TNFRAB1 (see, SEQ ID NOs: 1213 and 1213 for the sequences of the heavy and light chains of TNFRAB1, respectively), TNFRAB2 and TNFR2A3 (see, e.g, U.S. Patent Publication No. 2019/0144556 for descriptions of these antibodies).
  • TNFR2 antagonists also include antibodies and antibody fragments that contain the CDR-H3 sequence of TNFRAB1 (QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO: 1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223), or a CDR-H3 sequence with at least about 85% sequence identity thereto.
  • TNFRAB1 specifically binds residues 130-149, containing residues KCRPG of TNFR2, with a 40-fold higher affinity than residues 48-67, containing residues KCSPG of TNFR2 (see, e.g., U.S. Patent Publication No. 2019/0144556).
  • TNFRAB1 (see, SEQ ID NOs: 1212 and 1213 for heavy and light chains, respectively) is a murine antibody that antagonizes the TNF-TNFR2 interaction, and, in addition to binding the KCRPG sequence of TNFR2, also binds an epitope within residues 161-169 (CKPCAPGTF; SEQ ID NO: 1258) of TNFR2 (SEQ ID NO:4).
  • TNFRAB2 another antagonistic TNFR2 antibody, binds the epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO:851), as well as epitopes that include one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248), and 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR2.
  • CAPLRKCR epitope containing residues 137-144
  • epitopes that include one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248), and 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR2.
  • TNFR2A3 is a murine antagonistic human TNFR2 antibody that was discovered by immunization of a mouse with human TNFR2 and subsequent CDR mutagenesis, in which the CDR-H3 of the generated precursor antibody was replaced with the CDR-H3 sequence ARDDGSYSPFDYFG (SEQ ID NO: 1223).
  • TNFR2A3 binds to two distinct epitopes within human TNFR2; the first epitope includes residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and contains the KCRPG motif, and the second epitope is a downstream sequence that contains residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464).
  • replacement of the CDR-H3 sequence of a neutral anti-TNFR2 antibody i.e., an antibody that is neither antagonistic nor agonistic
  • an antagonistic TNFR2 antibody such as the CDR-H3 sequences of TNFRAB1, TNFRAB2 or TNFR2A3
  • converts the phenotype-neutral antibody to an antagonistic TNFR2 antibody such as a dominant antagonistic TNFR2 antibody, which is an antagonist that inhibits TNFR2 activation even in the presence of a TNFR2 agonist, such as TNF, or IL-2
  • a TNFR2 agonist such as TNF, or IL-2
  • TNFR2 antagonist antibodies or antigen-binding fragments thereof can contain the CDR-H1 sequences set forth in any of SEQ ID NOs: 1214, 1215, and 1231-1233; the CDR-H2 sequences set forth in any of SEQ ID NOs: 1216, 1224, and 1230; the CDR-H3 sequences set forth in any of SEQ ID NOs: 1217, 1223, and 1225-1229, or the CDR-H3 of TNFRAB1, corresponding to residues 99-112 of SEQ ID NO: 1212; the CDR-L1 sequences set forth in any of SEQ ID NOs: 1218 and 1234-1236, or the CDR-L1 sequence of TNFRAB1, corresponding to residues 24-33 of SEQ ID NO: 1213; the CDR-L2 sequences set forth in any of SEQ ID NOs: 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1, corresponding to residues 49-55 of SEQ ID NO:
  • Exemplary framework regions that can be used for the development of a humanized anti-TNFR2 antibody, containing one or more of the above CDRs include, without limitation, those described in U.S. Pat. Nos. 7,732,578 and 8,093,068, and in International Application Publication No. WO 2003/105782.
  • Another approach to engineering humanized anti-TNFR2 antagonistic antibodies is to align the sequences of the heavy chain variable region and light chain variable region of an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3, with the heavy chain variable region and light chain variable region of a consensus human antibody.
  • Consensus human antibody heavy chain and light chain sequences are known in the art (see e.g., the "VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, (1991); Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836). In this way, the variable domain framework residues and CDRs can be identified by sequence alignment.
  • exemplary variable domains of a consensus human antibody include the heavy chain variable domain set forth in SEQ ID NO: 1245, and the light chain variable domain set forth in SEQ ID NO: 1246, identified in U.S. Pat. No. 6,054,297 (see, e.g., U.S. Patent Publication No. 2019/0144556).
  • the CDR-H1 and CDR-H2 sequences of the exemplary consensus sequence of a human antibody heavy chain variable domain of SEQ ID NO: 1245 can be replaced, for example, with the corresponding CDR sequences of a phenotype- neutral, TNFR2-specific antibody, and the CDR-L1, CDR-L2 and CDR-L3 sequences of the exemplary consensus sequence of a human antibody light chain variable domain of SEQ ID NO: 1246 can be replaced with the corresponding CDR sequences of a phenotype-neutral, TNFR2-specific antibody, to produce humanized, antagonistic TNFR2 antibodies.
  • TNFR2 antagonists can be identified by screening for peptides that bind epitopes within TNFR2, such as those set forth in any one of SEQ ID NOs: 1247- 1464, by using techniques known in the art, for example, phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display, or any other methods known in the art, such as those described in U.S. Patent Publication No. 2019/0144556.
  • a human TNFR2 antagonist mAb when added to standard Treg expansion culture conditions, inhibits the expansion of Tregs and reduces their suppressive activity (see, e.g., Zou et al. (2016) Front. Immunol. 9:594).
  • Two potent, dominant anti-human TNFR2 antagonistic antibodies that outcompete TNF (the natural agonist of TNFR2), inhibit TNF-induced in vitro expansion of human Tregs, and can induce the death of Tregs in vitro.
  • the TNFR2 antagonists bind TNFR2 specifically through the F(ab) region, independently of the Fc region or the crosslinking of antibodies, and block the binding of TNF to TNFR2 by binding to the antiparallel dimers of TNFR2.
  • Tregs isolated from ovarian cancer tissues were found to be more sensitive to TNFR2 antagonist mAb-induced cell death, due to higher levels of TNFR2 expression on tumor-infiltrating Tregs.
  • the TNFR2 antagonists also induced the death of TNFR2 + OVCAR3 (ovarian cancer) tumor cells, which also express TNFR2.
  • thalidomide is a small molecule synthetic glutamic acid derivative with immunomodulatory and anti-inflammatory properties; thalidomide and its structural analogs, lenalidomide and pomalidomide, are classified as immunomodulatory drugs. Thalidomide and its analogs inhibit TNF synthesis by downregulating NF-excellentB, destroying TNF mRNA, and targeting reactive oxygen species and al-acid glycoprotein, and also, inhibit surface expression of TNFR2 on T cells by inhibiting intracellular TNFR2 transport to the cell surface.
  • thalidomide reduces the number and function of Tregs in patients with chronic lymphocytic leukemia, and, in patients with acute myeloid leukemia, combination therapy with lenalidomide and azacitidine downregulates TNFR2 expression on CD4 + T cells and reduces the number of TNFR2 + Tregs, enhancing effector immune function.
  • treatment with thalidomide and its analogs increased the number of Tregs, likely due to the elevated serum levels of TNF following treatment, indicating that the effects of thalidomide on TNFR2 + Tregs is disease specific (see, e.g., Zou et al. (2016) Front. Immunol. 9:594).
  • panobinostat a histone deacetylase inhibitor that can reduce FoxP3 expression and inhibit the suppressive activity of Tregs.
  • Combination therapy with panobinostat and azacitidine reduces the numbers of TNFR2 + Tregs in the blood and bone marrow of patients with acute myeloid leukemia, and the resulting increase in IFN ⁇ and IL-2 production by effector T cells results in a therapeutic effect in these patients (see, e.g., Zou et al. (2016) Front. Immunol. 9:594).
  • Cyclophosphamide a DNA alkylating agent commonly used as a cytotoxic chemotherapeutic in cancer treatment, can inhibit immunosuppressive function of Tregs at low doses, and depletes the maximally suppressive Tregs in mice bearing PROb colon cancer following the administration of a single dose, resulting in the activation of anti-tumor immune responses.
  • cyclophosphamide treatment depleted TNFR2 hi Tregs.
  • cyclophosphamide inhibited the growth of established CT26 tumors in mice, by blocking TNF-TNFR2 interaction and eliminating TNFR2-expressing Treg activity (see, e.g., Zou et al. (2016) Front. Immunol. 9:594).
  • Triptolide an immunosuppressive molecule isolated from the Chinese herb Tripterygium wilfordii, inhibits TNF and TNFR2 expression in the colon of a mouse colitis model, and also, decreases the number of Tregs and inhibits tumor growth in mice with melanoma (see, e.g., Zou et al. (2016) Front. Immunol. 9:594).
  • anti-TNF therapies which block TNF and inhibit its signaling via TNFR1 and TNFR2, are limited in therapeutic efficacy, tolerability, and safety.
  • Anti-TNF therapies ameliorate RA and other autoimmune and inflammatory diseases and conditions by preventing TNF signaling through TNFR1, and abrogating apoptotic and inflammatory pathways.
  • These anti-TNF therapies also block the beneficial effects of TNFR2 signaling, including the protective, pro-survival, regeneration-promoting and anti-inflammatory signaling pathways, as well as the TNFR2-associated expansion of immunosuppressive Tregs, resulting in serious, sometimes fatal, side effects, including serious infections.
  • TNF blocking therapies include congestive heart failure, liver injury, demyelinating disease/CNS disorders, lupus, psoriasis, sarcoidosis, and an increased susceptibility to the development of additional autoimmune diseases, as well as cancers, including lymphomas and solid malignancies.
  • Anti-TNF therapies have failed in the treatment of demyelinating and neurodegenerative diseases, and can exacerbate disease symptoms.
  • constructs including TNFR1 antagonist constructs, TNFR2 agonist constructs, multi-specific, such as bi-specific, TNFR1 antagonist/TNFR2 agonist constructs, and nucleic acids and methods for the selective inhibition of TNF signaling via TNFR1 (see, e.g., Figure 2, which depicts an exemplary bi-specific construct). Also provided are constructs and methods for selective inhibition of TNF signaling via TNFR1, including while maintaining or enhancing TNFR2 signaling. These constructs and methods provide improved therapeutic approaches for the treatment of diseases and disorders of the TNF/TNFR1 axis.
  • TNFR2 concomitant or sequential selective agonism of TNFR2 with TNFR1 antagonism has therapeutic effects, and can enhance the therapeutic index of selective TNFR1 antagonists, by activating desirable signaling pathways, such as anti- inflammatory pathways and NF-excellentB pathways that control cell survival and proliferation, and by inducing the expansion of immunosuppressive Tregs that remove excess autoreactive/effector T cells that result in tissue destruction, from the autoimmune microenvironment.
  • Sections 1 and 2 describe methods that target each of TNRF1 and TNRF2; section 3 provides an overview of constructs provided herein that solve the problems of prior approaches, particularly those that targeted TNFR1; and Section 4 describes the structure and components of constructs provided herein.
  • the TNF trimer binds to three TNFR1 chains as a preligand assembly complex, mediated by the preligand assembly domains (plads) of each monomeric TNFR. This differs from most receptor systems where ligand binding is required before clusters form on the surface of the cell.
  • the TNF receptors are single transmembrane glycoproteins with about 28% homology mostly in their extracellular domain with both receptors containing four tandemly repeated cysteine rich motifs. Their intracellular sequences are largely unrelated with almost no homology between each other, and early work indicated delineation of their signaling functions (Grell et al. (1994) J ImmoL 153(5): 1963-72).
  • TNFR1 and TNFR2 contain several motifs with known functional significance.
  • Each of TNFR1 and TNFR2 contains an extracellular pre- ligand-binding assembly domain (PLAD) domain (distinct from ligand binding regions) that precomplexes receptors. Conformational changes are induced when the trimeric TNF ligand binds to the TNFR trimer in the cell membrane, resulting in signal activation (MacEwan (2002) Br J Pharmacol. 135 (4) :855-875; and Lo et al. (2019) Sci Signal. 12(592) :eaav5637).
  • PAD pre- ligand-binding assembly domain
  • Monovalent antagonists such as single domain antibodies (dAbs or sdAbs), nanobodies (Nbs; camelid single domain antibodies), scFv fragments, and Fab fragments, on the other hand, bind to one TNFR1 molecule, and do not induce cross-linking or clustering of the receptor on cell surfaces, abrogating any activation of TNFR1 signaling.
  • Monovalent antagonists can bind to domain 1, 2, 3 or 4, or to an epitope spanning multiple domains, of the TNFR1 extracellular domain (see, e.g., U.S. Patent Nos. 9,028,817 and 9,028,822), but these existing antagonists were ineffective therapeutics. Among a variety of problems were the short serum-half-lives, and immunogenicity, and other problems. Selective blockade of TNFR1 can be achieved with TNFR1 antagonists with properties described and provided herein. 2. Selective Activation of TNFR2 with TNFR2 agonists
  • TNFR2 selective activation of TNFR2
  • TNFR2-specific agonists which can include, for example, TNFR2 agonistic antibodies and antigen-binding fragments thereof, and TNFR2-selective TNF muteins and fusion proteins thereof.
  • Antigen-binding fragments of antibodies that bind to the first and/or second epitope of human TNFR2 can be used.
  • the first epitope of TNFR2 includes amino acid residues 48-67 of SEQ ID NO:4, and the second epitope includes position 135 of SEQ ID NO:4, including, for example, residues 128-147, 130-149, 135-147, or 135-153, of SEQ ID NO:4 (see, e.g., International Application Publication No. WO 2014/124134; and U.S. Patent No. 9,821,010).
  • Other epitopes on TNFR2 have been identified and can be used to design antigen-binding fragments with TNFR2-selectivity, as discussed below.
  • TNFR2 agonists include TNFR2- selective TNF muteins and antibody fragments.
  • TNF mutein and antibody fragments that fused with multimerization domains, particularly dimerization or trimerization domains, as discussed below.
  • TNF mutein and antibody fragments that fused with multimerization domains, particularly dimerization or trimerization domains, as discussed below.
  • half-life extender proteins or peptides such as human serum albumin (with or without FcRn optimization, and with or without itself being PEGylated); and ADCC-inactivated/ FcRn optimized Fc domains of antibodies with or without PEGylation (reviewed, for example in Strohl (2015) BioDrugs 29(4):215-239).
  • Half-life extenders include, for example, PEGylation, modification of glycosylation, sialyation, PASylation (polymers of PAS amino acids about 100-200 residues in length), ELPylation (see, e.g., Floss et al. (2010) J.
  • TNFR1 antagonist constructs TNFR2 agonist constructs; Multi- Specific, Including Bi-Specific, TNFR1 Antagonist and TNFR2 Agonist Constructs
  • constructs for inhibiting TNRFR1 signaling/activity and/or for agonizing TNFR2 include constructs, discussed below, that are multi-specific, such as bi-specific that inhibit TNFR1 signaling and agonize TNFR2. Care is taken in designing these constructs, since bispecific antagonists TNFR1 or TNFR2 can inhibit the ability of TNF to induce activating changes in conformation of the resting trimeric TNFR, thus preventing its signaling. Other multimeric molecules risk the aggregation of receptors, thus forcing the TNFR to signal for cellular inflammation and apoptosis. Multi-specific constructs herein generally target different receptors, such as each of TNFR1 and TNFR2. By inhibiting TNFR1 signaling, and advantageously agonizing TNFR2 activity, this provides improved treatments of diseases, conditions, and disorders in which TNF is involved.
  • TNFR1 antagonist constructs include fusion protein constructs, such as TNFR1 antagonist-Fc fusion constructs.
  • TNFR1 antagonists that specifically target TNFR1, without antagonizing or without substantially antagonizing TNFR2, or that include or exhibit TNFR2 agonist activity can be selected, generated, or designed.
  • the TNFR1 antagonist constructs improve the therapeutic efficacy and safety of prior TNFR1 antagonists, including monovalent antagonists, such as the dAbs, scFvs and Fabs.
  • selective TNFR2 agonist constructs such as TNFR2-Fc fusion constructs that improve the therapeutic efficacy of prior TNFR2 agonists.
  • the half-life of the Fc fusion constructs increases the half- life of prior TNFR1 antagonists or TNFR2 agonists, which, for example, reduces the frequency of dosing, improves patient compliance, and improves the therapeutic index.
  • selective TNFR2 agonist constructs such as TNFR2-Fc fusion constructs that improve the therapeutic efficacy of prior TNFR2 agonists.
