CA3193273A1 - Methods and compositions to treat autoimmune diseases and cancer - Google Patents

Methods and compositions to treat autoimmune diseases and cancer

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Publication number
CA3193273A1
CA3193273A1 CA3193273A CA3193273A CA3193273A1 CA 3193273 A1 CA3193273 A1 CA 3193273A1 CA 3193273 A CA3193273 A CA 3193273A CA 3193273 A CA3193273 A CA 3193273A CA 3193273 A1 CA3193273 A1 CA 3193273A1
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construct
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tnfr2
tnfr1
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Harold Michael Shepard
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Enosi Therapeutics Corp
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Enosi Therapeutics Corp
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Abstract

Provided are molecular constructs that target tumor necrosis factor receptor 1 (TNFR1) and/or tumor necrosis factor receptor 2 (TNFR2). 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 thereof can ameliorate the disease, disorder, and condition or a symptom thereof. Among the constructs provided herein, are TNFR1 antagonist constructs that are engineered to inhibit TNFR1 function, and to eliminate any TNFR1 agonist activity. The constructs provided herein include agonists and antagonists of TNFR1 and TNFR2. TNFR1 antagonist constructs are engineered to inhibit TNFR1 function, and in some embodiments, are engineered to avoid agonist activity. Included also are agonists and antagonists of TNFR2. 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. Methods of treatment of the various diseases in which TNF and its receptors play a role also are provided.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

METHODS AND COMPOSITIONS TO TREAT AUTOIMMUNE DISEASES
AND CANCER
RELATED APPLICATIONS
Benefit of priority is claimed to U.S. provisional application Serial No.
63/071,313, filed August 27, 2020, entitled "METHODS AND COMPOSITIONS TO
TREAT AUTOIIVIMUNE DISEASES AND CANCER" to inventor H. Michael Shepard, and Applicant Enosi Life Sciences Corp.
This application is related to International PCT application No.
PCT/US2020/018739, filed February 19, 2020, published on August 27, 2020, as International PCT Publication No. WO 2020/172218, to inventor H. Michael Shepard, and Applicant Enosi Life Sciences Corp., entitled "ANTIBODIES AND
ENONOMERS." This application also is related to the U.S. Application. Serial No.
17/432,720, filed August 20, 2021, which is the US national stage application of PCT/US2020/018739, filed February 19, 2020, which claims benefit of provisional application serial no. 62/808,635, filed February 21, 2019.
Where permitted, the subject matter of each of these applications is incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED
ELECTRONICALLY
An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on August 26, 2021, is 1.629 megabytes in size, and is titled 5301SEQPC1.txt.
FIELD
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 BACKGROUND
Anti-TNF therapies/TNF-blockers (a type of biological Disease Modifying Anti-Rheumatic Drugs; DMARDs) typically are prescribed after the failure of conventional DMARDs. These therapies include monoclonal antibodies (mAbs), such as the chimeric mAb infliximab (Remicadec)); containing a murine variable region and a human IgG1 constant region, and the fully humanized mAbs (IgGls)
-2-adalimumab (sold, for example under the trademark Humirag), 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 Enbrer), which contains the extracellular receptor region that contains the binding site of human TNFR2 fused to the Fc of human IgG1 . 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 (MD; such as, Crohn's disease and ulcerative colitis).
Such therapies, however, 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 ofM 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. Other anti-rheumatic drugs, such as methotrexate and glucocorticoids, also can induce apoptosis in immune cells (see, e.g., Vigna-Perez et al. (2005) Cl/n. Exp.
Immunol. 141(2):372-380). These therapeutic 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 (DMARDs) [Updated 2020 Feb 27]. In: StatPearls [Internet]. Treasure Island (FL):
StatPearls Publishing; 2020 Jan. Available from:
(ncbi.nlm.nih.gov/books/NBK507863/). 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. 113(43):12304-12309; Zalevsky et al.
(2007)1
3 Immunol. 179:1872-1883; Zoran et at. (2019) Sci. Rep. 9:17231). Thus, the uses of these therapeutic agents, particularly for chronic diseases/conditions that require long-term administration, such as arthritis and inflammatory bowel disease (MD, are 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 at.
(2014) Arthritis & Rheumatology 66(10):2728-2738). Non-responsiveness also occurs in non-RA patients receiving anti-TNF therapeutics. Depending on the anti-TNF
agent, 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 at.
(2019) MABS 11(4):653-665). Thus, there is a need for therapies with improved therapeutic efficacy and safety.
SUMMARY
Provided are molecular constructs, and nucleic acids encoding them, that target tumor necrosis factor receptor 1 (TNFR1) and/or tumor necrosis factor receptor 2 (TNFR2). 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. Also included are agonists and antagonists of TNFR2. 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 Cells have two 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. Existing TNF
blockers limit their own efficacy because the Treg generator (TNFR2), which is anti-inflammatory, is turned down/off.
-4-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. Among 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. These constructs can be administered to treat indications where TNF blockers have failed. Among the constructs provided herein are 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.
Existing anti-TNF drugs block overzealous inflammation, which occurs in 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 Enbrer), 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.
As established in mouse models, 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. In some embodiments, the Kd is < 1 nM, and the t1/2 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,
-5-intravenously, intratumorally, intra-hepatically, topically, mucosally, intradermally, and any other suitable route.
Among the constructs provided herein are the following. Provided are constructs that are a tumor necrosis factor receptor 1 (TNFR1) antagonist construct of formula 1: (TNFR1 inhibitor)n¨linkerp¨ (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 chemical and/or physical bonds. The constructs can be fusion proteins.
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. For example, the activity modifier (or property modifier) is a modified Fc region or is human serum albumin. In the constructs, the TNFR1 inhibitor can be one that inhibits TNFR1 signaling, and/or the activity modifier increases serum half-life of the construct. For example, 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.
Also provided are multi-specific constructs. For example, provided are 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
-6-on a receptor. Among the 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. Provided are constructs that have Formula 2: (TNFR1 inhibitor).¨ (activity modifier)," ¨ (Linker (L))p ¨
(activity modifier),2_ (TNFR2 agonist)q, or (TNFR1 inhibitor).¨ (activity modifier)," ¨
(Linker (L))p ¨ (activity modifier)r2 _ (Treg expander)q, where: n= 1, 2, or 3, p= 1, 2, or 3, q= 0, 1 or 2, and each of rl and r2 is independently 0, 1, or 2; and the components can be in the order specified or any other order as long as the construct interacts with TNFR1 and TNFR2 to antagonize TNFR1 and agonize TNFR2, or has Treg expander activity. For example, included are constructs, among any of those provided herein, where the TNFR1 inhibitor moiety inhibits binding of TNFa binding to TNFR1 and/or inhibits signaling.
Also provided are constructs of formula 3a or 3b: (TNFR2 agonist or Treg expander).¨ linkerp¨ (activity modifier)., formula 3a, or (activity modifier)q¨ linkerp ¨(TNFR2 agonist or Treg expander)., 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.
In some embodiments, 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. Exemplary of linkers are those that increase serum half-life of the construct.
For example, the linker can have a sequence set forth in any of SEQ ID NOs:

or is a PEG moiety linker. In some embodiments, 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
-7-reduced ADCC and/or CDC activity, such as an Fc modified to have reduced or no ADCC activity.
Included among the constructs provided herein are those in which 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; and the activity modifier is any set forth in the sequence listing, known in the art, and/or set forth below.
Provided are 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. In such constructs that antibody or antigen-binding portion thereof, comprises a modification that improves a pharmacological property and/or structure of the construct.
In any of the constructs provided herein, 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. In the constructs provided herein, 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 TNFa binding to TNFR1. Among the constructs are those where the TNFR1 inhibitor is an antibody or antigen binding portion that does not inhibit binding of TNFa 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.
For example the knob mutation(s) is/are selected from among S354C, T366Y, T366W, and T394W by EU numbering; and 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. In some embodiments where the
-8-construct comprises an Fe, the Fe is from trastuzumab. The construct can be dimerized by fusion of the N-terminus with the C-terminus of trastuzumab.
In some embodiments in which the constructs comprise a linker the linker is or comprises a hinge region from an Fe region. For example, in the hinge region is from .. trastuzumab, and it is linked to the Fe region. The constructs include those that comprise a linker that is linked to the anti-TNFR1 antagonist antibody or antigen-binding portion thereof The linker can be linked to the anti-TNFR1 antagonist antibody or antigen-binding portion thereof, and directly or via a hinge region to an Fe region. The Fe region or modified Fe 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.
Also provided are constructs that binds to neonatal Fe receptor (FcRn). For example, provided are 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. For example, the FcRnBP contains 12-20 residues or 15 residues or residues. Exemplary of these are 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 Fe heterodimer, where one Fe monomer comprises holes, and the other comprises knobs, whereby the Fe .. dimer that results is a heterodimer.
Provided are constructs that are TNFR1 antagonist constructs that comprise: a TNFR1 inhibitor; an Fe dimer; and a Treg expander, where: the Fe dimer comprises two complementary Fe monomers; the TNFR1 inhibitor is linked to one of the Fe monomer, and the Treg expander is linked to the other Fe monomer. In such constructs the Treg expander can be a TNFR2 agonist. The can further comprise a second Treg expander linked to the same Fe monomer as the TNFR1 inhibitor, where the first and second Treg expanders are the same or different. The second Treg expander can be a TNFR2 agonist. In some embodiments, the Treg expanders are the same. The TNFR1 inhibitor can be one that inhibits or blocks TNFR1 signaling.
In .. some embodiments, the TNFR1 inhibitor binds to TNFR1 and blocks or inhibits TNFa binding and TNFR1 signaling. In some embodiments, the TNFR1 inhibitor binds to TNFR1, does not or interfere with TNFa binding, and blocks or inhibits
-9-TNFR1 signaling. In some embodiments of these constructs, wherein 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, VI-11-1 single domain antibody, or Fab of aTNFR2 agonist monoclonal antibody. In these constructs, the Treg expander can be a TNFR2 agonist that is a small molecule, or a nucleic acid aptamer, or a peptide aptamer.
Also provided are any of these constructs that is or also is a TNFR2 agonist.
The TNFR2 agonist is a construct of formula 3a or 3b, where: formula 3a is (Treg expander).¨ linkerp¨ (activity modifier)., and formula 3b is (activity modifier).-linkerp¨ (Treg expander).. In these formulae, 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. In any of these constructs, the Treg expander in the construct is a TNFR2 agonist. For example, the TNFR2 agonist stimulates or induces TNFR2 signaling. In other examples, the Treg expander is a TNFR2 agonist that is an scFv, VI-11-1 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. In the constructs that comprise all or a portion of trastuzumab, such as the Fc portion and/or Fc and hinge region or modified forms thereof, the construct can be dimerized by N-terminal fusion with the C-terminus of trastuzumab.
Provided are constructs that comprise a TNFR1 inhibitor moiety linked via a central PEG linker to one more Treg expanders, or that comprise at least two 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. Exemplary are those that have a structure selected from among formulae 4A to 4D:
-10-Formula 4A:
(CH2CH20)-0 1 n _____ I