  • the half-life of the Fc fusion constructs increases the half- life of prior TNFR1 antagonists or TNFR2 agonists, which, for example, reduces the frequency of dosing, improves patient compliance, and improves the therapeutic index.
  • Alternative candidate half-life extenders including PEGylating and fusion to peptides, are discussed above, and exemplary extenders are detailed below (reviewed in, Strohl (2015) BioDrugs 29(4)215-239, see also, Tan et al. (2016) Current Pharmaceutical Design 24:4932-4946), but also includes PEGylation using linear or branched PEG (see, e.g., Swierczewska et al. (2015) Expert Opin Emerg Drugs 20(4):53X-536).
  • the TNFR1 agonist constructs include an optional linker and an optional activity modifier. They can be assembled in any order.
  • the structure of TNFR1 antagonist constructs can be represented by the formulae 1 :
  • TNFR1 inhibitor n -linker p - (activity modifier) q , formula la, or
  • each of n and q is an integer, and each is independently 1, 2, or 3; p is 0, 1, 2 or 3; and an activity modifier is a moiety, such as a polypeptide, such as albumin, or an Fc that is modified to have reduced or no ADCC activity, that increases serum half-life of the TNFR1 inhibitor; and the TNFR1 inhibitor is a molecule, such as a polypeptide or small drug molecule that binds to TNFR1 and inhibits its activity.
  • the activity modifier is not a human serum albumin antibody or an unmodified Fc.
  • TNFR2 agonists of formula 3 (TNFR2 agonist) n -linker p - (activity modifier) q , where n, p and q, the linker, and the activity modifier, are as set forth for formula 1.
  • Such constructs can include a TNFR1 antagonist of the above formula or can have a structure as set forth in formula 2 below.
  • the bi-specific and multi-specific constructs selectively inhibit inflammatory and deleterious TNFR1 signaling, enhance protective and anti-inflammatory TNRFR2 signaling. They include moieties that provide for advantageous pharmacokinetic properties, including increased serum half- life and stability, and reduced peripheral clearance, compared with prior TNFR1 antagonists and TNFR2 agonists.
  • the linker can contain a plurality of components, such as a GS linker, a polymeric moiety, such as a PEG, or other such linker, or a hinge region, or other combinations of components, and the activity modifier is a moiety that modify the activity of the construct, such as an Fc region, or a modified Fc region, or a polypeptide the increases half-life, or resistance to endogenous inhibitors.
  • the components of formulae 1 and 2 can be polypeptides or can contain other molecules, such as small drugs that specifically bind or a chemical linker, or a non-peptidic activity modifier. Examples of each component are described below.
  • TNFR2 agonist (activity modifier) q -linker p -(TNFR2 agonist ) n , formula 5b, where each component is as defined above in formula 1 , and the TNFR2 agonist can be small molecule, or a polypeptide, such as an TNFR2 single chain antibody agonist or portion thereof.
  • TNFRl antagonist constructs Components of the TNFRl antagonist constructs, TNFR2 agonist constructs, and Multi-Specific, Including Bi-Specific, TNFRl Antagonist/TNFR2 agonist constructs
  • TNFRl inhibitor moiety (TNFRl antagonist)
  • the TNFRl inhibitor moiety in formula 1, above, and in the multi-specific molecules/constructs (formula 2, above) provided herein is any molecule, including a polypeptide or small molecule, that inhibits TNFR1 signaling. This includes a TNFR1 inhibitor that selectively inhibits TNFR1 signaling, without inhibiting TNFR2 signaling.
  • the TNFR1 antagonist construct In order to avoid receptor clustering, which agonizes TNFR1, the TNFR1 antagonist construct generally is monomeric/mono valent.
  • TheTNFRl antagonist inhibitor component of the construct can be one that is known to have TNFR1 antagonist activity, or can be identified, such as by selecting from a library, such as a phage library, an antibody library, or an aptamer library.
  • the TNFR1 inhibitor moieties are those that are modified or selected to have increased specificity or affinity for TNFR1 , and, have no or little (such that the adverse side effects from such activity are less than grade 2, and generally grade 1 or less based on the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system) agonist activity for TNFR1, and optionally also have agonist activity for TNFR2.
  • CCAE Common Terminology Criteria for Adverse Events
  • the TNFR1 inhibitor moiety can be provided as a single chain antibody or in any of the other forms described herein, including, such as linked to a half-life extender, such as any described above and below, such as a modified Fc region or Fc dimer, or to another moiety or moieties that increase(s) serum half-life.
  • the TNFR1 inhibitor component of the TNFR1 antagonist construct can be or can include a human domain antibody (dAb) that specifically binds to TNFR1.
  • the dAb can contain a variable-region heavy chain (V H ) or light chain (V L ) domain.
  • dAbs for use herein include, for example, dAbs designated DOMlh-574-208 (SEQ ID NO:54) (from DMS5541; see, SEQ ID NO:38), GSK1995057 (see, SEQ ID NO:55) and GSK2862277 (see, SEQ ID NO:56), as well as the dAbs set forth in any of SEQ ID NOs: 57-672; see, e.g.,: U.S. Patent Nos.: 9,028,817 and 9,028,822; U.S.
  • Vhh dAbs that contain a heavy chain. These dAbs can be linked directly or indirectly to a moiety, such as Fc or HSA, that increases serum half-life, and also that can impart other properties or activities to a construct.
  • the anti-TNFRl inhibitor component can be or include a nanobody.
  • exemplary of these are (Nbs) Nb 70 and/or Nb 96 (see, SEQ ID NOs: 683 and 684, respectively).
  • These dAbs and Nbs are surveyed for immunogenicity, and, if needed, using molecular modeling and mutagenesis, are modified to remove predicted immunogenic sequences. Immunogenic sequences can be eliminated by standard methods known in the art. For example, identify the potentially antigenic peptides, and make of conservative replacements of each amino acid to identify those that are not antigenic and that retain activity. Other methods are known (see, e.g., Schubert et al. (2016) PLoS Comput Biol.14(3)'. el005983), which describes a method for de- immunizing proteins).
  • the TNFR1 antagonist dAb portion can be the dAb set forth in any of SEQ ID NOs: 54-672, or a dAb with about or at least about 85%, 90%, 95%, 98%, 99%, or greater, sequence identity to a dAb set forth in any of SEQ ID NOs: 54-672, or a TNFR1 antagonist dAb known to those of skill in the art.
  • TNFR1 antagonists include, for example, antigen-binding antibody fragments.
  • the TNFR1 antagonist can be a Fab fragment, Fab’ fragment, single-chain Fv (scFv), disulfide-linked Fv (dsFv), Fd fragment, Fd’ fragment, single- chain Fab (scFab), hsFv (helix-stabilized Fv), a free light chain, or antigen-binding fragments of any of the above. It also can include linkers, such as GS linkers within the construct, for example, to increase flexibility.
  • the TNFR1 inhibitor portion of the antagonist can contain antigen-binding fragments from the TNFR1 antagonistic antibody designated ATROSAB.
  • the fragments include one or more (or all) of the heavy chain or light chain CDRs of ATROSAB, or CDRs that exhibits at least 85%, 90%, 95% or more sequence identity thereto (e. g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity).
  • the TNFR1 antagonist can contain the V H (residues 1-115 of SEQ ID NO:31) and/or V L (residues 1-113 of SEQ ID NO:32) of ATROSAB, or a V H or V L containing at least 85%, 90%, 95%, or more, sequence identity to the V H or V L of ATROSAB.
  • V H deoxyribonucleic acid
  • V L deoxyribonuentases 1-113 of SEQ ID NO:32
  • a V H or V L containing at least 85%, 90%, 95%, or more, sequence identity to the V H or V L of ATROSAB.
  • it can contain a dAb derived from ATROSAB.
  • the TNFR1 antagonist can contain other monovalent antibody fragments of ATROSAB, including, for example, Fab or scFv fragments, such as the ATROSAB Fab (FabATR) light and heavy chains set forth in SEQ ID NOs: 679 and 680, respectively, or the ATROSAB scFv (scFv IZI06.1) set forth in SEQ ID NO:673.
  • the scFv contains the V H domain, corresponding to residues 1-115 of the ATROSAB heavy chain (see, SEQ ID NOG 1), linked by a short peptide linker (e.g., GGGGSGGGGSGGSAQ, as in SEQ ID NO:673, or a linker set forth in any of SEQ ID NOs:813-834) to the V L domain, corresponding to residues 1-113 of the ATROSAB light chain (see, SEQ ID NO:32).
  • a short peptide linker e.g., GGGGSGGGGSGGSAQ, as in SEQ ID NO:673, or a linker set forth in any of SEQ ID NOs:813-834
  • the TNFR1 antagonist can contain variants of the ATROSAB scFV with increased affinity or selectivity or both for TNFR1, including scFv IG11, which includes or has the sequence set forth in SEQ ID NO:674, scFv T12B, containing the sequence set forth in SEQ ID NO:675, or scFv
  • the TNFR1 antagonist also can include the sequence of amino acid residues from the Fab 13.7 light and heavy chains (derived from scFV 13.7), as set forth in SEQ ID NOs: 681 and 682, respectively.
  • TNFR1 inhibitors in the TNFR1 antagonist construct also include TNF variants (muteins) that bind to TNFR1 to reduce or inhibit signaling.
  • TNF variants include, for example, TNF variants (muteins), such as, but not limited to, TNF variants containing one or more of the mutations L29S, L29G, L29Y, R31E, R31N, R32Y, R32W, S86T, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, and E146R, with reference to SEQ ID NO:2, which impart selectivity to TNFR1.
  • the TNFR1 antagonist can contain, for example, the TNFR1 -selective antagonistic TNF mutein derived from the mutein designated XProl595 (see, SEQ ID NO:701).
  • XProl595 contains the mutations VIM, R31C, C69V, Y87H, C101A and A1456R, with reference to SEQ ID NO:2.
  • TNFR1 -selective antagonistic TNF muteins are derived from XENP345 (see, SEQ ID NO:702), which contains the mutations I97T/A145R, with reference to SEQ ID NO:2; and the TNFR1 -selective antagonistic TNF mutein designated RlantTNF (see, SEQ ID NO:703), which contains the mutations A84S, V85T, S86T, Y87H, Q88N and T89Q, with reference to SEQ ID NO:2.
  • TNFR1 inhibitors to be used in the TNFR1 antagonists also include small molecule inhibitors that can be chemically conjugated to a linker.
  • the TNFR1 inhibitor (antagonist) moiety can be modified to improve its specificity/selectivity for TNFR1, and also, optionally can be modified to have TNFR2 agonist activity.
  • INF binds to TNFR1 with low pM affinity (Kd 19 pM); in general the antagonists herein have at least the same affinity as TNF, unless its activity is due to ‘locking’ the receptor in an inactive conformation, then it is not necessary since the receptors become locked.
  • TNFR1 antagonist constructs include those that specifically bind to TNFR1 with a KD value of less than or less than about 100 nM (e.g., less than or equal to: 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM).
  • nM e.g., less than or equal to: 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM
  • the TNFR1 antagonists specifically bind to TNFR1 with a KD value of less than 1 nM (e.g., less than or equal to: 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640
  • the TNFR1 antagonist constructs provided herein also are selected or designed so that they lack or have reduced binding for other TNFR superfamily members. For example, they are assessed to identify those that do not specifically bind to another TNFR superfamily member, such as TNFR2, using any suitable in vitro binding assay. Assays include, for example, ELISA-based methods.
  • the TNFR1 antagonist constructs can specifically bind to human TNFR1 or a TNFR 1 -derived peptide, with an affinity that is greater than the affinity for another family member or corresponding peptide thereof.
  • the increased affinity is, for example, at least or at least about 5 -fold greater (e.g., at least or equal to 5 -fold greater, 6-fold greater, 7-fold greater, 8-fold greater, 9-fold greater, 10-fold greater, 20-fold greater, 30-fold greater, 40-fold greater, 50-fold greater, 60-fold greater, 70- fold greater, 80-fold greater, 90-fold greater, 100-fold greater, 200-fold greater, 300- fold greater, 400-fold greater, 500-fold greater, 600-fold greater, 700-fold greater, 800-fold greater, 900-fold greater, 1,000-fold greater, 2,000-fold greater, 3,000-fold greater, 4,000-fold greater, 5,000-fold greater, 6,000-fold greater, 7,000-fold greater, 8,000-fold greater, 9,000-fold greater, 10,000-fold greater, or more), than the affinity of the TNFR1 antagonist for another TNFR superfamily member, such as TNFR2.
  • the TNFR1 antagonist constructs provided herein are those that exhibit high k on and low k off values upon interaction with TNFR1, consistent with high- affinity receptor binding.
  • the TNFR1 antagonist constructs provided herein can exhibit kon values in the presence of TNFR1 of greater than or equal to, or greater than, about 10 4 M -1 s -1 (e.g., greater than or equal to 1.0 x 10 4 M -1 s -1 ,1.5 x 10 4 M -1 s -1 , 2.0 x 10 4 M -1 s -1 , 2.5 x 10 4 M -1 s -1 , 3.0 x 10 4 M -1 s -1 , 3.5 x 10 4 M -1 s -1 , 4.0 x 10 4 M-
  • the TNFR1 antagonists provided herein can exhibit k off values, when complexed to TNFR1, of less than or equal to, or less than about 10 -3 s -1 (e.g., less than or less than about 1.0 x 10 -3 s -1 , 9.5 x 10 -4 s -1 , 9.0 x 10 -4 s -1 ,8.5 x 10 -4 s -1 , 8.0 x 10 -4 s -1 , 7.5 x 10 -4 s -1 , 7.0 x 10 -4 s -1 , 6.5 x 10 -4 s -1 , 6.0 x 10 -4 s -1 , 5.5 x 10 -4 s -1 , 5.0 x 10 -4 s -1 , 4.5 x 10 -4 s -1 , 4.0 x 10 -4 s -1 , 3.5 x 10 -4 s -1 , 3.0 x 10 -4 s -1 , 2.5
  • the C-terminus of the TNFR1 antagonist (TNFR1 inhibitor portion of the construct of formula 1 and also formula 2), such as any of the TNFR1 antagonist constructs described herein, can be linked, directly, or more generally via a linker or combination of linker elements, to an activity modifier, or fused with the N-terminus of an TNFR2 agonist (or to a small molecule TNFR2 agonist) via one or more linkers, as discussed below and elsewhere herein.
  • the N-terminus of the TNFR1 inhibitor moiety can be fused to the C-terminus of the TNFR2 agonist, or the C- terminus of the TNFR1 inhibitor moiety (or to a small molecule TNFR2 agonist) can be fused directly or via linker to the activity modifier or to a linker.
  • the linkers (L), discussed in more detail below, are any that improve pharmacological properties, including increasing stability and flexibility and decreasing steric hindrance, and optionally conferring additional properties on the constructs.
  • the linkers can include more than one component, where each component confers a particular property.
  • the TNFR1 antagonists can include any one or more of an Ig Fc region, and/or an antibody hinge region, and/or a short peptide linker, such as a glycine-serine linker.
  • the Fc regions are modified, for example, to eliminate or reduce ADCC activity, and/or to alter receptor binding, and/or for other such activities and properties.
  • Linkers as discussed below, also include chemical linkers.
  • the linker is a polyethylene glycol) (PEG) molecule, or a branched PEG molecule, such as those whose molecular mass is at or about 30 kDa or more.
  • PEG polyethylene glycol
  • branched PEG molecule such as those whose molecular mass is at or about 30 kDa or more.
  • TNFR2 agonist regulatory T cell generator constructs can be used for treating, among other diseases, disorders, and conditions, inflammation and autoimmune diseases, and also solid tumors.
  • Regulatory T cells Tregs
  • Tregs suppress autoimmunity, and have an immunosuppressive effect, such as in a tumor microenvironment.
  • the proliferation of Tregs is positively regulated by TNFR2, and the absence of TNFR2 correlates with reduced Treg numbers and worsened experimental arthritis.
  • a TNFR2 agonist construct thus, can be used for the treatment of many autoimmune diseases, other chronic inflammation, and other acute inflammatory conditions (e.g., SARS, COVID-19).
  • TNFR2 antagonist constructs suppress regulatory T cells and are used for the treatment of cancer and other hyperproliferative diseases (TNFR2 is a ‘checkpoint receptor’). Regulatory T cells accumulate in the tumor microenvironment and are responsible for suppressing the anti-tumor immune response.
  • the TNFR2 antagonist constructs are for treatment of cancers and other hyperproliferative diseases, such as Dupuytren's Contracture, and idiopathic lung fibrosis.
  • TNFR2 agonist constructs containing the TNFR2 agonists. These include TNFR2 agonists linked directly or via a linker to an activity modifier, and also include multi-specific constructs, such as bi- specific constructs that contain a TNRF1 antagonist and a TNFR2 agonist in various configurations with linkers with appropriate structures and properties. In some embodiments, the TNFR2 agonists are in bi-specific constructs.
  • the TNFR2 agonist particularly in the multi-specific, such as bi-specific, molecules/constructs provided herein selectively activates, or agonizes, TNFR2, without activating or without substantially activating TNFR1 and/or without interfering with the inhibition of TNFR1 signaling via the TNFR1 antagonist portion of the multi-specific, such as bi- specific, molecule.
  • the TNFR2 agonist can be any known to those of skill in the art, including agonist antibodies and antigen-binding portions thereof and single chain and other configuration derivatives of antibodies, and also can be small molecule agonists.
  • TNFR2 agonists also can be produced, such as by in silico design, and/or by preparing candidates and screening a library. For example, a phage library, or an antibody library, or an aptamer library can be screened to identify TNFR2 agonists.
  • TNFR2 agonist antibodies, or antigen-binding fragments thereof can be produced by screening libraries of antibodies and antigen-binding fragments thereof for functional molecules that bind to epitopes within TNFR2 and that selectively promote receptor activation. Exemplary of such methods and molecules are those described in International Application Publication No. WO 2017/040312.
  • TNFR2-selective agonists can include the elucidation of epitopes within TNFR2 that promote agonistic receptor-binding.
  • Epitope mapping analysis using linear peptides, and constrained cyclic and bicyclic peptides, derived from various regions of TNFR2 indicates that agonistic TNFR2 antibodies bind to epitopes from distinct regions of the TNFR2 polypeptide in a conformation-dependent manner.
  • one identified epitope of TNFR2 includes residues 56-60 (KCSPG) of SEQ ID NO:4.
  • the agonistic TNFR2 antibody MR2-1 binds to this epitope; it does not bind an epitope containing residues 142-146 (KCRPG) of SEQ ID NO:4.
  • Human TNFR2 can be selected to bind to an epitope (such as including residues 56-60 of SEQ ID NO:4).
  • a human TNFR2 agonist can be selected or designed to bind to an epitope within human TNFR2 that contains at least five discontinuous or continuous residues within residues 96-154 of SEQ ID NO:4 (CGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGF GVARPGT; SEQ ID NO:841), and/or can bind an epitope within residues 111-150 of SEQ ID NO:4 (TREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA; SEQ ID NO: 842), to which MR2-1 additionally binds.
  • the human TNFR2 agonist also can bind an epitope within residues 115-142 of SEQ ID NO:4 (NRICTCRPGWYCALSKQEGCRLCAPLRK; SEQ ID NO:843), and/or residues 122-136 of SEQ ID NO:4 (PGWYCALSKQEGCRL; SEQ ID NO:844), and/or residues 96-122 of SEQ ID NO:4 (CGSRCSSDQVETQACTR; SEQ ID NO:845), and/or an epitope within residues 101-107 of SEQ ID NO:4 (SSDQVET; SEQ ID NO:846; to which MR2-1 additionally binds), and/or an epitope within amino acids 48-67 of SEQ ID NO:4 (QTAQMCCSKCSPGQHAKVFC; SEQ ID NO:847), and/or an epitope containing residues 130-149 of SEQ ID NO:4
  • the TNFR2 agonist antibody and antigen-binding fragments thereof specifically bind to an epitope within, or containing the amino acid residues, of any one of SEQ ID NOs: 853-1211, whereby the antibody or antigen-binding fragment specifically binds human TNFR2, but does not specifically bind another TNFR superfamily member, particularly TNFR1.
  • the human TNFR2 agonist antibody or antigen-binding fragment thereof does not bind, or has impaired/reduced binding to other members of the TNFR superfamily, including TNFR1 (see, e.g., International Application Publication No. WO 2017/040312).
  • Epitopes within TNFR2 that can be used to screen for TNFR2 agonists include the peptides whose sequences are set forth in any of SEQ ID NOs: 853-1211. These peptides can be converted into cyclic and polycyclic formats (for example, by incorporating cysteine residues into the N- and C- terminal positions, or at various internal positions within the peptide chain), in order to confine the peptide fragments to distinct three-dimensional conformations, mimicking the structurally rigidified framework of TNFR2 and the conformational constraint of peptide fragments within TNFR2.
  • the cyclic and polycyclic peptide fragments can then be immobilized on a solid surface and screened for molecules that bind, for example, the TNFR2 agonistic antibody MR2-1, using ELISA.
  • peptides that contain residues within epitopes of TNFR2 that promote receptor activation can structurally pre-organize these amino acids such that they resemble the conformations of the corresponding peptide in the native protein.
  • Cyclic and polycyclic peptides thus obtained can be used to screen libraries of antibodies and antigen-binding fragments thereof in order to identify TNFR2 agonists for use herein.
  • the constrained peptides act as surrogates for epitopes within TNFR2 that promote receptor activation, and thus, antibodies or antigen-binding fragments generated using this screening technique bind to the corresponding epitopes in TNFR2 and are agonistic of receptor activity (see, e.g., International Application Publication No.
  • phage display is used.
  • the phage display library is contacted with under conditions in which specific binding occurs.
  • TNFR2-derived peptide(s) e.g., the peptides of any of SEQ ID NOS: 853-1194
  • Phage containing a TNFR2 -binding moiety form a complex with the target on the solid support, and non-binding phage are washed away.
  • Bound phage then are liberated from the target by changing the buffer to an extreme pH (pH 2 or 10), changing the ionic strength of the bugger, adding denaturants, or by other known means.
  • a protein elution can be performed (see, e.g., International Application Publication No. WO 2017/040312).
  • MR2-1 is an exemplary agonistic TNFR2 antibody that binds TNFR2 and potentiates TNFR2-mediated Treg cell proliferation.
  • MR2-1 binds osteoprotegerin, however, the heavy and/or light chain variable regions of this antibody, or specifically, the heavy and/or light chain CDRs of MR2-1 , can be modified to eliminate the capacity of the resulting antibody or fragment thereof to bind a TNFR superfamily member other than TNFR2, generating an agonistic TNFR2 antibody or antigen-binding fragment thereof. This can be achieved using genetic engineering and/or antibody library screening techniques, for example, as described in International Application Publication No. WO 2017/040312.
  • the TNFR2 agonist can contain an antigen-binding fragment of an agonistic human anti-TNFR2 antibody, such as MR2-1 and MAB2261, such as the commercially available MR2-1 from Hycult Biotech; and MAB2261 from R&D Systems.
  • an agonistic human anti-TNFR2 antibody such as MR2-1 and MAB2261, such as the commercially available MR2-1 from Hycult Biotech; and MAB2261 from R&D Systems.
  • the V H and V L domains of MR2-1 or MAB2261, or one or more of the CDRs contained therein is used to generate a TNFR2 agonist.
  • Such an agonist can contain a human domain antibody (dAb) that is specific for TNFR2; the dAb can contain a variable-region heavy chain (V H ) or light chain (V L ) domain of MR2-1 or MAB2261, or a V H or V L with at least or at least about 85%, 90%, 95%, or more, sequence identity to the V H or V L or MR2-1 or MAB2261, provided the resulting TNFR2 retains TNFR2 agonist activity.
  • V H variable-region heavy chain
  • V L light chain
  • the TNFR2 agonist also can contain other antigen-binding fragments derived from the MR2-1 or MAB2261 antibody, or sequences of amino acids with at least or at least about 85%, 90%, 95%, or more, sequence identity thereto, such as, for example, a Fab fragment, Fab' fragment, F(ab’)2 fragment, Fv fragment, disulfide-linked Fv (dsFv), Fd fragment, Fd’ fragment, single-chain Fv (scFv), single-chain Fab (scFab), hsFv (helix-stabilized Fv), minibody, diabody, anti-idiotypic (anti-Id) antibody, free light chains, or antigen-binding fragments of any of the above.
  • a Fab fragment, Fab' fragment, F(ab’)2 fragment Fv fragment, disulfide-linked Fv (dsFv), Fd fragment, Fd’ fragment, single-chain Fv (scFv), single-chain Fab (sc
  • Antibody fragments include combinations of any of the above fragments, such as, for example, tandem scFv, Fab- scFv (HC C-term, or LC C-term), Fab-(scFv)2 (C-term), scFv-Fab-scFv, Fab-Cw2- scFv, scFv fusions (C term, or N term), Fab-fusions (HC C-term, or LC C-term), scFv-scFv-dAb, scFv-dAb-scFv, dAb-scFv-scFv, and tribodies.
  • a TNFR2 agonist includes any of the dAbs whose sequences are provided herein or that are known in the art, with about or at least about 85%, 90%, 95%, or more, sequence identity thereto, and TNFR2 agonist activity.
  • the TNFR2 agonist can be the scFv of a TNFR2 agonistic monoclonal antibody, including any known in the art, or an scFv with about or at least about 85%, 90%, 95% or more than 95% sequence identity to such scFvs, provided the resulting construct retains TNFR2 agonist activity.
  • the TNFR2 agonist can be the Fab fragment of an TNFR2 agonistic monoclonal antibody or Fab thereof or a Fab with about or at least about 85%, 90%, 95% or more sequence identity, and TNFR2 agonist activity.
  • the TNFR2 agonist also can be or include a TNF mutein modified to bind to TNFR2 and to have agonist activity (see, e.g., SEQ ID NOs: 765-800).
  • TNFR2 agonists that contain a TNFR2-selective TNF mutein, such as, for example, a TNF variant with one or more of the TNFR2-selective mutations K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A
  • TNF variants with the mutations D143N/A145R bind to and agonize TNFR2, and can be used in the constructs provided herein.
  • a TNF mutein with the mutations S95C/G148C, and combinations with any of the others listed or known or identified, with reference to SEQ ID NO:2 also is a TNFR2- selective agonist that can be included in the constructs provided herein.
  • the TNFR2 agonists can contain fusions of single-chain TNFR2-selective TNF mutein trimers, with multimerization domains.
  • the primary ligand for TNFR2 is membrane-bound TNF (mcmTNF; also referred to herein as transmembrane TNF or tmTNF).
  • mcmTNF membrane-bound TNF
  • tmTNF transmembrane TNF
  • multimenzation domains such as dimerization or trimerization domains, generates hexameric or nonameric molecules, respectively, with respect to the TNF subunits; these hexamers and nonamers of TNF mimic membrane-bound TNF trimers and thus, activate TNFR2 signaling.
  • Dimerization domains include, for example, EHD2 (SEQ ID NO:808), discussed above.
  • EHD2 is derived from the heavy chain C H 2 domain of IgE and MHD2 (SEQ ID NO:811), which is derived from the heavy chain C H 2 domain of IgM.
  • Dimerization domains also include Fc domains, such as those derived from IgGl (see, SEQ ID NO: 10) and IgG4 (see, SEQ ID NO: 16), optionally including modifications, such as those that alter immune effector functions and/or enhance FcRn recycling.
  • Trimerization domains include, for example, the trimerization domains of chicken tenascin C (TNC) (SEQ ID NO: 805) and the trimerization domain of human TNC (SEQ ID NO:807). Dimerization and trimerization enhances TNFR2 signaling, and improves pharmacological properties of the constructs. For example, the half-life of a fusion protein is increased by increasing the molecular weight of the molecule, and/or by introducing FcRn recycling, for example, when the dimerization domain is an Fc.
  • the TNFR2 agonist can contain a TNF mutein (TNFmut) trimer chain, with any of the mutations described herein that impart selectivity for TNFR2 and/or reduce or eliminate binding to TNFR1.
  • TNFmut TNF mutein
  • Exemplary of such mutations are the replacements D143N/A145R, with reference to SEQ ID NO:2, fused with a multimerization domain (MD), such as a dimerization or trimerization domain.
  • MD multimerization domain
  • the multimerization domain can be fused to the N- or C-terminus of the TNF mutein trimer chain, and linkers are included between each TNF mutein, and between the TNF mutein trimer chain and the multimerization domain.
  • Such TNFR2 agonists have the formulae 4 and 5:
  • MD-Ll-TNFmut-L2-TNFmut-L3 -TNFmut (Formula 4) or TNFmut-Ll-TNFmut-L2-TNFmut-L3-MD (Formula 5), where MD is a multimerization domain (activity modifier); TNFmut is a TNFR2- selective TNF mutein, such as the mutein with the mutations D143N/A145R; and LI, L2 and L3 are linkers, described below, such as Gly-Ser linkers, that can be the same or different.
  • the multimenzation domain is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken TNC (SEQ ID NO: 805), the trimerization domain of human TNC (SEQ ID NO: 807), an IgGl Fc, or an IgG4 Fc.
  • the dimerization domain is an IgGl Fc or IgG4 Fc, it is the same Fc that is used to link the TNFR1 antagonist to the TNFR2 agonist, and not an additional Fc.
  • the IgGl or IgG4 Fc can be modified to enhance or eliminate immune effector functions, such as ADCC, ADCP and/or CDC activities, and/or to enhance FcRn binding.
  • the multimerization domains such as Fc regions, increases in vivo stability and serum half-life of the construct.
  • Fc regions for purposes herein, in the constructs of Formulae 1-5 or variations thereof, generally are modified to alter or modulate pharmacological properties or activities of the constructs. Fc modifications are discussed in more detail below. Any multimerization domains, known in the art, also are contemplated for use in the TNFR2 agonists herein.
  • the TNF muteins can be TNF variants with any one or more of the mutations that impart TNFR2-selectivity. Mutations, include, for example, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A
  • TNF variants with the mutations D143N/A145R are contemplated for use herein. Any other mutations that impart TNFR2-selectivity, known in the art, also are contemplated for use herein.
  • the TNF muteins can contain the full sequence of soluble TNF (i.e. , residues 1-157 of SEQ ID NO:2), or can contain a partial sequence of soluble TNF, such as, for example, residues 4-157, 9-157, or 12-157 of SEQ ID NO:2, of sufficient length to bind to and/or to agonize TNFR2.
  • the LI, L2, or L3 linkers can be the same or different.
  • the linkers can contain a short peptide linker, such as a GS linker.
  • the linkers also can contain all or a portion (at least 10, 15, or 20 contiguous residues) of the stalk region of TNF- ⁇ , containing the sequence of amino acids GPQREEFPRDLSLISPLAQAVRSSSRTPSDK (SEQ ID NO: 812), which corresponds to residues 57-87 of the full length sequence of TNF (transmembrane TNF), set forth in SEQ ID NO: 1.
  • a linker containing all or a portion, containing at least 10, 15, or 20 contiguous amino acid residues, of the stalk region can be between the N- or C-terminal TNF mutein and the multimerization domain.
  • All three linkers can be (GGGGS) n , where n is generally 1 -10 (SEQ ID NO: 1472), or other combination of Gly-Ser, or can contain mixtures of Gly-Ser resides, such as (GGGGS)n and all or a portion, containing at least 10, 15, or 20 contiguous amino acid residues, of the stalk region of TNF.
  • Exemplary linkers are set forth in SEQ ID NOs: 813-834, 1471 and 1472.
  • TNFR2 agonists include those that specifically bind to TNFR2 with a KD value of less than or equal to or less than about 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM).
  • nM e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 n
  • the TNFR2 agonists specifically bind to TNFR2 with a KD value of less than 1 nM (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630
  • the TNFR2 agonist is one that can induce the proliferation of Tregs (e.g., CD4 + , CD25 + FOXP3 + Tregs), for example, in vivo in a subject to which the agonist is administered, or, for testing purposes, in vitro in a sample containing Tregs that are contacted with the TNFR2 agonist.
  • Tregs e.g., CD4 + , CD25 + FOXP3 + Tregs
  • the proliferation of Tregs can be induced, for example, by or by about 0.00001% to 100.0% (e.g., 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2 %, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.
  • the TNFR2 agonist thus, can be used to promote Treg cell proliferation and can be administered to a mammalian subject, such as a human patient, with an autoimmune or chronic inflammatory disease or disorder, in order to attenuate the magnitude and duration of an immune response (e.g., quantity of CD8 + cytotoxic T lymphocytes produced in vivo in response to a self or non-threatening foreign antigen) in the patient.
  • a mammalian subject such as a human patient
  • an autoimmune or chronic inflammatory disease or disorder in order to attenuate the magnitude and duration of an immune response (e.g., quantity of CD8 + cytotoxic T lymphocytes produced in vivo in response to a self or non-threatening foreign antigen) in the patient.