I
CH 0-(OCH2CH2)170-CH21¨CH2 0-( _ 2CH 0 I

, I
CI-I2CH20 1),0 ,or (CH2CH20)-.
1 n _____ I

I , 0-(OCH2CH2)-0-CH2-C¨R' n I

I

, I

2 2 gior n is 1 to 5;
le is H or CH3, or CH2CH3 or other Cl-05 alkyl 0 is aTNFR1 inhibitor (TNFR1 antagonist);
0 is a Treg expander; or Formula 4B:
H
0-(CH2C1-120)n ......-)f-N 0 0 is a TNFR1 inhibitor (TNFR1 antagonist) ..:.:...
..... ' is a Treg expander;
n is 1 to 5; or Formula 4C:
-11-H
All All /NN
IMP N¨(CH2CH20), IMP

All mom IP. is a TNFR1 inhibitor (TNFR1 antagonist), or a Treg expander; and n is 1 to 5; or Formula 4D:
activity modifier et--(CH2CH20), or =
el¨(C H2CH20)e) wherein each te 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.
In TNFR1 antagonist constructs and other constructs provided herein, 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 Fe region, or is an Fc region that includes a hinge region or other linker; and the Fe region or Fe region with hinge region is an Fe that is modified to reduce or eliminate ADCC and/or CDC
activity.
Exemplary thereof are constructs where the Fe or modified Fe is an IgG Fe or is an IgG1 or IgG4 Fe, and/or are constructs that bind to neonatal Fe receptor (FcRn).
Exemplary of these constructs are those, where: 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.
Also provided are TNFR1 antagonist constructs of any of the formulae above and in the application that comprise: a) a TNFR1 inhibitor moiety that is a RECTIFIED SHEET (RULE 91) ISA/EP
-12-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). In such constructs, the TNFR1-selective antagonist selectively binds and inhibits signaling, but not TNFR2 signaling. As described for the constructs above, the TNFR1 inhibitor, linkers, and other components can be those as described above.
These include constructs where the TNFR1 inhibitor that is a selective antagonist comprises an antigen-binding fragment that selectively binds and inhibits signaling but not TNFR2 signaling. For example, 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. In any of the constructs described herein, the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody. For example, 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. Exemplary of 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 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.
Among the 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:
V1M, L295, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A845, V85T, 586T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L295/R32W,
-13-L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF, set forth in SEQ ID NO:2. For example, the TNFR1 inhibitor is a TNF
mutein that comprises the sequence of residues set forth in any one of SEQ ID NOs: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. For example, 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. Exemplary GS linkers for any of the constructs provided herein include those selected from among (GlySer),, where n= 1-10; (GlySer2); (Gly4Ser),, where n=
1-10;
(Gly3Ser),, where n= 1-5; (SerGly4),, where n= 1-5; (GlySerSerGly),, where n=
1-5;
GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS. Also included are 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.
-14-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 IgG1 or IgG4 Fc. The IgG1 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. For example, the IgG1 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 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 (Gly4Ser)3, and/or linkers that comprise (Gly4Ser)3 and SCDKTH (residues 217-222 of SEQ ID NO:31); and/or linkers that comprise (Gly4Ser)3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26; and/or those that comprise (Gly4Ser)3 and the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29. Exemplary of 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.
Provided herein are TNFR1 antagonist constructs. These 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.
-15-Other exemplary TNFR1-inhibiting constructs provided herein 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. These constructs include those that are a TNFR1 antagonist, 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 GS linker selected from among (GlySer),, where n= 1-10; (GlySer2); (Gly4Ser),, where n= 1-10; (Gly3Ser),, where n=
1-5; (SerGly4),, where n= 1-5; (GlySerSerGly),, where n= 1-5; GSGGSSGG;
GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG;
GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) a half-life extending moiety that is an IgG Fc. In these constructs, or any provided herein that include one or more of these components, the GS linker can be (GGGGS)3; and the IgG Fc can be the Fc of trastuzumab or the Fc of nivolumab.
Others of the constructs provided herein that are TNFR1 antagonist constructs include 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. In such constructs, 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.
Provided are any of the constructs provided herein that is a TNFR1 antagonist construct, 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;
-16-b) a first linker that is a GS linker selected from among (GlySer),, where n=

10; (GlySer2); (Gly4Ser),, where n= 1-10; (Gly3Ser),, where n= 1-5;
(SerGly4),, where n= 1-5; (GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS;
c) a second 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 d) a half-life extending moiety that is an IgG Fc.
In some embodiments, 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. In other embodiments, 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.
Provided are the constructs that are TNFR1 agonists that comprise:
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 GS linker selected from among (GlySer),, where n= 1-10; (GlySer2);
(Gly4Ser),, where n= 1-10; (Gly3Ser),, where n= 1-5; (SerGly4),, where n= 1-5;

(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) a half-life extending moiety that is a PEG molecule. 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..
Provided are the constructs that are TNFR1 agonist 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
-17-nanobody of SEQ ID NO: 683 or 684, or the TNF mutein of any of SEQ ID NOs:685-703;
b) a GS linker selected from among (GlySer)n, where n= 1-10; (GlySer2);
(Gly4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5;
(GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) a half-life extending moiety that is human serum albumin. Exemplary of the linkers are any described herein, such as where the GS linker is (GGGGS)3.
The primary amino acid sequence of any of the constructs provided herein (those described above, and below) can be optimized or modified to eliminate immunogenic sequences or immunogenic epitopes. For example, in constructs that contain an IgG Fc, 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. In such constructs and in any that contain an IgG Fc the knob mutation can be selected from among S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T3665, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering. These 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/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259IN308F, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, by EU numbering. 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).
RECTIFIED SHEET (RULE 91) ISA/EP

For example, in these TNFR1 antagonist constructs, 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, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, .. 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, 5228P/L235E, and 5228P/F234A/L235A, by EU numbering.
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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T3665, 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, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance one or more immune effector functions, wherein:
the immune effector function(s) is/are selected from among one or more of CDC, ADCC and ADCP; and the modification(s) to increase or enhance an immune effector function is/are selected from among one or more of:

in IgG1 : S239D, 1332E, S239D/I332E, S239D/A330L/1332E, S298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/S298A in the first heavy chain and S239D/A330L/1332E in the second heavy chain;
L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain;
A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A;
T256A/K290A/S298A/E333A/K334A; G23 6A; G236A/I332E;
G236A/S239D/I332E; G236A/S239D/A330L/1332E; 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.
In some embodiments, of any of the constructs that comprises an Fc region, the construct can comprise an IgG1 Fc that comprises one or more modifications to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb. For example, the .. modification or modifications that increase binding to FcyRIIb 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.
Also provided are 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. In some embodiments, the TNFR2 agonist is a TNFR2 selective agonist.

Provided are 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. In TNFR2 agonist constructs, 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 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.
Provided are 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. Exemplary of such antibodies are agonist anti-TNFR2 antibody is selected from 1VIR2-1 (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. In some embodiments, the TNFR2 agonist is a TNFR2-selective agonist. The selective agonist can comprise a TNFR2 agonist TNF mutein. Exemplary 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/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, and combinations of any of the preceding, all with reference to SEQ ID NO:2. For example a TNFR2 agonist is a TNF mutein comprising the mutations D143N/A145R.
In the TNFR2 agonist constructs, 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. The linker can be a glycine-serine (GS) linker, such as, but not limited to, a GS linker selected from among (GlySer),, where n= 1-10; (GlySer2); (Gly4Ser),, where n= 1-10;
(Gly3Ser),, where n= 1-5; (SerGly4),, where n= 1-5; (GlySerSerGly),, where n=
1-5;
GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS. 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 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. For example, a half-life extending moiety is an activity or property modifier. Exemplary of such as discussed above and also below, are IgG Fc, a polyethylene glycol (PEG) molecule, and human serum albumin (HSA), or portions or derivative of variants thereof For example, in some the IgG Fc is an IgG1 or IgG4 Fc. Exemplary of the IgG1 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 IgG1 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.