  • administration of the TNFR2 agonist to a human patient, or a population of Treg cells expanded ex vivo by treatment with the TNFR2 agonist can cause a reduction in the amount of secreted immunoglobulin (e.g., IgG) that is cross-reactive with a self or non-threatening antigen, for example, by or by about 0.00001 mg/mL to 10.0 mg/mL (e.g., 0.00001 mg/mL, 0.0001 mg/mL, 0.001 mg/mL, 0.01 mg/mL, 0.1 mg/mL, 1.0 mg/mL, or 10.0 mg/mL), or by 0.
  • IgG secreted immunoglobulin
  • 001 to 1.0 mg/mL e.g., 0.001 mg/mL, 0.005 mg/mL, 0.010 mg/mL, 0.050 mg/mL, 0.10 mg/mL, 0.20 mg/mL, 0.30 mg/mL, 0.40 mg/mL, 0.50 mg/mL, 0.60 mg/mL, 0.70 mg/mL, 0.80 mg/mL, 0.90 mg/mL, or 1.0 mg/mL
  • a subject not treated with the TNFR2 agonist e.g., 0.001 mg/mL, 0.005 mg/mL, 0.010 mg/mL, 0.050 mg/mL, 0.10 mg/mL, 0.20 mg/mL, 0.30 mg/mL, 0.40 mg/mL, 0.50 mg/mL, 0.60 mg/mL, 0.70 mg/mL, 0.80 mg/mL, 0.90 mg/mL, or 1.0 mg/mL
  • the TNFR2 agonists can decrease cytotoxic T- cell counts (e.g., levels of CD8 + T cells), for example, by or by about 0.00001 to 100.0% (e.g., 0.00001%, 0.00002 %, 0.00003%, 0.00004 %, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%
  • the TNFR2 agonist can be administered to a subject (e.g., a mammalian subject, such as a human) to treat an autoimmune or chronic inflammatory disease or disorder, such as those described herein. Treatment of a subject in this manner reduces the quantity of autoreactive CD8 + T-cells within the subject.
  • a subject e.g., a mammalian subject, such as a human
  • Treatment of a subject in this manner reduces the quantity of autoreactive CD8 + T-cells within the subject.
  • TNFR2 agonists provided herein can be assessed to identify those that lack specific binding for another TNFR superfamily member, particularly TNFR1. This can be achieved using any of a variety of in vitro binding assays, such as ELISA- based methods, known to those of skill in the art.
  • TNFR2 agonists include those that specifically bind to human TNFR2 or a TNFR2-derived peptide, such as the peptide fragment containing residues 48-67 of SEQ ID NO:4 within human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO:847), with an affinity that is, for example, at least or at least about 2-, 3-, 4-, or 5-fold greater (e.g., 5-fold greater, 6-fold greater, 7-fold greater, 8-fold greater, 9-fold greater, 10-fold greater, 20-fold greater, 30-fold greater, 40-fold greater, 50-fold greater, 60-fold greater, 70- fold greater, 80-fold greater, 90-fold greater, 100-fold greater, 200-fold greater, 300- fold greater, 400-fold greater, 500-fold greater, 600-fold greater, 700-fold greater, 800-fold greater, 900-fold greater, 1,000-fold greater, 2,000-fold greater, 3,000-fold greater, 4,000-fold greater, 5,000-fold greater,
  • the TNFR2 agonists provided herein include those that exhibit high k on and low k off values upon interaction with TNFR2, consistent with high-affinity receptor binding.
  • the TNFR2 agonists provided herein can exhibit k on values in the presence of TNFR2 of greater than or equal to, or greater than about 10 4 M -1 s -1 (e.g., greater than or greater than about 1.0 x 10 4 M -1 s -1 , 1.5 x 10 4 M -1 s -1 , 2.0 x 10 4 M- 1 s -1 ,2.5 x 10 4 M -1 s -1 , 3.0 x 10 4 M -1 s -1 , 3.5 x 10 4 M -1 s -1 , 4.0 x 10 4 M -1 s -1 , 4.5 x 10 4 M -1 s- 1 , 5.0 x 10 4 M -1 s -1 , 5.5 x 10 4 M -1 s -1 , 6.0 x 10 4 M
  • the TNFR2 agonists provided herein can exhibit k off values, when complexed to TNFR2 of less than or less than about 10 -3 s -1 (e.g., less than or less than about 1.0 x 10 -3 s -1 , 9.5 x 10 -4 s -1 , 9.0 x 10 -4 s -1 , 8.5 x 10 -4 s -1 , 8.0 x 10 -4 s -1 , 7.5 x 10 -4 s -1 , 7.0 x 10 -4 s -1 , 6.5 x 10 -4 s -1 , 6.0 x 10 -4 s -1 , 5.5 x 10 -4 s -1 , 5.0 x 10 -4 s -1 , 4.5 x 10 -4 s -1 , 4.0 x 10 -4 s -1 , 3.5 x 10 -4 s -1 , 3.0 x 10 -4 s- 1 , 2.5 x 10
  • a TNFR2 agonist is linked directly or indirectly via a linker to a TNFR1 antagonist, such as any described above, in any order or suitable configuration.
  • a TNFR2 agonist such as any of the TNFR2 agonists described herein, is fused with the C-terminus of an TNFR1 antagonist via one or more linkers, as discussed below and elsewhere herein.
  • the C-terminus of the TNFR2 agonist can be fused with the N-terminus of the TNFR1 antagonist.
  • the N-terminus of the multimerization domain is linked to the C-terminus of the TNFR1 antagonist
  • the TNFR2 agonist has the structure set forth in Formula 4
  • the C-terminus of the multimerization domain is linked to the N-terminus of the anti-TNFRl antagonist.
  • the linker (L), between the TNFR1 antagonist and the TNFR2 agonist can include any suitable linkers and combinations thereof, such as one or more of an Ig Fc region, and/or an antibody hinge region, and/or a short peptide linker, such as a glycine-serine linker, for example.
  • the linker is a poly(ethylene glycol) (PEG) molecule, or a branched PEG molecule, of 30 kDa or more.
  • PEG poly(ethylene glycol)
  • the multimerization domain is an Fc, then it is the same Fc that is used to link the TNFR1 antagonist to the TNFR2 agonist.
  • the TNFR1 antagonist constructs (such as formula 1), the multi-specific TNFR1 antagonists- TNFR2 agonist constructs (such as formula 2), and the TNFR2 agonist constructs (such as formulae 3-5), above, optionally include linkers, as well as activity modifiers.
  • the linkers have a variety of functions, including provision of additional or improved biological and pharmacological properties, and for structural purposes for linking a different molecules.
  • Exemplary linkers are Gly-Ser polypeptides, hinge regions (see, e.g., Tables 1-4 above, which set forth the sequences of various hinge regions, and combinations thereof).
  • Linker peptides are included as spaces between polypeptides, and can promote proper protein folding and stability of the polypeptides, improve protein expression, and enhance the bioactivity of the components of the constructs.
  • Peptide linkers primarily are designed to be unstructured, flexible peptides.
  • Linkers can be included as set forth in exemplary formulae 1-4, above. For example, in the bi- specific constructs provided, the components are fused via a linker (L) in an N- terminus to C-terminus, or C-terminus to N-terminus configuration.
  • the linker generally is a peptide linker, including a polypeptide, such as an Fc region, alone, or in combination with one or more other linkers, including, for example, short peptide linkers, such as a glycine-serine (GS) linker, and/or the hinge region of an immunoglobulin (Ig).
  • short peptide linkers such as a glycine-serine (GS) linker
  • Ig immunoglobulin
  • the C-terminus of the TNFR1 antagonist is fused to the N-terminus of the peptide linker(s), and the C- terminus of the peptide linker(s) is fused with the N-terminus of the TNFR2 agonist.
  • the C-terminus of the TNFR2 agonist is fused to the N- terminus of the peptide linker(s), and the C-terminus of the peptide linker(s) is fused with the N-terminus of the TNFR1 antagonist.
  • the linker provides increased molecular weight, increasing the stability and serum half-life, enhancing tissue retention, and reducing or decreasing peripheral elimination, thereby improving the therapeutic index of the molecule.
  • the linker also increases the flexibility of the molecule, allowing each portion of the molecule to interact with its target antigen/epitope, such as TNFR1 and TNFR2, as provided herein.
  • linker contains an Fc region of an immunoglobulin, generally a modified Fc region
  • additional properties can be imparted, including, for example, neonatal Fc receptor (FcRn) recycling, which further increases serum stability and half-life, and/or the enhancement or elimination of immune effector functions.
  • FcRn neonatal Fc receptor
  • Linkers for fusion proteins are well known to those of skill in the art. See, e.g., Chen et al (2013) Adv. Drug. Deliv. Rev. 65: 1357- 1369, entitled “Fusion Protein Linkers: Property, Design and Functionality.”
  • Linkers can be designed or can be from or based on linkers from naturally-occurring multi-domain proteins.
  • Empirical linkers designed by researchers are generally classified into 3 categories, according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers, which are used, for example, for delivering prodrugs that are activated by cleavage of the linker in situ.
  • linkers Besides the role in linking the functional domains together (as in flexible and rigid linkers) or releasing the free functional domain in vivo (as in in vivo cleavable linkers), linkers also can improve properties of the linked moieties. These include, for example, improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. Databases and methods for selecting linkers are known to those of skill in the art (see, e.g., George et al. (2002) “An analysis of protein domain linkers: their classification and role in protein folding," Protein Eng. 15: 871-879). a) Flexible linkers
  • Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. Flexible linkers are generally rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. They are suitable choices when certain movements or interactions (e.g., in an scFv) are required for fusion protein domains. In addition, although flexible linkers do not have rigid structures, they can serve as a passive linker to keep a distance between functional domains. The length of the flexible linkers can be adjusted to allow for proper folding or to achieve optimal biological activity of the fusion proteins.
  • Flexible linkers generally are composed of small, non-polar (e.g. Gly) or polar (e.g., Ser or Thr) amino acids as suggested by Argos (1990) J. Mol. Biol. 211(4):943-958.
  • the small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains.
  • Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties.
  • Exemplary flexible linkers are linkers that contain primarily or only stretches of Gly and Ser residues (“GS” linkers).
  • An example is a flexible linker that has the sequence of (Gly-Gly-Gly-Gly-Ser) n .
  • the length of this GS linker can be selected or chosen to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions.
  • Flexible linkers are also rich in small or polar amino acids such as Gly and Ser, and also can contain additional amino acids, such as Thr and Ala, to maintain flexibility, as well as polar amino acids, such as Lys and Glu, to improve solubility.
  • the SCDKTH hinge sequence and other hinge sequences can be replaced with, or preceded by, a short polypeptide linker.
  • exemplary of polypeptide linkers are (Gly-Ser) n amino acid sequences (GS linkers), with some Glu or Lys residues dispersed throughout to increase solubility.
  • the linker can be a poly- Gly peptide that is at least 2-18 residues in length, or longer, or a similar linker of the same length and flexibility.
  • Exemplary polypeptide linkers in the molecules provided herein include, but are not limited to (see SEQ ID NOs: 816-827 for Gly-Ser linkers): GSGS, GGGGS, or GGGGSGGGGSGGGGS, for example.
  • Another linker that provides similar performance is a (GGGGS)4 (SEQ ID NO:819) linker.
  • Gly and Ser rich flexible linker is GSAGSAAGSGEF (SEQ ID NO:828). This linker has been shown to maintain good solubility in aqueous solutions. Linkers that contain only glycine can be used. For example (Gly) 6 (SEQ ID NO: 1473) and (Gly) 8 (SEQ ID NO: 1474) linkers are known and shown to be stable against proteolytic enzymes digestion during protein purification from the expression organism.
  • While flexible linkers have the advantage of connecting functional domains passively and permitting certain degree of movements, the lack of rigidity of these linkers can be limiting.
  • Rigid linkers are chosen when the spatial separation of the domains is needed to preserve the stability or bioactivity of the fusion proteins.
  • Rigid linkers exhibit relatively stiff structures by adopting a-helical structures or by containing multiple Pro residues. The length of the linkers can be easily adjusted by changing the copy number to achieve an optimal distance between domains.
  • Alpha helix-forming linkers with the sequence of (EAAAK) n have been applied to the construction of many recombinant fusion proteins.
  • An a-helical structure is rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone.
  • the stiff a-helical linkers can act as rigid spacers between protein domains.
  • Another type of rigid linker has a Pro- rich sequence, (XP) n , where X designates any amino acid, and is generally Ala, Lys, or Glu.
  • X designates any amino acid
  • the presence of Pro in non-helical linkers increases stiffness, and allows for effective separation of the protein domains. Examples of such linkers are 33 -residue peptides containing repeating -Glu-Pro- and -Lys-Pro-.
  • linkers or design linkers Desirable properties and requisites therefor are known.
  • Flexible linkers are rich in small and/or hydrophilic amino acids such as Gly or Ser to provide the structural flexibility and have been use to connect functional domains that favor interdomain interactions or movements.
  • Rigid linkers may be used where sufficient separation of protein domains is needed.
  • Rigid linkers are designed or selected to be those that adopt a-helical structures or incorporate proline. Rigid linkers can keep protein moieties at a distance.
  • Flexible and rigid linkers are stable in vivo, and do not allow the separation of joined proteins.
  • Cleavable linkers permit the release of free functional domain in vivo via reduction or proteolytic cleavage. They generally are used for delivery of a prodrug to a target site.
  • an additional linker such as between the TNFR1 antagonist and/or the TNFR2 agonist portions, and the activity-modifying portion, such as the Fc portion, can be included; such linkers can contain, for example, all or a portion of the hinge sequence, sufficient to provide flexibility, of trastuzumab, including at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO:26), or all or a portion, containing a sufficient portion to provide flexibility, of the hinge region of nivolumab, with the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO:29) or a sequence having at least 98% or 99% sequence identity thereto, or any other suitable antibody hinge region or sequence known in the art.
  • GS linker only a GS linker is included.
  • Other short peptide linkers known in the art, also are contemplated for use in the bi-specific molecules provided herein.
  • the N- or C-terminal extensions from an Fc can be used as a linker.
  • a second Fc subunit which is or is not a fusion protein, can be included (see, e.g., Figure 2, and can be modified to contain knobs-in-holes (see discussion below). It will assemble within the mammalian cell expression system to form a knobs-in-hole mediated Fc dimer to create an Fc dimer, which further increases the serum half-life and stability of the molecule.
  • the second Fc subunit is fused with a second TNFR2 agonist, creating a bivalent antibody-like structure.
  • only one Fc subunit is included (an Fc monomer).
  • the linker is a chemical linker.
  • linkers that are non-cleavable moieties, chemical cross-linking reagents, and polypeptide modifying agents, such as polymeric molecules, including PEGylation moieties.
  • Chemical linkers are more amenable to creation of branched constructs and other structures that cannot be achieved with peptide linkers.
  • Exemplary linkers include non-cleavable linkers.
  • Non-cleavable linkers include, for example, amide linkers and amide and ester linkages with succinate spacers (see, e.g., Dosio et al., (2010) Toxins 3:848-883).
  • Exemplary chemical cross- linking linkers include, but are not limited to, SMCC (Succinimidyl-4-(N - maleimidomethyl)cyclohexane-l -carboxylate) and SIAB (Succinimidyl (4- iodoacetyl)aminobenzoate).
  • SMCC is an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane-stabilized spacer arm.
  • SIAB is a short, NHS-ester and iodoacetyl cross- linker for amine-to-sulfhydryl conjugation.
  • cross-linking reagents include, but are not limited to, thioether linkers, chemically labile hydrazone linkers, 4-mercaptovaleric acid, BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo- MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfonyl)benzoate), and bis-maleimide reagents, such as DTME, BMB, BMDB, BMH, BMOE, BM(PEO) 3 , and BM(PEO)4, which are commercially available (Pierce Biotechnology, Inc.).
  • Bis-maleimide reagents allow the attachment of a free thiol group of a cysteine residue of an antibody to a thiol-containing targeted agent, or linker intermediate, in a sequential or concurrent fashion.
  • Other thiol-reactive functional groups include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.
  • Other exemplary linkers and methods of use are well known to those of skill in the art, for example, the linkers and methods described in U.S. Patent Publication No. 2005/0276812, and in Ducry et al. (2010) Bioconjug. Chem. 21 :5-13.
  • Linkers optionally can be substituted with groups that modulate properties, such as solubility and reactivity.
  • a sulfonate substituent can increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with and antibody or drug moiety, and/or facilitate coupling reactions.
  • Linker reagents can also be obtained via commercial sources, such as Molecular Biosciences Inc. (Boulder, CO.), or synthesized in accordance with procedures described in Toki et al. (2002) J, Org. Chem. 67:1866-1872; U.S. Pat. No. 6,214,345; U.S. Publication Nos. 2003/130189, and 2003/096743; and International Application Publication Nos.
  • linker reagents such as DOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) can be prepared by the reaction of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated with isopropylchloroformate (Aldrich), following the procedure of Axworthy et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97(4): 1802-1807. DOTA- maleimide reagents react with the free cysteine amino acids of the cysteine engineered antibodies and provide a metal complexing ligand on the antibody (Lewis et al.
  • Chelating linker labelling reagents such as DOTA- NHS (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid mono (N- hydroxysuccinimide ester), are commercially available (Macrocyclics, Dallas, TX.).
  • the linker can be a dendritic type linker for covalent attachment of more than one moiety through a branching, multifunctional linker moiety to an antibody (see, e.g., Sun et al. (2002) Bioorganic &Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11 :1761-1768; King et al. (2002) Tetrahedron Letters 43: 1987-1990). If an antibody bears only one reactive cysteine thiol group, a multitude of other moieties can be attached through a dendritic linker. Exemplary dendritic linker reagents are known (see, e.g., U.S. Patent Publication No. 2005/0276812).
  • a chemical linker also can be an activity modifier for use in constructs herein
  • PEG molecules and branched PEG molecules, particularly those with a molecular weight of 30 kDa or more.
  • a PEG linker provides for the introduction of multispecificity and bivalency (in the case of TNFR2 agonists where receptor clustering enhances signaling), and increases the molecular weight of the molecule, which increases in vivo serum half-life.
  • PEG linkers also ameliorate difficulties in the re-engineering of antibodies, for example, by avoiding the introduction of non-natural structures that are degraded and cleared rapidly and/or cause immunogenicity. d.
  • portions or regions that modulate or alter the activity and/or pharmacological properties of the constructs are portions or regions that modulate or alter the activity and/or pharmacological properties of the constructs (see formula 1 and 2 above).
  • exemplary of such are Fc regions, modified Fc regions, other multimerization domains, dimers of the Fc and modified Fc, and other moieties, such as polymeric moieties, including polypeptides, such as half-life extending polypeptides, albumins, such as human serum albumin (HSA), and transferrin, and polymers, such as PEG, discussed elsewhere herein, that can increase serum half-life.
  • Activity modifiers can confer properties, such as, but not limited to, extending plasma half-life by decreasing access to proteases, decreasing renal filtration, and/or by altering the intracellular routing via receptor-mediated recycling; providing for absorption across epithelial bilayers by binding to receptors that undergo transcytosis; targeting in vivo sites that over-express or uniquely express specific receptors or antigens; and other properties, as exemplified in the discussion below, and also as known in the art.
  • the constructs can include, as an activity modifier, the Fc region of a human immunoglobulin, such as an IgG, for example, an IgGl Fc (SEQ ID NO: 10), an IgG2 Fc (SEQ ID NO: 12), an IgG3 Fc (SEQ ID NO: 14), or an IgG4 Fc (SEQ ID NO: 16).
  • the Fc is derived from an IgGl or IgG4 antibody.
  • the linker can include an IgGl kappa Fc region, such as the IgGl Fc derived from trastuzumab, containing the C H 2 and C H 3 domains of the trastuzumab heavy chain (see, e.g., residues 234-450 of SEQ ID NO:26; see, also, SEQ ID NO:27).
  • IgGl kappa Fc region such as the IgGl Fc derived from trastuzumab, containing the C H 2 and C H 3 domains of the trastuzumab heavy chain (see, e.g., residues 234-450 of SEQ ID NO:26; see, also, SEQ ID NO:27).
  • the Fc subunit in the bi-specific molecules provided herein also can be an IgG4 Fc, such as, for example, the IgG4 Fc derived from nivolumab (Opdivo®), containing the C H 2 and C H 3 domains of the nivolumab heavy chain (see, e.g., residues 224-440 of SEQ ID NO:29; see, also, SEQ ID NO:30).
  • IgG4 Fc such as, for example, the IgG4 Fc derived from nivolumab (Opdivo®), containing the C H 2 and C H 3 domains of the nivolumab heavy chain (see, e.g., residues 224-440 of SEQ ID NO:29; see, also, SEQ ID NO:30).
  • the Fc region can be mutated or modified, as discussed below, to eliminate, reduce, or enhance, immune effector functions, including, for example, any one or more of antibody-dependent cellular cytotoxicity (ADCC; also known as antibody- dependent cell-mediated cytotoxicity), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the construct is a bi-specific molecule is used to treat inflammatory and autoimmune diseases and conditions
  • immune effector functions are eliminated or reduced.
  • immune effector functions can be enhanced to improve the anti- tumor immune response and therapeutic efficacy.
  • the Fc region is modified to enhance FcRn recycling, to increase the in vivo serum stability and half-life of the molecules provided herein.
  • the Fc regions or domains are modified, particularly to decrease or eliminate ADCC.
  • Small molecule therapeutics such as antibody fragments (e.g., Fabs, scFvs, dAbs), are advantageous. They can be produced in high yields, and have other advantageous properties. They exhibit enhanced tissue penetration and target accessibility compared to monoclonal antibodies (mAbs), and they can prevent undesirable effects of mAbs, such as, for example, receptor clustering, the activation of immune effector functions, poor tissue penetration and lack of access to targets in poorly vascularized areas.
  • mAbs monoclonal antibodies
  • Small antibody fragments have poor pharmacokinetic properties. For example, due to their small size, dAbs and other antibody fragments are rapidly cleared by the kidneys, as molecules that are 50-60 kDa in size or smaller are subject to renal filtration. The rapid clearance and short elimination half-life of small antibody fragments, which can be less than a few hours, decreases the in vivo efficacy and necessitates frequent administration and/or continuous infusion.
  • the dAb(s) in the TNFR1 antagonist, TNFR2 agonist and combination/multi-specific constructs is/are fused to a linker that is or includes a half-life extender, such as, for example, the Fc region of an IgG, such as IgGl or IgG4.
  • the Fc can be a monomer or a dimer.
  • Fusion of small antibody fragments, such as dAbs, to the Fc region of an IgG molecule increases the size of the molecule, thereby protecting it from being cleared/excreted from the body, and mediates binding to the neonatal Fc receptor (FcRn) expressed on endothelial cells, which protects antibodies from lysosomal degradation and prolongs their in vivo half-life.
  • FcRn neonatal Fc receptor
  • the addition of an Fc can introduce unwanted properties, such as the induction of immune effector functions that can result in complement activation, the release of proinflammatory cytokines and cytotoxicity. Because TNFR1 is almost universally expressed, and TNFR2 is expressed by many tissues, it generally is not desirable to use ADCC-enhanced antibodies, but rather rely on the antagonist activity of the antibody for efficacy.
  • the Fc region in the TNFR1 antagonist, TNFR2 agonist, and multi-specific, such as bispecific, constructs is modified to improve pharmacokinetic and pharmacodynamic (i.e., pharmacological) properties, and to eliminate undesirable properties.
  • the Fc region is modified to take advantage of/enhance neonatal FcR recycling to increase the in vivo half-life, and/or is mutated to eliminate Fc-related immune effector functions, such as antibody- dependent cellular cytotoxicity (ADCC; also known as antibody-dependent cell- mediated cytotoxicity), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
  • ADCC antibody- dependent cellular cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • the construct is multi-specific, such as bispecific, such as embodiments in which it contains an TNFR1 antagonist and an TNFR2 agonist), and contains an Fc dimer
  • the dimer is mutated to introduce knobs-in-holes to prevent homodimerization.
  • Numerous modifications to Fc portions (or regions) are known to those of skill in the art (see, e.g., Li et al., (2014) Expert Opin Ther Targets 75:335-350). i. Modifications to the Fc portions a) Knobs-in-Holes
  • Bispecific antibodies include two distinct antigen-binding sites, allowing for an alternative therapeutic approach to conventional therapeutic monoclonal antibodies (mAbs), whereby, limitations associated with mAbs, such as receptor co-clustering, can be avoided. While small antibody fragments are easier and less expensive to produce in high yields, and can easily penetrate tissues, they are associated with limitations, such as poor stability, solubility, and pharmacokinetic properties. For example, their small size results in shorter serum half-lives, reduced tissue retention and rapid clearance from the blood through the kidneys. As a result, IgG-like bi-specific (bs) Abs, which do not have the same limitations, are advantageous.
  • bsAbs can include an Fc region to increase the serum half-life, and also, to permit effector functions where desirable.
  • the production of high yields of purified bsAbs, however, can be challenging, as homodimerization of the heavy chains must be prevented.
  • the “knobs-in-holes” (KiH; also known as “knobs-into-holes”) approach provides a solution to this problem.
  • the C H 3 domains of antibody (IgG) heavy chains are engineered for heterodimerization, to allow for the construction of Fc-containing bi-functional therapeutic molecules that will not self- associate.
  • the knobs-in-holes approach involves asymmetrically mutating interfacial residues in the C H 3 domains of the two parental heavy chains in a complementary manner.
  • “Knobs” are created by replacing amino acids with small side chains with amino acids with larger side chains, such as tyrosine or tryptophan, at the interface between C H 3 domains
  • “holes” are created by replacing amino acids with large side chains with amino acids with smaller ones, such as alanine or threonine.
  • the knob and hole variants heterodimerize by virtue of the knob inserting into a correspondingly designed hole on the partner C H 3 domain. Knob-knob association is prevented due to steric repulsion, and hole-hole homodimers are destabilized.
  • the knob mutation for example, can be S354C, T366Y, T366W, or T394W, and the hole mutation can be Y349C, T366S, L368A, F405A, Y407T, Y407A, or Y407V (all by EU numbering). It has been shown that knobs created towards the center of the dimer interface, such as at residue T366, are more disruptive to homodimer formation than those located near the edge of the dimer interface.
  • T366 on the first C H 3 domain is within hydrogen-bonding distance of residue Y407 on the second or partner C H 3 domain, thus, T366Y and Y407T represent a common knob-in-hole pair; this pair has been shown to generate heterodimers in yields of over 90% (see, e.g. , Ridgway et al. (1996) Protein Eng. 9(7):617-621).
  • the IgG Fc regions for example, in the bispecific TNFR1 antagonist/TNFR2 agonist constructs provided herein can be modified using the knobs-in-holes approach to generate heterodimerized molecules in high yields.
  • Table 6, below, shows the corresponding knob and hole mutations by Kabat numbering and sequential numbering, with reference to the sequence of the IgGl heavy chain constant domain set forth in SEQ ID NO:9. Any mutations known to those of skill in the art that introduce knobs-in-holes can be employed in constructs herein.
  • Fes modified to have “knobs-in-holes” as described above also can be employed with other bi-specific molecules to produce heterodimers.
  • U.S. Patent Publication No. 2010/0055093, and Jin et al. (2009) Mol. Med. 75: 11-20 describe bispecific “ligand” trap constructs that target EGF receptor family ligands, including one designated RB200, and another designated RB242.
  • a problem with those constructs, is that they are heterogeneous, and contain homodimers, and heterodimers, the latter of which are the intended therapeutic.
  • RB200 and RB242 are exemplary of the ligand traps that can be modified by replacing the Fc portions with modified Fc regions that have complementary knobs and holes, so that the resulting dimers all are heterodimers.
  • RB242 targets HER1 (EGFR), HER2, and HER3 ligands, and some HER4 ligands. It was designed so that it does not trap HER4-specific ligands because HER4 has roles in neuronal development that are not shared by other members of the EGFR family.
  • RB242 is composed of the extracellular domain (ECD) of HERl/ErbBl (amino acids 1 to 621 of SEQ ID NO:41) and HER3/ErbB3 (amino acids 1 to 621 of SEQ ID NO:45), fused with the Fc domain of human immunoglobulin G1 (IgGl) (HER1-HER3/Fc), and acts as a chimeric bispecific ligand trap.
  • the HER3/Fc component of RB242 contains a 6xHistidine tag on the COOH terminal (see, e.g., Jin et al. (2009) Mol. Med. 15: 11-20).
  • RB200 binds HERl/ErbBl ligands (EGF, TGF-a, HB-EGF, AR, BTC, EPR and EPG) and HER3/ErbB3 ligands (NRGl-a and NRG1-P3) with high affinity.
  • RB242 inhibits EGF-stimulated and NRGl-pi -stimulated tyrosine phosphorylation of HER family proteins (HER1, HER2 and HER3), and has shown potency in a variety of cell proliferation assays.
  • RB200 inhibits tumor growth in in vivo animal models.
  • the epidermal growth factor (EGF) ligand/receptor family plays a role in a variety of diseases, disorders, and conditions, including rheumatoid arthritis (RA).
  • the EGF family (ErbB and the human epidermal growth factor receptor (HER)) of cell-surface receptors belong to the receptor tyrosine kinase (RTK) superfamily and contain extracellular domains (ECDs) and an intracellular tyrosine kinase signaling domain.
  • EGF EGF receptor
  • HER2/ErbB2 HER3/ErbB3
  • HER4/ErbB4 which are activated by a large family of ligands, including EGF, transforming growth factor a (TGF-a), heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), ⁇ -cellulin (BTC), epiregulin (EPR), epigen (EPG) and neuregulin (NRG).
  • TGF-a transforming growth factor a
  • HB-EGF heparin-binding EGF-like growth factor
  • AR amphiregulin
  • BTC ⁇ -cellulin
  • EPR epiregulin
  • EPG epigen
  • NRG4 binds to HER4/ErbB4.
  • the epidermal growth factor receptor family is composed of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 (Erb-B2), HER3 (ErbB-3) and HER 4 (ErbB-4).
  • EGFR ErbB-1
  • HER2 Erb-B2
  • HER3 ErbB-3
  • HER 4 ErbB-4
  • mutations or amplification of one the family members is associated with worsened survival in cancer patients.
  • TNF signaling transactivates the EGFR signaling pathway by inducing the synthesis of epiregulin and heparin-binding EGF (HB-EGF) on macrophages, both growth factors that activate the EGFR).
  • HB-EGF epiregulin and heparin-binding EGF
  • the EGFR and HER2 are upregulated on synovial fibroblasts, thereby driving their proliferation.
  • EGFR, HER2 (ErbB2), and EGF-like growth factors are overexpressed, for example, in RA synovial fibroblasts and macrophages.
  • the TNF and the EGFR pathways cooperate in the progression of lupus and rheumatoid arthritis, and other autoimmune diseases.
  • constructs designated as “ligand traps.” The ligand trap constructs intercept most inflammatory growth factors of the EGFR family, thereby suppressing the growth of rapidly growing synovial fibroblasts in affected RA joints.
  • These ligand traps are for administration in combination therapy protocols with the TNF blocker constructs that are TNFR1- and/or TNFR2 -targeting constructs provided herein.
  • This combination therapy such as for rheumatoid arthritis, synergistically can combine to achieve disease regression.
  • the EGFR family of growth factors are overexpressed in hyperprolifera- tive/inflammatory diseases such as RA, and also is overexpressed in ovarian and other cancers. Elevated levels of the EGFR family and/or its cognate are a common component of multiple types of cancer. When overexpressed (or sometimes mutated) these receptors are causally associated with shorter survival in many kinds of malignancies.
  • targeted therapeutics that act via the EGFR family are (listed with generic name and exemplary trademark providing source) cetuximab (Erbitux®), panitumumab (Vectibix®), trastuzumab (Herceptin®), and pertuzumab (Perjeta®).
  • Small molecule inhibitors also target the intracellular tyrosine kinase activity of the EGFR family.
  • small molecules include lapatinib (Tykerb®), erlotinib (Iressa®), and neratinib (Nerlynx®). These drugs target only one of the EGFR family members, with the result that other members of the family can upregulate and compensate tumor growth.
  • an antibody vs. a single growth factor e.g., TGF-a, EGF, HB-EGF, and others
  • the ligand trap constructs provided herein address this by blocking HER1, HER2 and HER3 together. This results in pan-inhibition of the EGFR family on cancer cells. Ovarian cancer is among the cancers that are for treatment.
  • the ligand trap constructs provided herein are improved by optimizing heterodimer production, and FcRn recycling, using the Fc regions modified as described herein below for the TNFR1/TNFR2 constructs.
  • the ligand trap constructs are administered in combination therapy protocols with the TNFR1 antagonist constructs, and/or the TNFR2 agonist constructs, and/or the multi-specific TNFR1 antagonist/bi-specific constructions, and/or any other constructions provided herein for treatment of diseases, disorders, and conditions in which TNF plays a role as described herein and/or known to those of skill in the art.