In some embodiments of the TNFR2 agonist constructs, the TNFR2 agonist is monovalent; in others it is multivalent, such as bivalent or trivalent. The constructs can contain linkers as described herein. For example, the linker can comprise Gly-Ser, such as (Gly4Ser)3, or (Gly4Ser)3 and SCDKTH (residues 217-of SEQ ID NO:31), or (Gly4Ser)3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26, or (Gly4Ser)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.
Also provided are TNFR2 agonist constructs that 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 GS linker selected from among (GlySer)n, where n= 1-10; (GlySer2);
(Gly4Ser)n, where n= 1-10; (Gly3Ser)n, where n= 1-5; (SerGly4)n, where n= 1-5;

(GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) an activity modifier that is a half-life extending moiety that is an IgG
Fc.
As above, in exemplary embodiments, the GS linker can be (GGGGS)3; and the IgG

Fc is the Fc of trastuzumab or the Fc of nivolumab.
Other 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;
RECTIFIED SHEET (RULE 91) ISA/EP

and the IgG Fe is the Fe of trastuzumab, or all or a portion of the hinge sequence of nivolumab; and the IgG Fe is the Fe of nivolumab.
In other embodiments, the TNFR2 construct, comprises:
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 first linker that is a GS linker selected from among (GlySer)n, where n=

10; (GlySer2); (Gly4Ser)n, where n= 1-10; (Gly3Ser)n, where n= 1-5;
(SerGly4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS;
c) a second 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 d) an activity modifier that is a half-life extending moiety that is an IgG
Fe.
Exemplary of such constructs are those in which the first GS linker is (GGGGS)3, and the second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID
NO:31); and the IgG Fe is the Fe of trastuzumab. In other embodiments, the first linker is (GGGGS)3, the second linker comprises all or a portion of the hinge sequence of nivolumab; and the IgG Fc is the Fe of nivolumab.
In some embodiments, the construct is a TNFR2 agonist construct, comprising:
a) the TNFR2 agonist that comprises an antigen-binding fragment of an agonist human anti-TNFR2 antibody selected from MR2-1 or MAB2261;
b) a linker comprising:
i) a GS linker selected from among (GlySer)n, where n= 1-10;
(GlySer2); (Gly4Ser)n, where n= 1-10; (Gly3Ser)n, where n= 1-5; (SerGly4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5; GSGGSSGG;
GSSSGSGSGSSG; GSSSGSGSGSSGG; 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 IgG1 or IgG4 Fc, a PEG molecule, and human serum albumin (HSA), wherein:
the IgG1 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 30 kDa.
In some embodiments, 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/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2;
b) a linker comprising:
i) a GS linker selected from among (GlySer)n, where n= 1-10;
(GlySer2); (Gly4Ser)n, where n= 1-10; (Gly3Ser)n, where n= 1-5; (SerGly4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5; GSGGSSGG;
GSSSGSGSGSSG; GSSSGSGSGSSGG; 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 IgG1 or IgG4 Fc, a PEG molecule, and human serum albumin (HSA), wherein:
the IgG1 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 30 kDa.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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 nivolumab, 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.
Provided are 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 Dom lh-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 DOM1h-131-206 of SEQ ID NO:59 and variants thereof that have at least 95%, 96%, 97%, 98%, 99% sequence identity thereto, and have TNFR1 antagonist activity; or e) combinations of any of a)-d); or f) humanized sequences of any of a)-f) or where a sufficient portion of the construct for administration to a human is humanized, where a sufficient portion is sufficient to eliminate or reduce any immune response to the construct when administered to a human..
The constructs provided herein that are TNFR1 constructs can further comprise a TNFR2 agonist or the construct can be a TNFR2 agonist construct. In the constructs that comprise a TNFR2 agonist, 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. In the constructs that contain TNFR2 agonist, it can be a TNFR2-selective agonist. These constructs can comprise a modified IgG Fc. For example, 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). Exemplary of such modifications are:
a) 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;
b) 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, V2591/V308F, V2591/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, by EU numbering; and c) 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, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G23 7A, 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.
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:
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, V2591/V308F, V2591/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance 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 increase or enhance immune effector functions is selected from among one or more of:
in IgGl: S239D, 1332E, S239D/I332E, S239D/A330L/1332E, S298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/S298A in the first heavy chain and S239D/A330L/1332E in the second heavy chain;

L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A;
T256A/K290A/S298A/E333A/K334A; G23 6A; G236A/I332E;
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.
The constructs provided herein that are TNFR2 agonist construct can comprise a modified IgG1 Fc, such as where the Fc is modified to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb, which can include modifications that increase binding to FcyRIIb. Exemplary of such 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.
Provided are constructs that are or comprise a TNFR2 agonist construct hat selectively activates or agonizesTNFR2, without activating or antagonizing TNFR1.
These 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:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III);
MD is a multimerization domain and each is/are the same or different;
TNFmut is a TNFR2-selective TNF mutein; and 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/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, with reference to SEQ ID NO:2, such as, for example, the TNFR2-selective mutations D143N/A145R. In these constructs, 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. For example, the multimerization domain is an IgG1 Fc or an IgG4 Fc and the IgG1 Fc or IgG4 Fc also is a half-life extending moiety.
These constructs contain linkers, including any described herein and any known to those of skill in the art. Exemplary of these constructs are those where the Li, L2 and/or L3 linkers are independently selected from among (GGGGS),, where n = 1-5, and all or a portion of the stalk region of TNF (SEQ ID NO:812) or a variant thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto. These constructs include those where the linker between the TNFR2 agonist and the half-life extending moiety is: a GS linker selected from among (GlySer)., where n= 1-10; (GlySer2);
(Gly4Ser)., where n= 1-10; (Gly3Ser)., where n= 1-5; (SerGly4)., where n= 1-5;
(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or 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; or a combination thereof. The half-life extending moiety can be selected from among: an IgG1 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.
Provided are constructs that are or comprise a TNFR2 agonist construct. These constructs include those that comprise the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III), where:
a) MD is a multimerization domain; TNFmut is a TNFR2-selective TNF
mutein; and Ll, 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: 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);
ii) Li, L2 and L3 each are (GGGGS)n, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a mixture thereof;
and iii) the TNF muteins comprise the TNFR2-selective mutations D143N/A145R;
b) a half-life extending moiety selected from among:
an IgG1 Fe that is the Fe of human IgGl, set forth in SEQ ID NO:10, or the Fe of trastuzumab, set forth in SEQ ID NO:27;
an IgG4 Fe that is the Fe of human IgG4 set forth in SEQ ID NO:16, or the Fe of nivolumab, set forth in SEQ ID NO:30;
a PEG molecule that is at least or at least about 30 kDa in size; and human serum albumin (HSA); and c) a linker between the TNFR2-selective agonist and the half-life extending moiety, wherein the linker comprises:
a GS linker selected from among (GlySer)n, where n= 1-10; (GlySer2);
(G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5;

(GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; or RECTIFIED SHEET (RULE 91) ISA/EP

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; or a combination thereof The constructs include TNFR2 agonist constructs, comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III), wherein 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:
i) the MD is selected from an IgG1 Fc or an IgG4 Fc;
ii) L2 and L3 in Formula II, and Li and L2 in Formula III each independently is (GGGGS)., where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a combination thereof;
iii) each of Li in Formula II and L3 in Formula III is independently selected from among:
a GS linker selected from among (GlySer),, where n= 1-10;
(GlySer2); (Gly4Ser),, where n= 1-10; (Gly3Ser),, where n= 1-5;
(SerGly4),, where n= 1-5; (GlySerSerGly),, where n= 1-5;
GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; or 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; or a combination thereof; and iv) the TNF muteins comprise the TNFR2-selective mutations D143N/A145R. In these constructs the MD can be selected from:
an IgG1 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 IgG1 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 IgG1 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 IgG1 Fc of trastuzumab, where the linker between the MD and the adjacent TNF mutein comprises (Gly4Ser)3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26. In some embodiments, the MD
is the IgG1 Fc of trastuzumab, and the linker between the MD and the adjacent TNF
mutein comprises (Gly4Ser)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 (Gly4Ser)3 and all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
The constructs herein, including the agonists constructs, can be modified to eliminate immunogenic sequences, such as those immunogenic to humans. Provided .. herein are 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.
In constructs provided herein that are TNFR2 agonist constructs and that comprises a modified IgG Fc, 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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T3665, 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, 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, V2591/V308F/M428L, E294del/T307P/N434Y, and 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 IgG1 : L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, 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.
Provided are 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, 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, V2591/V308F, V2591/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance 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 increase or enhance immune effector functions is selected from among one or more of:
in IgG1 : S239D, 1332E, S239D/I332E, S239D/A330L/1332E, S298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/S298A in the first heavy chain and S239D/A330L/1332E in the second heavy chain;
L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A;
T256A/K290A/S298A/E333A/K334A; G23 6A; G236A/I332E;
G236A/S239D/I332E; G236A/S239D/A330L/1332E; 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.
Provided are any of the foregoing TNFR2 agonist constructs of any of claims that comprise an IgG1 Fc that is modified to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb. Exemplary of such are those where the modifications that increase binding to FcyRIIb 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.
The 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:
(TNFR1 inhibitor), ¨ Linker (L)p ¨ (TNFR2 agonist)q (Formula I), or (TNFR1 inhibitor), ¨ Linker (L)p ¨(TNFR2 agonist)q, or (TNFR1 inhibitor), ¨ (TNFR2 agonist)q¨ Linker (L)p, or (TNFR2 agonist)q¨ (TNFR1 inhibitor), ¨ Linker (L)p, or any of the above, comprising an optional activity modifier, where: n= 1 or 2, p= 1, 2, or 3, and q= 1 or 2; the TNFR1 inhibitor interacts with TNFR1 to inhibit its activity;
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; and the linker, for example, increases solubility of the construct, or increases flexibility, or alters steric effects of the construct. These 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. Exemplary of such constructs are those of a)-c) as follows:
a) the TNFR1 inhibitor is selected from among:
i) an antigen-binding fragment of a human anti-TNFR1 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 NOs: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
mutein comprising a soluble TNF molecule, with one or more amino acid replacements that confer selective inhibition of TNFR1 and are selected from among:
V1M, L295, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A845, V85T, 586T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L295/R32W, L295/586T, R32W/586T, L295/R32W/586T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF, set forth in SEQ ID NO:2;
b) the linker is selected from:
i) a GS linker selected from (GlySer)n, where n= 1-10; (GlySer2);
(Gly4Ser)n, where n= 1-10; (Gly3Ser)n, where n= 1-5; (SerGly4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG;
GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS;
and/or ii) all or a portion of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26, or all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29;
and iii) an IgG1 or IgG4 Fc, wherein:
the IgG1 Fc is selected from the IgG1 Fc of human IgGl, set forth in SEQ ID NO:10, or the IgG1 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 Fe includes one or more modifications to introduce knobs-into-holes, and/or increase or enhance neonatal Fe receptor (FcRn) recycling, and/or reduce or eliminate immune effector functions; and c) the TNFR2 agonist is selected from:
i) an antigen-binding fragment 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; or ii) an antigen-binding fragment of an agonistic human anti-TNFR2 antibody selected from 1VIR2-1 or MAB2261; or iii) 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/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, with reference to SEQ ID NO:2; or iv) a single-chain TNFR2-selective TNF mutein trimer, comprising the mutations D143N/A145R, wherein the TNF muteins are linked by (GGGGS)n, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID
NO:812); or v) a TNFR2-selective agonist comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-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);
Li, L2 and L3 each are (GGGGS),, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a mixture thereof; and the TNF muteins comprise the TNFR2-selective mutations D143N/A145R.
Other such constructs include those that are multi-specific TNFR1 antagonist/TNFR2 agonist constructs, where:
a) 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 NOs: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-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2.
Other such multi-specific constructs are those where:
a) 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 NOs: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, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2.
Other such multi-specific constructs are those where:
a) 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 NOs: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, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, with reference to SEQ ID
NO:2.
Other such multi-specific constructs are those where:
a) 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 NOs: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, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, and any combination of the preceding mutations, with reference to SEQ ID NO:2.
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.
Exemplary of the Fc that comprise knobs-into-holes modifications are:
the knob mutation is selected from among one or more of 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T3665, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering.

Other examples are multi-specific constructs that comprise an Fc, such as where the 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, V2591/V308F, V2591/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, by EU numbering. 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 IgG1 and/or IgG4:
in IgGl: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A, by EU
numbering; and/or in IgG4: L235E, F234A/L235A, 5228P/L235E, and 5228P/F234A/L235A, by EU numbering.
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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T3665, 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, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V2591/V308F, V2591/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance 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 increase or enhance immune effector functions is selected from among one or more of:
in IgG1 : S239D, 1332E, S239D/I332E, S239D/A330L/1332E, S298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/S298A in the first heavy chain and S239D/A330L/1332E in the second heavy chain;
L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A;
T256A/K290A/S298A/E333A/K334A; G23 6A; G236A/I332E;
G236A/S239D/I332E; G236A/S239D/A330L/1332E; 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.
Other of such multi-specific constructs are those where: the construct that comprises an IgG1 Fc that is modified to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb. Exemplary of such are those where the modifications that increase binding to FcyRIIb 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.

Also provided are constructs that are a multi-specific TNFR1 antagonist/TNFR2 agonist, the TNFR1 antagonist is monovalent; and the TNFR2 agonist is monovalent. Also provided are 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.
In some embodiments, 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-TNFR1 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 NOs: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 TNFR1 and are selected from among:
V1M, L295, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A845, V85T, 586T, Y87H, Q88N, T89Q, I97T, C101A, Al 45R, El 46R, L295/R32W, L295/586T, R32W/586T, L295/R32W/586T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A845/V85T/586T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF, set forth in SEQ ID NO:2;
b) the linker is a branched chain PEG molecule that is at least or at least about kDa in size; and c) the TNFR2 agonist is selected from:
30 i) an antigen-binding fragment 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; or ii) an antigen-binding fragment of an agonistic human anti-TNFR2 antibody selected from 1VIR2-1 or MAB2261; or iii) 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/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, with reference to SEQ ID NO:2; or iv) a single-chain TNFR2-selective TNF mutein trimer, comprising the mutations D143N/A145R, wherein the TNF muteins are linked by (GGGGS)n, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID
NO:812); or v) a TNFR2-selective agonist comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-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);
Li, L2 and L3 each are (GGGGS)n, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a mixture thereof; and the TNF muteins comprise the TNFR2-selective mutations D143N/A145R.
Also provided are multi-specific constructs where 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. Provided are 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. Uses of multi-specific 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.
Also provided are 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 signaling in its etiology. Exemplary of 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. These include a diseases, disorders, and conditions selected from: rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), spondyloarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (II3D), uveitis, fibrotic diseases, endometriosis, lupus, multiple sclerosis (MS), congestive heart failure, cardiovascular disease, myocardial infarction (MI), atherosclerosis, metabolic diseases, cytokine release syndrome, septic shock, sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), SARS-CoV-2, influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, Alzheimer's disease, Parkinson's disease, Behcet's disease, Dupuytren's disease, Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS), pancreatitis, type I diabetes, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, graft rejection, graft versus host disease (GvHD), lung inflammation, pulmonary diseases and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, genetically inherited diseases with TNF/TNFR1 as the causative pathologic mediator, periodic fever syndrome, or cancer. In particular, constructs provided herein, such as, but not limited to, the TNFR1 antagonist constructs, can be used in uses, methods of treatment, and compositions for the treatment of rheumatoid arthritis.
Also provided herein are 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).¨linkerp¨ (activity modifier)q, or linkup¨ (activity modifier)q_(TNFR2 antagonist)., 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 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; and 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.
In these constructs, 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. 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 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. For example, 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). In some embodiments of the TNFR2 antagonist constructs, 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. 2019/0144556 for descriptions of these antibodies);
antibodies and antibody fragments and single chain forms that contain the CDR-H3 sequence of TNFRAB1 (QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO:1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID
RECTIFIED SHEET (RULE 91) ISA/EP

NO:1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO:1223), or a CDR-H3 sequence with at least about 85% sequence identity thereto. 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.
In some embodiments, 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).
In some embodiments, 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 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-L2 sequence of TNFRAB1, corresponding to residues 49-55 of SEQ ID NO:1213; and/or the CDR-L3 sequences set forth in any of SEQ ID NOs: 1221, 1222, and 1241-1244, or the CDR-L3 sequence of TNFRAB1, corresponding to residues 88-96 of SEQ ID NO:1213; and/or CDR-H1 and CDR-H2 sequences of the consensus sequence of a human antibody heavy chain variable domain of SEQ ID NO:1245 replaced with the corresponding CDR sequences of a phenotype-neutral, TNFR2-specific antibody, and/or the CDR-L1, CDR-L2 and CDR-L3 sequences of the sequence of a human antibody light chain variable domain of SEQ ID NO:1246 replaced with the corresponding CDR sequences of a phenotype-neutral, TNFR2-specific antibody, to produce humanized, antagonistic TNFR2 antibodies. For example, the construct comprises a TNFR2 antagonist specifically binds to an epitopes within TNFR2 set forth in any one of SEQ ID NOs:1247-1464. In some embodiments, the TNFR2 antagonist specifically binds to an epitope(s) selected from among:
(a) one or more 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, and do not bind to the epitope containing residues KCSPG corresponding to residues 56-60 of SEQ ID NO:4;
and/or (b) one or more 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.
In some embodiments, the TNFR2 antagonist construct comprises a TNFR2 antagonist that is a small molecule. For example, the TNFR2 antagonist is thalidomide or an analog thereof, such as lenalidomide and pomalidomide.
In some embodiments, the TNFR2 antagonist construct comprises a TNFR2 antagonist that that reduces FoxP3 expression and inhibits the suppressive activity of Tregs. Exemplary of such antagonists is a hi stone 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.
The claims set forth below and in the priority application, and subject matter thereof are incorporated by reference into this Summary.
BRIEF DESCRIPTION OF THE DRAWINGS
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.
Figures 3A-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 3A 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. In embodiments provided herein, 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. Again 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.
.. DETAILED DESCRIPTION
Outline A. DEFINITIONS
B. OVERVIEW OF CONSTRUCTS AND METHODS
C. TUMOR NECROSIS FACTOR (TNF) AND CHRONIC INFLAMMATORY
AND AUTOIMMUNE DISEASES AND DISORDERS
1. Tumor Necrosis Factor (TNF) 2. Tumor Necrosis Factor Receptors (TNFRs) a. TNFR1 b. TNFR2 3. Regulatory T Cells (Tregs) and Their Role in the Autoimmune Microenvironment 4. Autoimmune / Inflammatory Diseases Mediated by or involving TNF
a. Arthritis i. Rheumatoid Arthritis and other types of arthritis b. Inflammatory Bowel Disease (IBD) and Uveitis c. Fibrotic Diseases d. Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS) e. Other Diseases Mediated by or involving TNF
i. Neurodegenerative Diseases a) Alzheimer's Disease b) Parkinson's Disease c) Multiple Sclerosis (MS) Endometriosis iii. Cardiovascular Disease iv. Acute Respiratory Distress Syndrome (ARDS) v. Severe Acute Respiratory Syndrome (SARS) and D. THERAPIES FOR RHEUMATOID ARTHRITIS AND OTHER CHRONIC
INFLAMMATORY AND AUTOIMMUNE DISEASES AND DISORDERS
1. Conventional Synthetic Disease Modifying Anti-Rheumatic Drugs (csDMARDs) 2. Anti-TNF Therapies/TNF Blockers E. THERAPEUTICS FOR TARGETING TNFR1/INFR2 1. TNFR1-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-TNFR1 dAb-Anti-Albumin dAb Fusion Constructs b) Domain antibody fragments designated G5K1995057 and GSK2862277 Nanobodies (Nbs) iv. Anti-TNFR1 Nanobody-Anti-Albumin Nanobody Fusion Constructs c. Dominant-Negative Inhibitors of TNF (DN-TNFs)/T'NF
Muteins 2. TNFR2-Selective Agonists a. TNFR2 agonistic Antibodies b. TNFR2-Selective TNF Muteins and Fusions Thereof 3. Anti-TNFR2 Antagonistic Antibodies and Small Molecule Inhibitors F. SELECTIVE TARGETING OF THE TNFR1 AND/OR TNFR2 AXIS
1. Selective Blockade of TNFR1 with TNFR1 antagonists 2. Selective Activation of TNFR2 with TNFR2 agonists 3. TNFR1 antagonist constructs, TNFR2 agonist constructs; Multi-Specific, Including Bi-Specific, TNFR1 Antagonist and TNFR2 Agonist Constructs 4. Components of the TNFR1 antagonist constructs, TNFR2 agonist constructs, and Multi-Specific, Including Bi-Specific, TNFR1 Antagonist/TNFR2 agonist constructs a. TNFR1 inhibitor moiety (TNFR1 antagonist) b. TNFR2 Agonist Constructs and TNFR2 Antagonist Constructs RECTIFIED SHEET (RULE 91) ISA/EP