  • PEGylation which is increases the serum half-life of small protein therapeutics, has a disadvantage. PEGylation can decrease potency or activity of a protein therapeutic, can result in heterogeneity, and can result immunoreactivity of the protein. Other approaches involve fusion to albumin, which can improves protein circulation by increasing the molecular weight and reducing renal clearance.
  • Serum half-life also can be increased by fusion to Fc portion of IgGs.
  • the long circulating half-life of approximately 2-3 weeks, and slow clearance rate, of IgGs results at least in part, from their interaction with the neonatal Fc receptor (FcRn), which binds IgGs with high affinity at acidic pH, and releases them at neutral or higher pH.
  • FcRn neonatal Fc receptor
  • FcRn binds to the Fc portion (within the C H 2-C H 3 domains) of pinocytosed IgGs in the acidic ( ⁇ pH 6) endosome in a 2: 1 FcRmlgG configuration (bivalent interaction), traffics them away from the lysosomal degradation pathway and to the cell surface, and recycles them back into circulation after exposure to the extracellular physiological pH ( ⁇ 7.4), at which the Fc-FcRn complex dissociates. Poor binding to FcRn at acidic pH results in trafficking of an antibody to the lysosome where it is degraded.
  • Recycling receptors such as FcRn
  • FcRn also provide a route for the transport of IgGs across the epithelium (transcytosis) and into the blood stream. Leveraging the interaction with FcRn can improve protein transport across epithelial barriers, such as in the gut and the lungs, allowing for noninvasive administration. Residues in the Fc C H 2 and C H 3 domains are involved in FcRn binding, and their mutation in mAbs has been shown to affect the in vivo serum half- life. The circulation and delivery of small protein therapeutics can be improved by fusing them to the Fc domain of IgG, such that the resulting fusion proteins bind to FcRn and take advantage of the IgG serum stabilization pathway.
  • Fusion with an Fc domain also increases the molecular weight of the therapeutic, reducing renal clearance, but can be undesirable due to the potentially reduced tissue penetration and specific activity of the fusion protein.
  • FcRnBPs short FcRn-binding peptides
  • fusion with an FcRnBP increases the molecular weight by approximately 3 kDa, in comparison to fusions with Fc or albumin, which increase the molecular weight by approximately 50-70 kDa (see, e.g., Datta-Mannan el al. (2019) Biotechnol. J. 14: 1800007; Sockolosky et al. (2012) Proc. Natl. Acad. Sci. USA 109(40): 16095- 16100).
  • FcRnBP-Fab constructs For example, short (16 residue) linear and cyclic FcRnBPs (see, e.g., SEQ ID NOs: 48-51) have been fused to the C-terminus, N-terminus, or both, of Fab heavy and light chains (FcRnBP-Fab constructs), with 1-4 FcRnBPs per Fab.
  • FcRnBP-Fab constructs Studies of the pharmacokinetics in cynomolgus monkeys have shown that the FcRn binding of FcRnBP-Fab constructs increases as the number of peptides fused to the Fab increases.
  • fusion with an IgG Fc increases the half-life of small protein therapeutics by taking advantage of FcRn binding, and also by increasing the molecular weight of the therapeutic, such that it is less rapidly cleared from the body, for example, by the kidneys.
  • residues within the Fc region can be mutated to increase the affinity for FcRn, generally by greater than 30-fold, further increasing the in vivo half-life.
  • the Fc region spanning the interface of the C H 2 and C H 3 domains interacts with FcRn.
  • Human Fc residues identified to play a role in FcRn binding include, for example, L251, M252, 1253, S254, L309, H310, Q311, L314, E380, N434, H435 and Y436 (by EU numbering, see Table 1). Mutations in residues located at the Fc-FcRn interface, including M252, S254, T256, H433, N434 and Y436 (by EU numbering), improve the stability of the human FcRn-IgGl complex.
  • the replacements M252Y/S254T/T256E and H433K/N434F/Y436H result in an 11-fold and 6.5-fold improvement in binding to human FcRn at pH 6.0 relative to the wild- type IgGl, respectively, with efficient release at pH 7.4.
  • the combination of these replacements results in a 57-fold increase in binding affinity to FcRn.
  • Additional mutations in IgGl Fc that showed an improvement in binding to FcRn include, for example, M252W, M252Y, M252Y/T256Q, M252F/T256D, E380A, and N434F/Y436H (see, e.g, Dall’Acqua etal. (2002) J. Immunol. 169:5171-5180).
  • the triple substitution M252Y/S254T/T256E when introduced into the C H 2 domain of MED 1-524, a humanized anti -respiratory syncytial virus (RSV) mAb, increased the serum half-life of the mAb approximately 4-fold in cynomolgus monkeys when compared to unmodified MEDI-524.
  • RSV respiratory syncytial virus
  • YTE reduced its ADCC activity and its binding to human Fc ⁇ RIIIA (F158 allotype).
  • the ADCC activity of MED 1-522- YTE can be restored, and increased in comparison to unmodified MEDI-522, by introduction of the ADCC-enhancing replacements S239D/A330L/I332E (by EU numbering), indicating that the replacements YTE provide a reversible mechanism to modulate the ADCC function of a human IgGl (see, e.g., Dall’Acqua et al. (2006) J. Biol. Chem. 281(33):23514-23524).
  • the mean clearance i.e., the volume of serum antibody cleared per unit of time
  • the elimination half-life was ⁇ 1.8-fold longer for the M428L mutant and ⁇ 1.9-fold longer for the T250Q/M428L mutant, compared to unmodified antibody.
  • the mutations M428L and T250Q/M428L are expected to have similar effects in human IgGl, IgG3 and IgG4 antibodies (see, e.g., Hinton et al. (2004) J. Biol. Chem. 279(8):6213-6216).
  • the modifications T250R/M428L were shown to result in selective binding to FcRn at pH 6.0, and a 2.8-fold decreased degradation of serum IgG2 and IgGl in rhesus monkeys (see, e.g. , Saxena et al. (2016) Front. Immunol. 7:580).
  • the mutation N434A (by EU numbering), when introduced into the human anti-HER2 IgGl trastuzumab, resulted in ⁇ 4-fold higher affinity towards human FcRn over unmodified antibody at pH 6, but negligible binding at pH 7.4.
  • the N434A variant had increased exposure, decreased clearance ( ⁇ 2-fold) and increased half-life ( ⁇ 2-fold) compared to the wild-type antibody when tested in vivo in cynomolgus monkeys.
  • the N434A mutation also counters the poor FcRn affinity that can result from the introduction of mutations that increase binding to Fc ⁇ Rs; N434A is typically added to the mutations S298A/E333A/K333 A to create a variant with enhanced Fc ⁇ R binding and normal or improved FcRn binding.
  • Fc mutations that improve FcRn binding also include N434Y, E294del/T307P/N434Y and T256N/A378V/S383N/N434Y.
  • the E294 deletion results in higher sialylation of the N297 glycan on the Fc, which increases antibody half-life in vivo. Indicating that sialylation also plays a role in regulating serum half-life (see, e.g., Saunders, K. O. (2019) Front. Immunol. 10: 1296).
  • the replacements M428L/N434S resulted in an 11- fold increase in affinity to FcRn at pH 6.0, and extended the in vivo serum half-life in cynomolgus monkeys from 9.7 days to 31.1 days, representing a 3.2-fold improvement.
  • the M428L/N434S modification resulted in similar increases in FcRn binding and half-life extension when introduced into the anti-EGFR antibody cetuximab, which is rapidly cleared due to receptor-mediated internalization.
  • the above-identified mutations, and other such mutations, can be introduced into the IgG Fc region in constructs provided herein. These include constructs, such as those of Formulae 1 and 2, in which the linker includes an Fc or an Fc dimer, depending upon the structure of the construct.
  • the IgG Fc regions in constructs herein, such bispecific TNFR1 antagonist/TNFR2 agonist constructs, and the TNFR1 antagonist constructs provided herein are modified to enhance neonatal FcR recycling to increase in vivo half-life. This can be effected by mutating residues at the interface of the C H 2 and C H 3 domains of IgG Fc, which are responsible for binding to FcRn.
  • Exemplary Fc modifications that increase binding to FcRn include, but are not limited to, one or more of T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y, T256N/A378V
  • Table 7 shows the corresponding mutations by Kabat numbering and sequential numbering, with reference to the sequence of the IgGl heavy chain constant domain set forth in SEQ ID NO:9. Other modifications, known in the art to confer enhanced or increased FcRn binding also are contemplated for use herein. Table 7: c) Enhancement of or Reduction/Elimination of
  • IgG subclasses There are four human IgG subclasses that differ in effector functions, circulating half-life and stability.
  • IgGl has Fc effector functions, is the most abundant IgG subclass, and is the most commonly used subclass in FDA-approved therapeutic proteins.
  • IgG2 is deficient in Fc effector functions, but dimerizes with other IgG2 molecules, and is unstable due to scrambling of disulfide bonds in the hinge region.
  • IgG3 has Fc effector functions, and a very long, rigid hinge region.
  • IgG4 is deficient in Fc effector functions, has a shorter circulating half-life than the other subclasses, and the IgG4 dimer is biochemically unstable due to the presence of a single disulfide bond in the hinge region, which leads to the exchange of H chains between different IgG4 molecules.
  • Fc regions from IgG2 and IgG4 do not possess effector functions, and can be used in instances where effector functions are not required or would be detrimental, for example, in the context of autoimmune and inflammatory diseases and disorders.
  • Most approved therapeutic mAbs belong to the human IgGl subclass, and can interact with the humoral and cellular components of the immune system.
  • antibodies engage the humoral immune response via interaction with complement protein Clq, which initiates the complement cascade, resulting in the formation of the membrane attack complex which induces cytolysis in the target cell (i.e. , complement-dependent cytotoxicity (CDC)), and engage the cellular immune response by interaction with Fc gamma receptors (Fc ⁇ Rs).
  • the Fc ⁇ Rs include the Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) classes that differ in their cell surface expression and Fc binding affinities.
  • the five activating Fc ⁇ Rs include the high affinity Fc ⁇ RI that can bind monovalent antibodies, and the lower affinity Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa and Fc ⁇ RIIIb, which require avidity-based interactions.
  • Fc ⁇ RIIb is the only inhibitory receptor.
  • IAMs immunoreceptor tyrosine-based activation motifs
  • the hinge and proximal C H 2 amino acid sequence (lower hinge-upper C H 2 domain region), and glycosylation of the conserved N297 residue (by EU numbering) in the C H 2 domain Asn-X-Ser/Thr glycosylation motif of the Fc region, mediate the interactions of antibodies with Fc ⁇ Rs and complement protein Clq.
  • Antibody/Fc engineering has been used to modify the immune effector functions of antibodies by altering their binding to Clq and various Fey receptors.
  • the CDC, ADCC and ADCP activities of therapeutic mAbs can thus be increased or decreased, depending on the application.
  • the efficacy of anti-cancer mAbs depends in part on their induction of Fc ⁇ R effector functions.
  • the effector function includes the activation of natural killer (NK) cells via Fc ⁇ RIIIa and the subsequent ADCC activity and release of inflammatory cytokines, the induction of macrophage-mediated ADCP via interactions with multiple Fc ⁇ Rs, and the recruitment and activation of other immune cells, such as neutrophils, the primary receptor for NK cell-mediated ADCC.
  • Fc ⁇ RIIIa has two polymorphic variants: one with VI 58, which has a higher affinity for IgGl ; and one with Fl 58, with a lower affinity for IgGl .
  • Residues in the lower hinge and proximal C H 2 regions of IgGs have been determined to be critical for binding to Fc ⁇ Rs.
  • Residues that are within 5 angstroms from the Fc ⁇ R:Fc interface for Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb and Fc ⁇ RIIIb include the residues (by EU numbering) P232, E233, L234, L235, G236, G237, P238, S239 (corresponding to residues P115-S122, with reference to SEQ ID NO:9), D265, V266, S267, H268, E269, D270 (corresponding to residues D148-D153, with reference to SEQ ID NO:9), Y296, N297, S298, T299 (corresponding to residues Y179-T182, with reference to SEQ ID NO:9), and N325, K326, A327, L328, P329, A330, P331 and 1332 (corresponding to residues N
  • Fc modifications that increase the IgGl affinity for and binding to Fc ⁇ RIIIa, and/or enhance ADCC function include the replacements (by EU numbering): F243 L/R292P/Y300L/V305I/P396L, L235 V/F243 L/R292P/Y300L/P396L, F243L/R292P/Y300L, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A, and the combinations of L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in one heavy chain and D270E/K326D/A330M/K334E in the opposing heavy chain, and L234Y/G
  • the mutations A327Q/P329A (interact with Fc ⁇ RI), D265A/S267A/H268A/D270A/K326A/S337A (interact with Fc ⁇ RIIa), G236A (interacts with Fc ⁇ RIIa), and T256A/K290A/S298A/E333A/K334A (interact with Fc ⁇ RIIIa), result in high affinity interactions with Fc ⁇ Rs.
  • Fc modifications that increase binding to Fc ⁇ RIIa and Fc ⁇ RIIIa, and enhance ADCC and ADCP include (by EU numbering) G236A/I332E, G236A/S239D/I332E (also increases binding to Fc ⁇ RI), and G236A/S239D/A330L/I332E (see, e.g., Wang et al. (2016) Protein Cell 9(l):63-73; Saxena et al (2016) Front. Immunol. 7:580; and Saunders, K. O. (2019) Front. Immunol. 10: 1296).
  • Glyco-engineering of IgGs which contain a conserved N-linked glycosylation site at residue N297 in the C H 2 domain, can enhance Fc effector function. Glycosylation of N297 is essential for maintaining Fc conformation and mediating its interactions with Fc ⁇ Rs (and Clq).
  • the glycan present at residue N297 typically has two N-acetylglucosamine (GlcNAc), three mannose, and two more GlcNAc linked to the mannose, to form a biantennary complex glycan. Additional fucose, galactose, sialic acid and GlcNAc can be added to the core glycan structure.