c. Linkers i. Peptide Linkers a) Flexible linkers b) Rigid linkers ii. Chemical Linkers d. Activity modifiers i. Modifications to the Fc portions a) Knobs-in-Holes b) Modifications that Enhance Neonatal Fc Receptor (FcRn) 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 / 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 5. Prediction and Removal of Immunogenicity in Protein Therapeutics a. B-cell and T-Cell Epitopes b. In Silico Epitope Prediction Methods i. In Silico Prediction of B-Cell Epitopes ii. In Silico Prediction of T-Cell Epitopes iii. Peptide-MHC Class II Binding Prediction c. In Vitro Epitope Prediction Methods i. In Vitro B-cell Epitope Prediction Methods ii. In Vitro T-Cell Epitope Prediction Methods MHC/HLA Binding Assays iii. In Vitro T-Cell Assays d. In Vivo Epitope Prediction Methods e. Removal of Predicted B-cell and T-cell Epitopes (De-immunization) G. PAN-GROWTH FACTOR TRAP POLYPEPTIDES
1. Receptor Tyrosine Kinases (RTKs) a. Human Epidermal Growth Factor Receptor (HER) Family b. Diseases Associated with the Human Epidermal Growth Factor Receptor (HER) Family and their Ligands 2. Pan-Growth Factor Inhibition a. RB242 Ligand Trap b. RB200 and RB242 for the Treatment of Autoimmune Disease c. RB242 Ligand Trap 3. Optimized Multi-Specific, such as Bi-Specific, Growth Factor Trap Constructs a. The Extracellular Domain (ECD) Polypeptides b. Modifications to the Extracellular Domains c. The Multimerization Domain d. Modifications to the Fc Domains i. Introduction of Knobs-in-Holes ii. Modifications that Enhance Neonatal Fc Receptor (FcRn) Recycling iii. Effector Functions 4. Compositions, Therapeutic Uses and Methods of Treatment a. Pharmaceutical Compositions b. Therapeutic Uses and Methods of Treatment 5. Combination Therapies H. ASSESSING TNFR1 ANTAGONIST AND TNFR1 ANTAGONIST/TNFR2 AGONIST CONSTRUCT ACTIVITY AND EFFICACY
1. Disease Activity Score (DA528) 2. SOMAscan Proteomic Analysis and other proteomic tools for quantifying analytes 3. Transcriptome Analysis to Predict Responsiveness to Therapy and to select subjects likely to benefit from treatment 4. L929 Cytotoxicity Assay 5. HeLa IL-8 Assay 6. HUVEC Assay 7. Quantification and Evaluation of Treg Cell Activity 8. Evaluation of Binding Properties of the TNFR1 antagonist/TNFR2 Agonist Constructs 9. Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) Assays 10. Disease Models a. Collagen-Induced Arthritis (CIA) b. Rheumatoid Arthritis Synovial Membrane Mononuclear Cell Cultures c. Tg197 Mouse Model of Arthritis d. AARE Mouse Model of Arthritis/IBD
e. Humanized TNF/TNFR2 Mice I. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING TNFR1 AGONIST CONSTRUCTS

1. Isolation or Preparation of Nucleic Acids Encoding TNFR1 Antagonist and TNRF2 Agonist Polypeptides 2. Generation of Mutant or Modified Nucleic Acids and Encoding Polypeptides 3. Vectors and Cells 4. Expression a. Prokaryotic Cells b. Yeast Cells c. Insects and Insect Cells d. Mammalian Expression Cells e. Plants 5. Purification 6. Additional Modifications a. PEGylation b. Albumination c. Purification Tags 7. Nucleic Acid Molecules and Gene Therapy J. COMPOSITIONS, FORMULATIONS AND DOSAGES
1. Formulations 2. Administration of the TFNR1 Antagonist Constructs, TNFR2 Agonist Constructs, the Multi-specific, such as Bi-Specific, Constructs and Nucleic acids 3. Administration of Nucleic Acids Encoding Polypeptides (Gene Therapy) K. THERAPEUTIC USES AND METHODS OF TREATMENT
1. Treatment of Chronic Inflammatory/Autoimmune Diseases and Disorders 2. Treatment of Neurodegenerative and Demyelinating Diseases and Disorders 3. Treatment of Cancer and other Immunosuppressing Diseases, Disorders, and Conditions 4. Combination Therapies L. EXAMPLES
A. DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a RECTIFIED SHEET (RULE 91) ISA/EP

URL or other such identifier or address, it is understood that such identifiers can change, and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, 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.
As used herein, 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.
As used herein, 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.
As used herein, a construct that is a TNFR2 antagonist construct, is a construct that comprises a TNFR2 antagonist.
As used herein, 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.
As used herein, "tumor necrosis factor," "tumor necrosis factor alpha,"
"TNF," "TNF-alpha," "TNF-a" and "TNFa" 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 tumorigenesis/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
(solTNF), which contains 157 amino acids; membrane-bound and soluble forms of TNF are biologically active. 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. The uncontrolled or dysregulated production of TNF is associated with several 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.
As used herein, a "TNF mutein" or "TNF-a 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 (TNFa) by one or more amino acids. Generally, such 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. For example, as described herein, there are TNFR1-selective antagonistic TNF muteins, and TNFR2-selective agonistic TNF muteins.
As used herein, 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, 586Q, 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.
As used herein, 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.
As used herein, "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.
As used herein, "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. Thus, 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.
As used herein, "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.
As used herein, a "substitution" or "replacement" 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.
Thus, 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. For example, a modified polypeptide having a modification in the amino acid at the 100th 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, Tyr100G1u, or 100E. Y100 can be used to indicate that the amino acid at the modified 100th position is a tyrosine. For purposes herein, since 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.
As used herein, "at a position corresponding to," or recitation that nucleotides or amino acid positions "correspond to" nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence Listing, 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.
By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, HG., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carrillo et al.
(1988) SIAIviI Applied Math 48:1073).
As used herein, 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. By aligning the sequences of polypeptides or nucleic acids, 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.
As used herein, recitation that 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. For example, when the activity of an antibody is compared to another antibody, 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.
As used herein, 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. For example, side effects associated with the administration of an anti-TNF antibody, such as adalimumab (sold, for example, under the trademark Humirac)), are known to one of skill in the art, and some are described herein. 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. Thus, "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. 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).
As used herein, a "property" of a polypeptide, such as an antibody, 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.
As used herein, an "activity" or a "functional activity" of a polypeptide, such as an antibody, 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. For an antibody (including antibody fragments), 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.
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).
For example, for an antibody polypeptide, 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.
As used herein, "bind," "bound," and grammatical variations thereof, refers to the participation of a molecule in any attractive interaction with another molecule, resulting in a stable association in which the two molecules are in close proximity to one another. 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. Exemplary bonds are antibody-antigen interactions and receptor-ligand interactions. When an antibody "binds" a particular antigen, "bind" 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.

As used herein, "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.
As used herein, "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 (ka or kan) and/or dissociation (kd or koff), half maximal effective concentration (EC50) values, and/or thermodynamic data (e.g., Gibbs free energy (AG), enthalpy (AH), entropy (-TAS), and/or calculating association (Ka) or dissociation (Ka) constants. EC50, also called the apparent Ka, is the concentration (e.g., ng/mL) of antibody, where 50% of the maximal binding is observed to a fixed amount of antigen. Typically, EC50 values are determined from sigmoidal dose-response curves, where the EC50 is the concentration at the inflection point.
A high antibody affinity for its substrate correlates with a low EC50 value, and a low affinity corresponds to a high EC50 value. Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, such as ELISA, followed by curve-fitting analysis.
As used herein, "affinity constant" refers to an association constant (Ka) used to measure the affinity of an antibody for an antigen. The higher the affinity constant, the greater the affinity of the antibody for the antigen. Affinity constants are expressed in units of reciprocal molarity (i.e., M-'), 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 Ka. The dissociation constant is the reciprocal of the association constant, i.e., Ka = 1/Ka.
Hence, an affinity constant also can be represented by the Ka. 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. Op/n. 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).
Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g., BIAcore 2000, BIAcore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans.
27:335).
Methods for calculating affinity are well-known, such as methods for determining EC50 values, or methods for determining association/dissociation constants. For example, in terms of EC50, high binding affinity means that the antibody specifically binds to a target protein with an EC50 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 (Ka) of 10' M or lower, such as 10' M, 108 M, 10"9M, 10-10 NI--, 1041 M, or 1042 M, or lower. In terms of equilibrium association constant (Ka), high binding affinity is generally associated with Ka values of greater than or equal to about 106 M-1, greater than or equal to about 107M1, greater than or equal to about 108 M"1, or greater than or equal to about 109M-1, 1010 A4-1, 1011 A4-1, or 1012 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. For example, such 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 at., (1949) Ann N.Y. Acad. Sc., 51:660, or by curve fitting analysis, for example, using a Parameter Logistic nonlinear regression model using the equation: y = ((A-D)/(1+((x/C)13))) + D, where A is the minimum asymptote, B is the slope factor, C is the inflection point (EC50), and D is the maximum asymptote.
As used herein, "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.
As used herein, "specificity for a target," such as TNFR1, 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 107-108M-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.
As used herein, "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.
Typically, an antibody that immunospecifically binds (or that specifically binds), for example, to TNFR1, is one that binds to TNFR1 with an affinity constant (Ka) of about or lx 107 M-1 or lx 108 M-1 or greater (or a dissociation constant (Ka) of lx 10' M or lx 10' M
or less). 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.
As used herein, "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.
As used herein, "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. In some instances, a modified, or variant, polypeptide can retain an activity that is increased compared to a target or unmodified polypeptide. In some cases, 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. In other embodiments, 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, 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.
As used herein, 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. For example the BIAcore system (GE Healthcare Life Sciences) is an exemplary commercial system.
As used herein, "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. Hence, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). For example, 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, VH chains, chains, and VH-CH1-CH2-CH3 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, VL chains and VL-CL chains). Each heavy chain (H and H') pairs with one light chain (L and L', respectively). Typically, antibodies minimally include all or at least a portion of the variable heavy (VH) chain and/or the variable light (VL) 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.
For purposes herein, the term "antibody," unless otherwise specified, includes full-length antibodies and portions thereof, including antibody fragments, such as, for example, anti-TNFR1, antibody fragments. Antibody fragments, include, but are not limited to, for example, Fab fragments, Fab' fragments, F(a1302 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFvs), single-chain Fabs (scFab), hsFy (helix-stabilized Fv), single domain antibodies (dAbs, or sdAbs), minibodies, diabodies, anti-idiotypic (anti-Id) antibodies, nanobodies and camelid antibodies, free light chains, VHH 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-CH2-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. The 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).
As used herein, 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.
As used herein, reference to 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.
As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g.,VH-CH1-CH2-CH3, or VH-CH1-CH2-CH3 -CH4), two full-length light chains (VL-CL), and hinge regions, such as human antibodies produced by antibody secreting B cells, and antibodies with the same domains that are produced synthetically.
As used herein, a "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.
As used herein, 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. For human therapeutics, 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. For example, 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.
As used herein, "antibody fragment" or "antibody portion" 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.
Examples of 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 amino acids, and typically, at least about or at least 110, 120, 150, 170, 180, or 200 amino acids.
As used herein, an "Fv antibody fragment" is composed of one variable heavy domain (VH) and one variable light (VI) domain, linked by noncovalent interactions.
As used herein, a dsFy (disulfide-linked Fv) refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair.
As used herein, an "scFv fragment" refers to an antibody fragment that contains a variable light chain (VL) and variable heavy chain (VH), 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.
As used herein, "diabodies" are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs, and preferentially dimerize.
As used herein, "triabodies" are trimeric scFv; they contain three peptide chains, each of which contains one VH domain and one VL domain joined by a short .. linker (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain; triabodies typically trimerize.
As used herein, 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 VL and CL), and another chain containing a variable domain of a heavy chain (VH) and one constant region domain of the heavy chain (CH1).
As used herein, 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.
As used herein, a Fab' fragment is a fragment containing one half (i.e., one heavy chain and one light chain) of the F(ab')2 fragment.
As used herein, an Fd fragment is a fragment of an antibody containing a variable domain (VH) and one constant region domain (CH1) of an antibody heavy chain.
As used herein, an Fd' fragment is a fragment of an antibody containing one heavy chain portion of a F(ab')2 fragment.

As used herein, an Fv' fragment is a fragment containing only the VH and VL
domains of an antibody molecule.
As used herein, hsFy (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).
As used herein, a "domain antibody," "single domain antibody," "sdAb," or "dAb," used interchangeably, refers to a monomeric small antibody fragment that contains a variable domain of the heavy chain (VH) or of the light chain (VL) of an antibody. 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). There are three complementarity determining regions (CDRs) on each VH and each VL. Each dAb contains three out of the six CDRs, which are the highly diversified loop regions that bind to the target antigen, from a VH-VL pair in an antibody.
As used herein, 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.
As used herein, 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. For example, 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. Alternatively, 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. Typically, a polypeptide contains a plurality of domains. For example, 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. Those of skill in the art are familiar with polypeptide domains and can identify them by virtue of structural and/or functional homology with other such domains. For exemplification herein, definitions are provided, but it is understood that it is well within the skill in the art to recognize particular domains by name. If needed, appropriate software can be employed to identify domains.
As used herein, 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 VH, VL, CH, CL, and portions thereof, such as CDRs, including CDR1, CDR2 and CDR3, or antigen-binding portions, such as antibody combining sites.
As used herein, a "structural region" of a polypeptide is a region of the polypeptide that contains at least one structural domain.
As used herein, 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 (VL) and one constant region domain (CL), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.
As used herein, a "variable domain," with reference to an antibody, 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. Each light chain and each heavy chain has one variable region domain (VL and VH, 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).
As used herein, "hypervariable region," "HV," "complementarity-determining region," "CDR" and "antibody CDR" are used interchangeably to refer to one of a plurality of portions within each variable region that together form an antigen-binding site of an antibody. Each 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.
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).
As used herein, "portion thereof," with reference to an antibody heavy or light chain, or variable heavy or light chain, 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 (VH) and variable light (VI) chains sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody containing all 6 CDRs. Generally, a sufficient antigen-binding site requires the CDR3 of the heavy chain (CDRH3). It typically further requires the CDR3 of the light chain (CDRL3). As described herein, one of skill in the art knows and can identify the CDRs based on Kabat or Chothia numbering (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, NIH Publication No. 91-3242; and Chothia, C. et at.

(1987)1 Mol. Biol. 196:901-917).
As used herein, "framework regions" or "FRs" 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.
As used herein, 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 (CH) domains, which include, CH1, CH2, CH3 and CH4. Full-length IgA, IgD and IgG isotypes contain CH1, CH2 and CH3 domains and a hinge region, while IgE and IgM contain CH1, CH2, CH3 and CH4 domains. CH1 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.
As used herein, 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 CH1 and CH2 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. Two interchain disulfide bonds within the hinge region stabilize the interaction between the two heavy chains.
In some embodiments provided herein, 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.
As used herein, 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 (CH2 and CH3, also referred to as Cy2 and Cy3), or the last three constant region immunoglobulin domains of IgE and IgM (CH2, CH3 and CH4). Optionally, an Fc domain can include all or part of the flexible hinge region, which is N-terminal to these domains. For IgA and IgM, the Fc can include the J chain. For an exemplary Fc domain of IgG, Fc contains immunoglobulin domains CH2 and CH3, and optionally, all or part of the hinge between CH1 and CH2 (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. For purposes herein, 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.
As used herein, "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.
As used herein, "Kabat numbering" refers to the index numbering of the IgG1 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, NIH Publication No. 91-3242); it permits easy comparison among antibodies, similar to way chymotrypsin numbering permits comparison among proteases. One of skill in the art can identify regions of the constant region using Kabat numbering.
As used herein, "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 IgG1 Kabat antibody as set forth in Kabat, E. A. et at. (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. For example, one of skill in the art can identify regions of the constant region using EU numbering. For example, 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 CH1 domain of IgG1 corresponds to residues 118-215 (EU numbering) or 114-223 (Kabat numbering); CH2 corresponds to residues 231-340 (EU numbering) or 244-360 (Kabat numbering); CH3 corresponds to residues 341-447 (EU numbering) or 361-478 (Kabat numbering).
The following tables define the numbering for the IgG1 and IgG4 heavy chain constant domains, and the Ig kappa light constant domain, by EU, Kabat, and sequential numbering. Table 1 shows the IgG1 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 CH1, CH2 and CH3 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. In Table 2, the top row (bold) 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 CH1, CH2 and CH3 domains, as well as the hinge region.

Table 1. IgG1 Heavy Chain Constant Domain by EU, Kabat and Sequential Numbering Residue Numbering Residue Numbering Residue Numbering Domain Eu Sequential IgG1 Domain Eu Sequential IgG1 Do-EU main Sequential IgG1 Kabat (SEQ ID Kabat (SEQ ID Segue Kabat (SEQ ID
Se-Index Sequence Id Index nex NO: 9)NO: 9) nce NO:9) quence G

Q

P

R

E

P

Q

V

Y

T

L

P

P

S

R

D

E

L

T

K

N

Q

V

S

L

T

C

L

V

K

G

F

Y

P

S

D

A

V

E

W

E

S

N

G

Hinge 216 226 99 E CH2 329 348 212 P CH3 Hinge 217 227 100 P CH2 330 349 213 A CH3 Hinge 218 228 101 K CH2 331 350 214 P CH3 Hinge 219 232 102 S CH2 332 351 215 I CH3 Hinge 220 233 103 C CH2 333 352 216 E CH3 Hinge 221 234 104 D CH2 334 353 217 K CH3 Hinge 222 235 105 K CH2 335 354 218 T CH3 Hinge 223 236 106 T CH2 336 355 219 I CH3 Hinge 224 237 107 H CH2 337 357 220 S CH3 Hinge 225 238 108 T CH2 338 358 221 K
Hinge 226 239 109 C CH2 339 359 222 A
Hinge 227 240 110 P CH2 340 360 223 K
Hinge 228 241 111 P
Hinge 229 242 112 C
Hinge 230 243 113 P
Table 2. Kabat and EU Numbering of Ig Kappa Light Chain Constant Domain R T V AAP S V F I F PPSD

EQLKSG T A S V V CLLN

NF YPR E A K V QWK VDN

AL QSGN S Q E S V T EQD

SK DST Y S L S S T L TLS

HQG LS S P V T K S F NR G

EC

Table 3. IgG4 Heavy Chain Constant Domain by EU, Kabat and Sequential Numbering Residue Numbering Residue Numbering Residue Numbering Segue ntial I
(2, Domain Eu Sequential IgG4 Domain EIndex u Kabat Sequential IgG4 Domain EU Ka (SEQ "-gs--(SEQ ID (SEQ ID Se-Index Kabat Sequence Sequence Index bat ID
NO: 15) NO: 15) quence NO:15 ) N

Hinge 216 226 99 E CH2 329 348 209 P CH3 439 470 319 K
Hinge 217 227 100 S CH2 330 349 210 S CH3 440 Hinge 218 228 101 K CH2 331 350 211 S CH3 441 472 321 L
Hinge 219 229 102 Y CH2 332 351 212 I CH3 442 473 322 S
Hinge 220 230 103 G CH2 333 352 213 E CH3 443 474 323 L
Hinge 224 237 104 P CH2 334 353 214 K CH3 444 475 324 S
Hinge 225 238 105 P CH2 335 354 215 T CH3 445 476 325 L
Hinge 226 239 106 C CH2 336 355 216 I CH3 446 477 326 G
Hinge 227 240 107 P CH2 337 357 217 S CH3 447 478 327 K
Hinge 228 241 108 S CH2 338 358 218 K
Hinge 229 242 109 C CH2 339 359 219 A
Hinge 230 243 110 P CH2 340 360 220 K
As used herein, the phrase "derived from," when referring to antibody fragments derived from another antibody, such as a monoclonal antibody, refers to the engineering of antibody fragments (e.g., Fab, F(ab'), F(ab')2, single-chain FIT (scFv), Fv, dsFy, dAb,_diabody, Fd and Fd' fragments) that retain the binding specificity of the original antibody. 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. Generally, the derived antibody fragment shares the identical, or substantially identical, heavy chain variable region (VH) and light chain variable region (VI) of the parent antibody, such that the antibody fragment and the parent antibody bind the same epitope.