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Abstract

L'invention concerne des constructions moléculaires ciblant le récepteur 1 du facteur de nécrose tumorale (TNFR1) et/ou le récepteur 2 du facteur de nécrose tumorale (TNFR2). Les constructions sont destinées au traitement de maladies, de troubles et d'états dans lesquels ces récepteurs et/ou TNF sont impliqués dans l'étiologie ou dans lesquels leur inhibition ou leur activation peut améliorer la maladie, le trouble, et l'état ou un symptôme associé. Parmi les constructions fournies dans l'invention, se trouvent des constructions antagonistes de TNFR1 qui sont conçues pour inhiber la fonction de TNFR1, et pour éliminer toute activité agoniste de TNFR1. Les constructions de l'invention comprennent des agonistes et des antagonistes de TNFR1 et TNFR2. Des constructions antagonistes de TNFR1 sont conçues pour inhiber la fonction de TNFR1, et dans certains modes de réalisation, sont conçues pour éviter une activité agoniste. L'invention concerne également des agonistes et des antagonistes de TNFR2. Les agonistes de TNFR2 augmentent la fonction des lymphocytes T régulateurs pour contrôler une inflammation aiguë ou chronique. Les antagonistes de TNFR2 diminuent la fonction des lymphocytes T régulateurs augmentant ainsi l'immunité, et sont destinés au traitement du cancer et de certaines maladies d'immunodéficience. L'invention concerne également des méthodes de traitement des diverses maladies dans lesquelles le TNF et ses récepteurs jouent un rôle.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023168426A1 (fr) 2022-03-03 2023-09-07 Enosi Therapeutics Corporation Compositions et cellules contenant des mélanges de protéines de fusion oligo-trap (ofps) et leurs utilisations
WO2024123701A3 (fr) * 2022-12-05 2024-07-11 Fzata, Inc. Traitements de la douleur
WO2025049818A1 (fr) 2023-08-29 2025-03-06 Enosi Therapeutics Corporation Antagonistes tnfr1 dépourvus d'activité agoniste et leurs utilisations
US12472266B2 (en) 2019-02-21 2025-11-18 Enosi Therapeutics Corporation Antibodies and enonomers

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118340891A (zh) * 2024-04-03 2024-07-16 暨南大学附属第一医院（广州华侨医院） Pdi抑制剂在制备抑制sting激活的产品中的应用

Citations (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5422104A (en) 1990-11-21 1995-06-06 Hoffmann-La Roche Inc. TNF-muteins
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
WO1996030347A1 (fr) 1995-03-30 1996-10-03 Pfizer Inc. Derives de quinazoline
WO1996033980A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1996033978A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1996033979A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1997038983A1 (fr) 1996-04-12 1997-10-23 Warner-Lambert Company Inhibiteurs irreversibles de tyrosine kinases
US5753230A (en) 1994-03-18 1998-05-19 The Scripps Research Institute Methods and compositions useful for inhibition of angiogenesis
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
WO1998043960A1 (fr) 1997-04-03 1998-10-08 American Cyanamid Company 3-cyano quinolines substituees
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1999006378A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs irreversibles de tyrosines kinases
WO1999006396A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs bicycliques irreversibles de tyrosine kinases
WO1999009016A1 (fr) 1997-08-01 1999-02-25 American Cyanamid Company Derives de quinazoline substitues et leur utilisation en tant qu'inhibiteurs de la tyrosine kinase
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
WO2002088172A2 (fr) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Composes pentapeptidiques et leurs utilisations
WO2003026577A2 (fr) 2001-09-24 2003-04-03 Seattle Genetics, Inc. P-aminobenzyl ether dans des agents d'administration de medicaments
US20030096743A1 (en) 2001-09-24 2003-05-22 Seattle Genetics, Inc. p-Amidobenzylethers in drug delivery agents
WO2003043583A2 (fr) 2001-11-20 2003-05-30 Seattle Genetics, Inc. Traitement des troubles immunologiques au moyen des anticorps anti-cd30
WO2003105782A2 (fr) 2002-06-17 2003-12-24 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Greffage par specificite d'un anticorps murin sur une charpente humaine
WO2004032828A2 (fr) 2002-07-31 2004-04-22 Seattle Genetics, Inc. Conjugues anticorps anti-cd20-medicament pour le traitement du cancer et des troubles immunitaires
WO2004058820A2 (fr) 2002-12-27 2004-07-15 Domantis Limited Fusion de fc
WO2004081026A2 (fr) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
US20050276812A1 (en) 2004-06-01 2005-12-15 Genentech, Inc. Antibody-drug conjugates and methods
WO2006038027A2 (fr) 2004-10-08 2006-04-13 Domantis Limited Antagonistes et leurs methodes d'utilisation
WO2007049017A2 (fr) 2005-10-24 2007-05-03 Domantis Limited Antagonistes du recepteur 1 du facteur de necrose des tumeurs servant a traiter des maladies respiratoires
US7229619B1 (en) 2000-11-28 2007-06-12 Medimmune, Inc. Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
WO2008149148A2 (fr) 2007-06-06 2008-12-11 Domantis Limited Polypeptides, domaines variables d'anticorps et antagonistes
WO2008149144A2 (fr) 2007-06-06 2008-12-11 Domantis Limited Polypeptides, domaines variables d'anticorps et antagonistes
US20100055093A1 (en) 2006-06-12 2010-03-04 Receptor Biologix Inc. Pan-cell surface receptor-specific therapeutics
US7732578B2 (en) 2001-07-12 2010-06-08 Arrowsmith Technology Licensing Llc Super humanized antibodies
WO2010094720A2 (fr) 2009-02-19 2010-08-26 Glaxo Group Limited Polypeptides, domaines variables d'anticorps, et antagonistes améliorés, tous étant des anti-tnfr1
US20100221213A1 (en) 2002-03-13 2010-09-02 Shishan Ji Y-shape branched hydrophilic polymer derivatives, their preparation methods, conjugates of the derivatives and drug molecules, and pharmaceutical compositions comprising the conjugates
WO2011006914A2 (fr) 2009-07-16 2011-01-20 Glaxo Group Limited Antagonistes, utilisations, et procédés pour inhiber partiellement le tnfr1
WO2011051217A1 (fr) 2009-10-27 2011-05-05 Glaxo Group Limited Polypeptides anti-tnfr1 stables, domaines variables de l'anticorps, et antagonistes
US8093068B2 (en) 2006-10-26 2012-01-10 Centocor, Inc. Methods for use in human-adapting monoclonal antibodies
WO2012104322A1 (fr) 2011-01-31 2012-08-09 Fairchild Semiconductor Corporation Transistor à jonction bipolaire au carbure de silicium comprenant des zones de blindage et son procédé de fabrication
WO2012172070A1 (fr) 2011-06-17 2012-12-20 Glaxo Group Limited Antagonistes du récepteur 1 du facteur de nécrose tumorale
WO2014124134A1 (fr) 2013-02-07 2014-08-14 The General Hospital Corporation Procédés d'expansion ou de déplétion de lymphocytes t régulateurs
US9028817B2 (en) 2009-07-14 2015-05-12 Glaxo Group Limited Stable anti-TNFR1 polypeptides, antibody variable domains and antagonists
US9028822B2 (en) 2002-06-28 2015-05-12 Domantis Limited Antagonists against TNFR1 and methods of use therefor
US9081017B2 (en) 2005-04-11 2015-07-14 Cambridge Enterprise Limited Methods for identifying modulators of tumor necrosis factor receptors
WO2015104322A1 (fr) 2014-01-09 2015-07-16 Glaxosmithkline Intellectual Property Development Limited Traitement de maladies inflammatoires avec des antagonistes anti-tnfr1 non compétitifs
US20150239951A1 (en) 2012-09-10 2015-08-27 Xencor Methods of Treating Neurological Diseases
WO2017040312A1 (fr) 2015-08-28 2017-03-09 The General Hospital Corporation Anticorps agonistes anti-récepteurs de type ii du facteur de nécrose tumorale
US20190144556A1 (en) 2016-05-13 2019-05-16 The General Hospital Corporation Antagonistic anti-tumor necrosis factor receptor superfamily antibodies
WO2019226750A1 (fr) 2018-05-23 2019-11-28 Kantum Diagnostics, Inc. Procédés de traitement et de prévention d'une inflammation rénale par inhibition de la signalisation de l'udp-hexose
US20200102362A1 (en) 2017-04-06 2020-04-02 Universität Stuttgart Tumor necrosis factor receptor (tnfr) binding protein complex with improved binding and bioactivity
WO2020172218A1 (fr) 2019-02-21 2020-08-27 Enosi Life Sciences Corp. Anticorps et énomomères

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102321155A (zh) * 2008-12-19 2012-01-18 华中科技大学 Tnf结合肽和tnfr1封闭肽及其在治疗溃疡性结肠炎中的应用
PE20141522A1 (es) * 2011-08-17 2014-11-17 Glaxo Group Ltd Proteinas y peptidos modificados
EP3221362B1 (fr) * 2014-11-19 2019-07-24 F.Hoffmann-La Roche Ag Anticorps anti-récepteur de transferrine et procédés d'utilisation
AR105634A1 (es) * 2015-09-18 2017-10-25 Chugai Pharmaceutical Co Ltd Anticuerpos que se unen a il 8 y sus usos

Patent Citations (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5422104A (en) 1990-11-21 1995-06-06 Hoffmann-La Roche Inc. TNF-muteins
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
US5753230A (en) 1994-03-18 1998-05-19 The Scripps Research Institute Methods and compositions useful for inhibition of angiogenesis
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
WO1996030347A1 (fr) 1995-03-30 1996-10-03 Pfizer Inc. Derives de quinazoline
WO1996033978A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1996033979A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1996033980A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1997038983A1 (fr) 1996-04-12 1997-10-23 Warner-Lambert Company Inhibiteurs irreversibles de tyrosine kinases
WO1998043960A1 (fr) 1997-04-03 1998-10-08 American Cyanamid Company 3-cyano quinolines substituees
WO1999006378A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs irreversibles de tyrosines kinases
WO1999006396A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs bicycliques irreversibles de tyrosine kinases
WO1999009016A1 (fr) 1997-08-01 1999-02-25 American Cyanamid Company Derives de quinazoline substitues et leur utilisation en tant qu'inhibiteurs de la tyrosine kinase
US7229619B1 (en) 2000-11-28 2007-06-12 Medimmune, Inc. Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
WO2002088172A2 (fr) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Composes pentapeptidiques et leurs utilisations
US7732578B2 (en) 2001-07-12 2010-06-08 Arrowsmith Technology Licensing Llc Super humanized antibodies
WO2003026577A2 (fr) 2001-09-24 2003-04-03 Seattle Genetics, Inc. P-aminobenzyl ether dans des agents d'administration de medicaments
US20030130189A1 (en) 2001-09-24 2003-07-10 Senter Peter D. P-amidobenzylethers in drug delivery agents
US20030096743A1 (en) 2001-09-24 2003-05-22 Seattle Genetics, Inc. p-Amidobenzylethers in drug delivery agents
WO2003043583A2 (fr) 2001-11-20 2003-05-30 Seattle Genetics, Inc. Traitement des troubles immunologiques au moyen des anticorps anti-cd30
US20100221213A1 (en) 2002-03-13 2010-09-02 Shishan Ji Y-shape branched hydrophilic polymer derivatives, their preparation methods, conjugates of the derivatives and drug molecules, and pharmaceutical compositions comprising the conjugates
WO2003105782A2 (fr) 2002-06-17 2003-12-24 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Greffage par specificite d'un anticorps murin sur une charpente humaine
US9028822B2 (en) 2002-06-28 2015-05-12 Domantis Limited Antagonists against TNFR1 and methods of use therefor
WO2004032828A2 (fr) 2002-07-31 2004-04-22 Seattle Genetics, Inc. Conjugues anticorps anti-cd20-medicament pour le traitement du cancer et des troubles immunitaires
US20060083747A1 (en) 2002-12-27 2006-04-20 Domantis Limited Fc fusion
WO2004058820A2 (fr) 2002-12-27 2004-07-15 Domantis Limited Fusion de fc
WO2004081026A2 (fr) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2005035572A2 (fr) 2003-10-08 2005-04-21 Domantis Limited Compositions d'anticorps et procedes
US20050276812A1 (en) 2004-06-01 2005-12-15 Genentech, Inc. Antibody-drug conjugates and methods
WO2006038027A2 (fr) 2004-10-08 2006-04-13 Domantis Limited Antagonistes et leurs methodes d'utilisation
US9081017B2 (en) 2005-04-11 2015-07-14 Cambridge Enterprise Limited Methods for identifying modulators of tumor necrosis factor receptors
WO2007049017A2 (fr) 2005-10-24 2007-05-03 Domantis Limited Antagonistes du recepteur 1 du facteur de necrose des tumeurs servant a traiter des maladies respiratoires
US20100034831A1 (en) 2005-10-24 2010-02-11 Argenta Discovery Limited Agents that Bind a Target in Pulmonary Tissue for Targeting Respiratory Diseases
US20100055093A1 (en) 2006-06-12 2010-03-04 Receptor Biologix Inc. Pan-cell surface receptor-specific therapeutics
US8093068B2 (en) 2006-10-26 2012-01-10 Centocor, Inc. Methods for use in human-adapting monoclonal antibodies
WO2008149144A2 (fr) 2007-06-06 2008-12-11 Domantis Limited Polypeptides, domaines variables d'anticorps et antagonistes
WO2008149148A2 (fr) 2007-06-06 2008-12-11 Domantis Limited Polypeptides, domaines variables d'anticorps et antagonistes
WO2010094720A2 (fr) 2009-02-19 2010-08-26 Glaxo Group Limited Polypeptides, domaines variables d'anticorps, et antagonistes améliorés, tous étant des anti-tnfr1
US9028817B2 (en) 2009-07-14 2015-05-12 Glaxo Group Limited Stable anti-TNFR1 polypeptides, antibody variable domains and antagonists
WO2011006914A2 (fr) 2009-07-16 2011-01-20 Glaxo Group Limited Antagonistes, utilisations, et procédés pour inhiber partiellement le tnfr1
US20120107330A1 (en) 2009-07-16 2012-05-03 Adriaan Allart Stoop Antagonists, uses & methods for partially inhibiting tnfr1
WO2011051217A1 (fr) 2009-10-27 2011-05-05 Glaxo Group Limited Polypeptides anti-tnfr1 stables, domaines variables de l'anticorps, et antagonistes
WO2012104322A1 (fr) 2011-01-31 2012-08-09 Fairchild Semiconductor Corporation Transistor à jonction bipolaire au carbure de silicium comprenant des zones de blindage et son procédé de fabrication
WO2012172070A1 (fr) 2011-06-17 2012-12-20 Glaxo Group Limited Antagonistes du récepteur 1 du facteur de nécrose tumorale
US20150239951A1 (en) 2012-09-10 2015-08-27 Xencor Methods of Treating Neurological Diseases
WO2014124134A1 (fr) 2013-02-07 2014-08-14 The General Hospital Corporation Procédés d'expansion ou de déplétion de lymphocytes t régulateurs
US9821010B2 (en) 2013-02-07 2017-11-21 The General Hospital Corporation Methods for expansion or depletion of T-regulatory cells
WO2015104322A1 (fr) 2014-01-09 2015-07-16 Glaxosmithkline Intellectual Property Development Limited Traitement de maladies inflammatoires avec des antagonistes anti-tnfr1 non compétitifs
WO2017040312A1 (fr) 2015-08-28 2017-03-09 The General Hospital Corporation Anticorps agonistes anti-récepteurs de type ii du facteur de nécrose tumorale
US20190144556A1 (en) 2016-05-13 2019-05-16 The General Hospital Corporation Antagonistic anti-tumor necrosis factor receptor superfamily antibodies
US20200102362A1 (en) 2017-04-06 2020-04-02 Universität Stuttgart Tumor necrosis factor receptor (tnfr) binding protein complex with improved binding and bioactivity
WO2019226750A1 (fr) 2018-05-23 2019-11-28 Kantum Diagnostics, Inc. Procédés de traitement et de prévention d'une inflammation rénale par inhibition de la signalisation de l'udp-hexose
WO2020172218A1 (fr) 2019-02-21 2020-08-27 Enosi Life Sciences Corp. Anticorps et énomomères

Non-Patent Citations (157)

* Cited by examiner, † Cited by third party
Title
"Fundamental Immunology", 1989, RAVEN PRESS, pages: 332 - 336
"National Biomedical Research Foundation", 1979, article "Atlas of Protein Sequence and Structure", pages: 353 - 358
2001, EUR. J. IMMUNOL., vol. 31, pages 2566 - 2573
ANONYMOUS: "Proof of therapeutic activity of selective TNFR targeting in an in vivo model of acute neurodegeneration", 27 October 2016 (2016-10-27), XP055870810, Retrieved from the Internet <URL:https://www.uni-stuttgart.de/en/university/news/all/Proof_of_therapeutic_activity_of_selective_TNFR_targeting_in_an_in_vivo_model_of_acute_neurodegeneration-00003/> [retrieved on 20211208] *
ARNDT ET AL., J. MOL. BIOL., vol. 7, no. 312, 2001, pages 221 - 228
AXWORTHY ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 97, no. 4, 2000, pages 1802 - 1807
BAKER ET AL., SELF/NONSELF, vol. 1, no. 4, 2010, pages 314 - 322
BAKER ET AL., SELFLNONSELF, vol. 1, no. 4, 2010, pages 314 - 322
BAKER ET AL., SELJLNONSELF1, no. 4, 2010, pages 314 - 322
BAN ET AL., MOLECULAR AND CELLULAR THERAPIES, vol. 3, 2015, pages 7
BANERJEE ET AL., JOURNAL OF DRUG DELIVERY, 2012
BEIGIER-BOMPADRE ET AL., SCAND. J. IMMUNOL., vol. 57, 2003, pages 221 - 228
BENJAMIN ET AL.: "StatPearls [Internet", January 2020, STATPEARLS PUBLISHING, article "Disease Modifying Anti-Rheumatic Drugs (DMARDs"
BIOCHEM., vol. 11, no. 9, 1972, pages 1726 - 1732
BLIIML ET AL., ARTHRITIS & RHEUMATISM, vol. 62, no. 6, 2010, pages 1608 - 1619
BLIIML ET AL., INTERNATIONAL IMMUNOLOGY, vol. 24, no. 5, 2012, pages 275 - 281
BULLIMORE, MED HYPOTHESES, vol. 60, 2003, pages 84 - 88
CARRILLO ET AL., SIAMJ. APPLIED MATH, vol. 48, 1988, pages 1073
CHEN ET AL., ADV. DRUG. DELIV. REV., vol. 65, 2013, pages 1357 - 1369
CHEN ET AL., J. IMMUNOL., vol. 190, no. 3, 2013, pages 1076 - 1084
CHEN ET AL.: "Fusion Protein Linkers: Property, Design and Functionality", ADV. DRUG. DELIV. REV., vol. 65, 2013, pages 1357 - 1369, XP028737352, DOI: 10.1016/j.addr.2012.09.039
CHOI ET AL., METHODS MOL. BIOL., vol. 1529, 2017, pages 375 - 398
CHOPRA ET AL., J. EXP. MED., vol. 213, no. 9, 2016, pages 1881 - 1900
CHOTHIA, C., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CLYNES ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 652 - 656
CORDY ET AL., CLIN. EXP. IMMUNOL., vol. 182, 2015, pages 139 - 148
COX ET AL., EUR. J. IMMUNOL., vol. 24, 1994, pages 827 - 836
DALL'ACQUA ET AL., J. BIOL. CHEM., vol. 281, no. 33, 2006, pages 23514 - 23524
DALL'ACQUA ET AL., J. IMMUNOL., vol. 169, 2002, pages 5171 - 5180
DALL'ACQUA ET AL., J. IMMUNOL., vol. 177, 2006, pages 1129 - 1138
DATTA-MANNAN ET AL., BIOTECHNOL, vol. 14, 2019, pages 1800007
DATTA-MANNAN ET AL., BIOTECHNOL. J., vol. 14, 2019, pages 1800007
DE GROOT, A. S., MOISE, L., CURR. OPIN. DRUGDISCOV. DEVEL., vol. 10, no. 3, 2007, pages 332 - 340
DE GROOT, A. S.MOISE, L., CURR. OPIN. DRUG DISCOV. DEVEL., vol. 10, no. 3, 2007, pages 332 - 340
DINGMAN ET AL., J. PHARM. SCI., vol. 108, no. 5, 2019, pages 1637 - 1654
DONG ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 113, no. 43, 2016, pages 12304 - 12309
DONG ET AL., PROC. NATL. ACAD. SCI. USA, vol. 113, no. 43, 2016, pages 12304 - 12309
DONG YUN ET AL: "Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 113, no. 43, 25 October 2016 (2016-10-25), pages 12304 - 12309, XP055870814, ISSN: 0027-8424, Retrieved from the Internet <URL:https://www.pnas.org/content/pnas/early/2016/10/07/1605195113.full.pdf> DOI: 10.1073/pnas.1605195113 *
DOSIO ET AL., TOXINS, vol. 3, 2010, pages 848 - 883
DUCRY ET AL., BIOCONJUG. CHEM., vol. 21, 2010, pages 5 - 13
EDELMAN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 63, 1969, pages 78 - 85
ENEVER ET AL., PROTEIN ENGINEERING, DESIGN & SELECTION, vol. 28, no. 3, 2015, pages 59 - 66
ENGLEBIENNE, ANALYST, vol. 123, 1998, pages 1599
ESPERITO SANTO ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 464, 2015, pages 1145 - 1150
ESPERITO SANTO ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 464, pages 1145 - 1150
EVAN ET AL., MOLECULAR AND CELLULAR BIOLOGY, vol. 5, 1985, pages 3610 - 3616
FAUSTMAN ET AL., FRONT. IMMUNOL., vol. 4, 2013, pages 478
FIELD ET AL., MOL. CELL. BIOL., vol. 8, 1988, pages 2159 - 2165
FISCHER ET AL., ANTIBODIES, vol. 4, 2015, pages 48 - 70
FISCHER ET AL., PLOS ONE, vol. 6, no. 11, 2011, pages e27621
FISCHER ROMAN ET AL: "Targeting sTNF/TNFR1 Signaling as a New Therapeutic Strategy", ANTIBODIES, vol. 4, no. 1, 6 March 2015 (2015-03-06), pages 48 - 70, XP055870813, DOI: 10.3390/antib4010048 *
FLOSS ET AL., J. TRENDS BIOTECHNOL., vol. 28, no. 1, 2010, pages 37 - 45
GEORGE ET AL.: "An analysis of protein domain linkers: their classification and role in protein folding", PROTEIN ENG, vol. 15, 2002, pages 871 - 879, XP002374925, DOI: 10.1093/protein/15.11.871
GOLD ET AL., PLOS ONE, vol. 5, no. 12, 2010, pages e15004
GOMPELS ET AL., ARTHRITIS RESEARCH & THERAPY, vol. 13, 2011, pages R161
GOODALL ET AL., PLOS ONE, vol. 10, no. 9, 2015, pages e0137065
GRECO ET AL., ARTHRITIS RESEARCH & THERAPY, vol. 17, 2015, pages 93
GRELL ET AL., J. IMMOL., vol. 153, no. 5, 1994, pages 1963 - 72
GRIBSKOV ET AL., NUCL. ACIDS RES., vol. 14, 1986, pages 6745
HAGA ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 105, 2008, pages 7809 - 7814
HAMANO ET AL., EUR. J. IMMUNOL., vol. 41, 2011, pages 2010 - 2020
HAN ET AL., SCI REP, vol. 4, 2014, pages 4387
HINTON ET AL., J. BIOL. CHEM., vol. 279, no. 8, 2004, pages 6213 - 6216
HOLLAND ET AL., J CLIN IMMUNOL, vol. 33, no. 7, 2013, pages 1192 - 203
HOLT ET AL., TRENDS IN BIOTECHNOLOGY, vol. 21, no. 11, 2003, pages 484 - 490
HORIUCHI ET AL., RHEUMATOLOGY (OXFORD, vol. 49, 2010, pages 1215 - 1228
HUANGMILLER, ADV. APPL. MATH., vol. 12, 1991, pages 337 - 357
INOUE MASAKI ET AL: "Structural optimization of a TNFR1-selective antagonistic TNF[alpha] mutant to create new-modality TNF-regulating biologics", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 295, no. 28, 12 May 2020 (2020-05-12), US, pages 9379 - 9391, XP055870808, ISSN: 0021-9258, Retrieved from the Internet <URL:https://www.jbc.org/action/showPdf?pii=S0021-9258(17)48960-5> DOI: 10.1074/jbc.RA120.012723 *
J. BIOL. CHEM., vol. 243, 1968, pages 3557 - 59
JIN ET AL., MOL. MED., vol. 15, no. 1-2, 2009, pages 11 - 20
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, pages: 91 - 3242
KALLIOLIASIVASHKIV, NAT. REV. RHEUMATOL., vol. 12, no. 1, 2016, pages 49 - 62
KEFFER ET AL., EMBO J, vol. 10, no. 13, 1991, pages 4025 - 4031
KEFFER ET AL., EMBO J., vol. 10, no. 13, 1991, pages 4025 - 4031
KING ET AL., TETRAHEDRON LETTERS, vol. 43, 2002, pages 1987 - 1990
KIPRIYANOV: "Methods in Molecular Biology", vol. 207, 2003, article "Recombinant Antibodies for Cancer Therapy Methods and Protocols", pages: 3 - 25
KITAGAKI ET AL., J. ATHEROSCLER. THROMB., vol. 19, no. 1, 2012, pages 36 - 46
KOCZAN ET AL., ARTHRITIS RESEARCH & THERAPY, vol. 10, 2008, pages R50
KONINCKX, HUM REPROD, vol. 23, 2008, pages 2017 - 2023
KUO ET AL., SCI. TRANSL. MED., vol. 11, 2019, pages 491
LEWIS ET AL., BIOCONJ. CHEM., vol. 9, 1998, pages 72 - 86
LI ET AL., EXPERT OPIN THER TARGETS, vol. 18, 2014, pages 335 - 350
LIS ET AL., ARCH MED SCI, vol. 10, no. 6, 2014, pages 1175 - 1185
LOETSCHER, J. BIOL. CHEM., vol. 268, no. 35, 1993, pages 26350 - 26357
LUCCHINO ET AL., RHEUMATOLOGY (OXFORD), vol. 59, no. 6, 2020, pages 1200 - 1203
MACEWAN, BR J PHARMACOL, vol. 135, no. 4, 2002, pages 855 - 875
MALMQVIST, BIOCHEM. SOC. TRANS., vol. 27, 2000, pages 335
MCALPINE ET AL., NEUROBIOL. DIS., vol. 34, no. 1, 2009, pages 163 - 177
MCCANN ET AL., ARTHRITIS & RHEUMATOLOGY, vol. 66, no. 10, 2014, pages 2728 - 2738
MCCOY ET AL., J. NEUROSCI., vol. 26, no. 37, 2006, pages 9365 - 9375
MCDERMOTT ET AL., BMC SYSTEMS BIOLOGY, vol. 10, 2016, pages 93
MORTON ET AL., SCI SIGNAL, vol. 12, no. 592, 2019, pages eaav5637
NANCHAHAL ET AL., EBIOMEDICINE, vol. 33, 2018, pages 282 - 288
NEEDLEMAN ET AL., J. MOL. BIOL., vol. 48, 1970, pages 443
PABORSKY, PROTEIN ENGINEERING, vol. 3, 1990, pages 547 - 553
POTOCNAKOVA ET AL., JOURNAL OF IMMUNOLOGY RESEARCH, 2016
PREVOO ET AL., ARTHRITIS & RHEUMATISM, vol. 38, no. 1, 1995, pages 44 - 48
PROUDFOOT ET AL., THORAX, vol. 73, 2018, pages 723 - 730
RAVETCH ET AL., ANNU. REV. IMMUNOL, vol. 9, 1991, pages 457 - 492
RICHMYSZKA, CURR. OPIN. BIOTECHNOL, vol. 11, 2000, pages 54
RICHTER ET AL., MABS, vol. 11, no. 1, 2019, pages 166 - 177
RICHTER ET AL., MABS, vol. 11, no. 4, 2019, pages 653 - 665
RICHTER ET AL., PLOS ONE, vol. 8, no. 8, 2013, pages e72156
RICHTER, F.: "Thesis", 2015, UNIVERSITAT STUTTGART, article "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB"
RIDGWAY ET AL., PROTEIN ENG, vol. 9, no. 7, 1996, pages 617 - 621
RUSPI ET AL., CELLULAR SIGNALING, vol. 26, 2014, pages 683 - 690
SAMA ET AL., PLOS ONE, vol. 7, no. 5, 2012, pages e38170
SANCHEZ-TRINCADO ET AL., JOURNAL OF IMMUNOLOGY RESEARCH, 2017
SANTI ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 109, 2012, pages 6211 - 6216
SARUP ET AL., MOL. CANCER THER., vol. 7, no. 10, 2008, pages 3223 - 3236
SAUNDERS, K. O., FRONT. IMMUNOL., vol. 10, 2019, pages 1296
SAXENA ET AL., FRONT. IMMUNOL., vol. 7, 2016, pages 580
SCATCHARD ET AL., ANN N.Y. ACAD. SCI., vol. 51, 1949, pages 660
SCHELLENBERGER ET AL., NAT BIOTECHNOL., vol. 27, no. 12, 2009, pages 1186 - 90
SCHMIDT ET AL., ARTHRITIS & RHEUMATISM, vol. 65, no. 9, 2013, pages 2262 - 2273
SCHUBERT ET AL., PLOS COMPUT BIOL, vol. 14, no. 3, 2018, pages e1005983
SHARMA ET AL., FRONT IMMUNOL, vol. 9, 2018, pages 883
SHEN ET AL., AILMENT PHARMACOL. THER., vol. 21, 2005, pages 251 - 258
SHIBATA ET AL., J. BIOL. CHEM., vol. 283, no. 2, 2008, pages 998 - 1007
SOCKOLOSKY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 109, no. 40, 2012, pages 16095 - 16100
SPENCE ET AL., CURR DIAB REP, vol. 16, no. 11, 2016, pages 110
STEELAND ET AL., J. BIOL. CHEM., vol. 290, no. 7, 2015, pages 4022 - 4037
STEELAND ET AL., SCI. REPORTS, vol. 7, 2017, pages 14248
STEELAND SOPHIE ET AL: "TNFR1 inhibition with a Nanobody protects against EAE development in mice", SCIENTIFIC REPORTS, vol. 7, no. 1, 20 October 2017 (2017-10-20), XP055870789, Retrieved from the Internet <URL:http://www.nature.com/articles/s41598-017-13984-y> DOI: 10.1038/s41598-017-13984-y *
STELLA ET AL.: "Directed Drug Delivery", 1985, HUMANA PRESS, article "Prodrugs: A Chemical Approach to Targeted Drug Delivery", pages: 247 - 267
STROHL, BIODRUGS, vol. 29, no. 4, 2015, pages 215 - 239
SUN ET AL., BIOORGANIC & MEDICINAL CHEMISTRY, vol. 11, 2003, pages 1761 - 1768
SUN ET AL., BIOORGANIC &MEDICINAL CHEMISTRY LETTERS, vol. 12, 2002, pages 2213 - 2215
SUN ET AL., COMPUT. MATH METHODM., 2013
SUN ET AL., COMPUT. MATHMETHODM., 2013
SWIERCZEWSKA ET AL., EXPERT OPIN EMERG DRUGS, vol. 20, no. 4, 2015, pages 531 - 536
TAN ET AL., CURRENT PHARMACEUTICAL DESIGN, vol. 24, 2018, pages 4932 - 4946
TANAKA ET AL., AGING CELL, vol. 17, 2018, pages e12799
TOKI ET AL., J. ORG. CHEM., vol. 67, 2002, pages 1866 - 1872
TOMLINSON ET AL., J. MOL. BIOL., vol. 227, 1992, pages 776 - 798
TORREY ET AL., SCI. SIGNAL., vol. 10, 2017, pages eaaf8608
UDALOVA ET AL., MICROBIAL SPECTRUM, vol. 4, no. 4, 2016
V. BERGER ET AL: "An anti-TNFR1 scFv-HSA fusion protein as selective antagonist of TNF action", PROTEIN ENGINEERING, DESIGN AND SELECTION, vol. 26, no. 10, 4 September 2013 (2013-09-04), GB, pages 581 - 587, XP055279872, ISSN: 1741-0126, DOI: 10.1093/protein/gzt044 *
VANAMEE ET AL., TRENDS IN MOLECULAR MEDICINE, vol. 23, no. 11, 2017, pages 1037,1046
VIGNA-PEREZ ET AL., CLIN. EXP. IMMUNOL., vol. 141, no. 2, 2005, pages 372 - 380
WANG ET AL., PROTEIN CELL, vol. 9, no. 1, 2018, pages 63 - 73
WATSON ET AL.: "Molecular Biology of the Gene", 1987, THE BENJAMIN/CUMMINGS PUB. CO., pages: 224
WEERATNA ET AL., IMMUN. INFLAMM. DIS., vol. 4, no. 2, 2016, pages 135 - 147
WELLS ET AL., ANN. RHEUM. DIS., vol. 68, 2009, pages 954 - 960
WILMAN, BIOCHEMICAL SOCIETY TRANSACTIONS, 615TH MEETING BELFAST, vol. 14, 1986, pages 375 - 382
WRIGHT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 106, no. 45, 2009, pages 19078 - 19083
YANG ET AL., FRONT. IMMUNOL., vol. 9, 2018, pages 594
YANG ET AL., ONCOL LETT, vol. 14, no. 2, 2017, pages 2393 - 2398
YANG ET AL., ONCOL LETT, vol. 16, no. 3, 2018, pages 2911 - 291
YANIK ET AL., BIOL. BLOOD MARROW TRANSPLANT, vol. 8, 2002, pages 395 - 400
YARDEN ET AL., NAT. REV. MOL. CELL BIOL., vol. 2, 2001, pages 127 - 137
YEUNG ET AL., J. IMMUNOL., vol. 182, 2009, pages 7663 - 7671
YING ET AL., MABS, vol. 6, no. 5, 2014, pages 1201 - 1210
ZALEVSKY ET AL., J. IMMUNOL., vol. 179, 2007, pages 4239 - 1883
ZALEVSKY ET AL., NAT. BIOTECHNOL., vol. 28, no. 2, 2010, pages 157 - 159
ZHANG ET AL., THORAC CANCER, vol. 10, no. 3, 2019, pages 437 - 444
ZORAN ET AL., SCI. REP., vol. 9, 2019, pages 17231

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2023168426A1 (fr) 2022-03-03 2023-09-07 Enosi Therapeutics Corporation Compositions et cellules contenant des mélanges de protéines de fusion oligo-trap (ofps) et leurs utilisations
WO2024123701A3 (fr) * 2022-12-05 2024-07-11 Fzata, Inc. Traitements de la douleur
WO2025049818A1 (fr) 2023-08-29 2025-03-06 Enosi Therapeutics Corporation Antagonistes tnfr1 dépourvus d'activité agoniste et leurs utilisations

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