As used herein, 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.
As used herein, the term "epitope" 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.
As used herein, "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, for example, 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. 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.
Methods for identifying such regions are known, including computer programs, which are designed for identifying the variable and non-variable regions of immunoglobulins.
Hence, in general, 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.
As used herein, 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. Generally, 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, for example, 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.
As used herein, "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).
As used herein, 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. Thus, 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.
As used herein, 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.
As used herein, a "linker," "linker unit," or "link," 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.
As used herein, a "linker peptide" or "spacer 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.
As used 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.
Typically, 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. 8:2159-2165);
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.
As used herein, 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.
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).
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)).
As used herein, "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. 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. For RNA, the uracil base is uridine.
As used herein, 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.
As used herein, "operably linked," with reference to nucleic acid sequences, regions, elements or domains, means that the nucleic acid regions are functionally related to each other. For example, 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. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) 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. For example, 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. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

As used herein, "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.
As used herein, the 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.
As used herein, "polypeptide" refers to two or more amino acids covalently joined. The terms "polypeptide" and "protein" are used interchangeably herein.
As used herein, a "peptide" refers to a polypeptide that is from 2 to about or 40 amino acids in length.
As used herein, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids.
For purposes herein, 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). As used herein, the 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.
As used herein, "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. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968), and adopted at 37 C.F.R. 1.821 - 1.822, abbreviations for amino acid residues are shown in Table 4:

TABLE 4 ¨ Table of Correspondence SYMBOL
1-Letter 3-Letter AMINO ACID
Tyr Tyrosine Gly Glycine Phe Phenylalanine Met Methionine A Ala Alanine Ser Serine Ile Isoleucine Leu Leucine Thr Threonine V Val Valine Pro Proline Lys Lysine His Histidine Gln Glutamine Glu Glutamic acid Glx Glutamic Acid and/or Glutamine Tip Tlyptophan Arg Arginine Asp Aspartic acid Asn Asparagine Asx Aspartic Acid and/or Asparagine Cy s Cy steine X Xaa Unknown or other All sequences of amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase "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. Furthermore, 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. In a peptide or protein, 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 at., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.
Co., p. 224).
Such substitutions can be made in accordance with the exemplary substitutions set forth in Table 5 as follows:

Table 5. Exemplary Conservative Amino Acid Substitutions Original Residue Conservative Substitution Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Tip (W) Tyr Tyr (Y) Tip; Phe Val (V) Ile; Leu Other substitutions also are permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.
As used herein, "naturally occurring amino acids" refer to the 20 L-amino acids that occur in polypeptides.
As used herein, the term "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-stereoisomers of amino acids. Exemplary 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), 0-alanine/f3-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 (EtAsn), Hydroxylysine (Hyl), allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline (4Hyp), Isodesmosine (Ide), allo-Isoleucine (Aile), N-Methylglycine, sarcosine (MeGly), N-Methylisoleucine (MeIle), 6-N-Methyllysine (MeLys), N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine (Orn).
As used herein, a "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.
As used herein, a "DNA segment" is a portion of a larger DNA molecule having specified attributes. For example, 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.
As used herein, the term "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"). The term nucleotides is used for single- and double-stranded molecules where the context permits.
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.
As used herein, 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.
As used herein, "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.
As used herein, 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.
As used herein, 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-TNFR1 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. Also contemplated are 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.
As used herein, 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.
As used herein, "primary sequence" refers to the sequence of amino acid residues in a polypeptide or the sequence of nucleotides in a nucleic acid molecule.
As used herein, "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.
As used herein, 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 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. Generally, 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 at. (1970)1 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.psi.iastate.edu/aat/align/align.html.
As used herein, 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.
For purposes herein, 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. Whether any two 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). Generally, for purposes herein sequence identity is determined using computer algorithms based on global alignment, such as the Needleman-Wunsch Global Sequence Alignment tool available from NCBUBLAST
(blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&Page TYPE=BlastHome); LAlign (William Pearson implementing the Huang and Miller algorithm (Adv. Appl. Math.

(1991) 12:337-357)); and the program from Xiaoqui Huang, available at deepc2.psi.iastate.edu/aat/align/align.html. Typically, 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.
As used herein, the term "identity" represents a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one non-limiting example, "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., 10 out of 100) of amino acids or nucleotides in the test polypeptide or polynucleotide differ from those of the reference polypeptide or polynucleotide. Similar comparisons can be made between a test and reference polynucleotide. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence, or they can be clustered in one or more locations of varying length, up to the maximum allowable, e.g., 10/100 amino acid difference (approximately 90% identity). Differences also can be due to deletions or truncations of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. Depending on the length of the compared sequences, at the level of homologies or identities above about 85-90%, the result can be independent of the program and gap parameters set;
such high levels of identity can be assessed readily, often without relying on software.
As used herein, 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.
As used herein, "coupled" or "conjugated" means attached via a covalent or noncovalent interaction.
As used herein, the phrase "conjugated to an antibody" or "linked to an antibody" or grammatical variations thereof, when referring to the attachment of a moiety to an antibody or antigen-binding fragment thereof, such as a diagnostic or therapeutic moiety, 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.
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. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. (1991) Annu. Rev. Immunol, 9:457-492. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed (see, e.g.,U U.S. Patent Nos. 5,500,362 and 5,821,337).Exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model, such as that disclosed in Clynes et at. (1998) Proc. Natl. Acad. Sci. USA 95:652-656.
As used herein, complement-dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When such antibodies are bound to a surface antigen on 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.
As used herein, antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which effector cells with phagocytic potential, such as monocytes and macrophages, internalize target cells. Once phagocytosed, the target cell resides in a phagosome, which fuses with a lysosome for degradation of the target cell via an oxygen-dependent or independent mechanism.

As used herein "therapeutic activity" refers to the in vivo activity of a therapeutic polypeptide. Generally, 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.
As used herein, the term "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.
As used herein, 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.
As used herein, "treating" a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain static following treatment. Hence, 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.
As used herein, treatment means amelioration of a symptom or manifestation of a disease, disorder, or condition.
As used herein, "prevention" or "prophylaxis," 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.
As used herein, 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.
As used herein, 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.
As used herein, 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.
As used herein, "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.
As used herein, 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. Thus, a prophylactically effective amount can be administered in one or more administrations.
As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, 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.
As used herein, 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 Belfast, 14:375-382; and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et at., (ed.), pp. 247-267, Humana Press, 1985).
As used herein, an "anti-cancer agent" refers to any agent that is destructive or toxic to malignant cells and tissues. For example, 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.
As used herein, an "anti-angiogenic agent" or "angiogenesis inhibitor" is a compound that blocks, or interferes with, the development of blood vessels.
As used herein, 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.
As used herein, 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. For example, 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. For example, 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 (TNFa), thus inducing TNFR2-mediated signaling. 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.
As used herein, a TNFR2-selective agonist is a TNFR2 agonist that does not or substantially does not result in TNFR1 signaling activity.
As used herein, 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.
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.
Generally, a pan-growth factor trap targets at least two different HER receptors, such as via ligand binding and/or interaction with the receptors.
As used herein, 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). For purposes herein, reference to an "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.
As used herein, "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 Fe 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 Fe monomer) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
As used herein, "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. For example, subdomain II in HER1, HER3 and HER4, can interact with subdomain IV, forming a tethered, inactive structure. When in a tethered state, 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. Upon ligand binding to a tethered (monomeric) form, 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.
As used herein, HER (ErbB)-related diseases, HER-associated diseases, or HER-mediated disease, 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.

As used herein, the term "subject" refers to an animal, including a mammal, such as a human being.
As used herein, a "patient" refers to a human subject.
As used herein, "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.
As used herein, a "composition" refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof As used herein, 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. Thus, included are agents that prevent proteins from degradation from other components in the compositions. Exemplary of such agents are amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, inhibitors, or substrates and other agents as described herein.
As used 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.
As used herein, "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.
As used herein, 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.
As used herein, a "unit dose form" refers to physically discrete units suitable for human and animal subjects, and packaged individually, as is known in the art.
As used herein, a "single dosage formulation" refers to a formulation for direct administration.
As used herein, a "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.
As used herein, 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.
As used herein, a "fluid" 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.
As used herein, 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.
Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
As used herein, a "cellular extract" or "lysate" refers to a preparation or fraction which is made from a lysed or disrupted cell.
As used herein, a "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.
As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide, containing "an immunoglobulin domain" includes polypeptides with one or a plurality of immunoglobulin domains.
As used herein, the term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive.
As used herein, 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.
As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).
For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

B. OVERVIEW OF CONSTRUCTS AND METHODS
Autoimmune disease occurs when the body's immune system attacks itself The resulting inflammation and tissue destruction is initiated by an inflammatory hormone called tumor necrosis factor (TNF). There are more than 100 types of autoimmune disease; overall, about 75% of those with an autoimmune disease are women. Prior drugs for autoimmune disease have adverse side effects, including infections, heart problems, and other diseases and disorders, 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 Remicadeg), adalimumab (sold as Humirag), and etanercept (sold as Enbrelg) 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. Provided herein a variety of constructs that address the problems with the prior art TNF blockers. Types of constructs identified by their activity, and detailed and provided herein, are summarized in the following table:
Type of Construct Disease to be Treated Action u A toimmune disease TNFR1 Antagonists . Specific blockade of TNFR1; spares TNFR2 and acute inflammation . Traps 9 growth factors from the EGF
family Rheumatoid arthritis Growth Factor traps (EGFR and HER3; and dimerization and cancer dependent HER2) . Cancer checkpoint Inhibition of tumoral suppressor Treg function TNFR2 Antagonists inhibitor thus increases active immunity Inflammation and Induces proliferation of Treg to reduce TNFR2 Agonists fibrosis inflammation Provided are constructs for treatment of TNF-mediated diseases, disorders, and conditions, or diseases, disorders, and conditions in which TNF plays a role in the etiology, or in which interference with TNFR1 signaling has an ameliorative effect.

For example, 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.
The 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.
Hence, provided are TNFR1 antagonist constructs, TNFR2 agonist constructs, and multi-specific, such as bi-specific constructs that include TNFR1 antagonist and TNFR2 agonist activity. The 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. Hence a construct as provided herein includes at least two moieties: 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.
Among 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 Ka < 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).
Also provided are multi-specific, such as bi-specific, constructs that contain a TNFR1 antagonist moiety, linked directly, or via a linker, to a TNFR2 agonist moiety.
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. These constructs solve problems associated with the administration of other therapies, such as anti-TNF
therapies ("TNF Blockers," (examples include Etanercept, adalimumab (Humira()), Infliximab), because these constructs increase 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. In addition, 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 at. (2007) J Immunol. 179:4239-48).
There are numerous differences between the activity of exemplary TNFR1 antagonist constructs provided herein and existing approved TNF Blockers: TNF
Blockers, such as etanercept, adalimumab, infliximab, is that they are not specific for TNFR1. 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. For example, the inflammatory cytokine El is not blocked by JAK
inhibitors, the inflammatory cytokine IL6 is blocked by JAK inhibitors (a second line use for rheumatoid arthritis treatment), and IL10, which is anti-inflammatory, is not blocked by JAK inhibitors. Constructs provided herein, in contrast, combine the effectiveness of TNFR1 and TNF inhibitor therapies with the benefits of TNFR2 agonists that eliminate or reduce the adverse effects of anti-TNFR1/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. For example, the structure of the TNFR1 antagonist constructs provided herein can be represented by the formulae 1:
(TNFR1 inhibitor),¨linkerp¨ (activity modifier)q, Formula la, or (activity modifier)q¨linkerp¨ (TNFR1 inhibitor), 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.
Also provided are multi-specific constructs. The structure of the multi-specific, such as, bi-specific, constructs provided herein is represented by the following formula (Formula 2):
(TNFR1 inhibitor).¨ (activity modifier)d ¨ (Linker (L)p ¨ (activity modifier)r2_ (TNFR2 agonist)ch where n= 1, 2, or 3, p= 1, 2, or 3, q= 0, 1 or 2, and each of rl and r2 is independently .. 0, 1, or 2. As with the constructs of formulae 1 the order of components can be varied and there can be additional linkers as needed. The constructs 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.

Also provided are TNFR2 agonist constructs that have formulae 3:
(TNFR2 agonist).¨linkerp¨ (activity modifier)q, formula 3a, or (activity modifier)q¨linkerp¨ (TNFR2 agonist),, formula 3b, where n, p and q are as set forth for formula 1, and the linkers and activity modifier are as described in formula 1.
The components, which are discussed in detail in the following sections, of formulae 1-3 can be polypeptides or other molecules, such as small drugs that specifically bind to or interact with the targeted receptor. Each component of the constructs/molecules provided herein is described in turn in sections below.
The properties of each component of the constructs provided herein is discussed in detailed in sections below. The components of the constructs, thus, include, but are not limited to, the following components, which are discussed in detail in Sections that follow:
1. TNFR1 Antagonists 2. TNFR2 Agonists 3. Linkers a. Glycine-Serine Linkers b. Hinge Regions c. chemical linkers 4. Activity modifiers a. Modified Fcs b. Polypeptides and other moieties that confer improved or altered pharmacological properties, such as increased serum half-life, resistance to degradation by endogenous proteases, and other such properties.
Other constructs, detailed in Sections that follow, also are provided.
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. Such diseases, conditions, and disorders, which include inflammatory diseases, including autoimmune diseases, are discussed in the section that follows.
Also provided are pharmaceutical 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.
C. TUMOR NECROSIS FACTOR (TNF) AND CHRONIC
INFLAMMATORY AND AUTOIMMUNE DISEASES AND
DISORDERS
This section describes the role that tumor necrosis factor (TNF) and/or its receptors play in inflammatory and autoimmune diseases, particulars of exemplary diseases, and problems with existing therapies, and shows how the constructs provided herein address these problems.
1. Tumor Necrosis Factor (TNF) Tumor necrosis factor (TNF; see e.g., SEQ ID NO:1; also referred to as TNF
alpha, TNF-a, or TNFa) 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. In humans, 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. Transmembrane human TNF contains 233 amino acids, and contains a cytoplasmic domain, corresponding to residues 1-35, a transmembrane RECTIFIED SHEET (RULE 91) ISA/EP

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).
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 (fl3D). 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. In rheumatoid arthritis (RA), TNF is over-expressed in synovial fluids and in the synovial membrane, while expression of TNF
receptors (TNFRs) is up-regulated in the synovial membrane. For example, 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., BlUml et at. (2010) Arthritis & Rheumatism 62(6):1608-1619; Keifer et al. (1991) EMBO 10(13):4025-4031; Esperito Santo et al. (2015) Biochem. Biophys. Res. Commun. 464:1145-1150; Bluml et at. (2012) International Immunology 24(5):275-281; Dong et al. (2016) Proc. Natl. Acad. Sci. USA
113(43):12304-12309).
As discussed further below, 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 (Bluml et at. (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 at. (2007)1 Immunol.

179:1872-1883). Thus, TNF signaling through TNFR1 and TNFR2, effects different outcomes, depending on the receptor type.
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 (fl3D;
e.g., Crohn's disease, ulcerative colitis). The use of TNF blockers, which block TNF
and prevent signaling via both TNFR1 and TNFR2, is associated with an increased risk of serious infections, such as tuberculosis and listeriosis, due to immunosuppression. TNF blockers not only block detrimental inflammatory signaling via TNFR1, but also block beneficial, immune-regulatory signaling via TNFR2.
As a result, the use of TNF blockers, particularly in the case of chronic diseases/conditions that require long-term administration, such as arthritis or fl3D, 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. Thus, there is a need for 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.
2. Tumor Necrosis Factor Receptors (TNFRs) Homotrimers of TNF bind to and signal through two specific, high-affinity homotrimeric receptors, TNFR1 (TNF receptor type 1; also known to as TNFRI, p55, p60, CD120a, TNF receptor superfamily member 1A, and TNFRSF1A), and TNFR2 (TNF receptor type 2; also known as TNFRII, p'75, p80, CD120b, TNF receptor superfamily member 1B, and TNFRSF1B). TNFR1 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.

In vivo, 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. Membrane-bound 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. 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. For example, 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. As described herein, 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. Promotion of TNF signaling via TNFR2 can be effected by administering a TNFR1 antagonist (see, e.g., Faustman et at.
(2013) Front. Immunol. 4:478).
a. 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 at. (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 correspond to the extracellular domain, residues 212-232 correspond to the transmembrane domain, and residues 233-455 correspond to the cytoplasmic domain.
Within the extracellular 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. 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. The binding of TNF to TNFR1 also initiates proinflammatory cascades through MAPK (mitogen-activated protein kinase; e.g., p38, JNK, ERK) and NF-KB (nuclear factor kappa-light-chain-enhancer of activated B
cells) signaling pathways. 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. For example, 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.

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. Additionally, 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):60137065;
McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Ruspi et al.
(2014) Cellular Signaling 26:683-690; Faustman and Davis (2013) Front.
IrnmunoL
4:478; Bluml etal. (2012) International Immunology 24(5):275-281; Dong etal.
(2016) Proc. Natl. Acad. Sci. USA 113(43):12304-12309).
b. 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. TNFR2 signaling via TRAF2 promotes cell survival and proliferation through NF-KB and activator protein 1 (AP1) activation, and has been associated with PI3K-PKB/Akt-mediated repair and migration. As discussed elsewhere herein, 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. 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. For example, 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 RECTIFIED SHEET (RULE 91) ISA/EP

proliferation. TNFR2 agonism 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. In animal models of type I diabetes, elimination of autoreactive T cells using low-dose TNF results in the regeneration of .. pancreatic tissue. 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. In experimental colitis, the lack of TNFR2 expression on CD4+ T cells accelerates the onset of disease and increases the severity of inflammation, while in experimental autoimmune encephalitis (EAE), symptoms are exacerbated in TNFR2-deficient mice (see, e.g., Schmidt et at.
(2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et at. (2015) PLoS ONE
10(9):e0137065; McCann et at. (2014) Arthritis & Rheumatology 66(10):2728-2738;
Ruspi et at. (2014) Cellular Signaling 26:683-690; Faustman and Davis (2013) Front.
.. Immunol. 4:478; Bluml et al. (2012) International Immunology 24(5):275-281;
Dong et at. (2016) Proc. Natl. Acad. Sci. USA 113(43):12304-12309). 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-KB and hampers TNFR2 signaling pathways in Tregs (see, e.g., Yang et at. (2018) Front.
Immunol.
9:784).

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-KB pathways to control cell survival and proliferation. In general, 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. As discussed above and elsewhere herein, it has been shown that 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, for example, show that TNFR1 is associated with inflammatory neurodegeneration, while TNFR2 is associated with neuroprotection.
The selective inhibition of TNFR1, or the selective activation of TNFR2, has .. been demonstrated in a mouse model of NMDA-induced acute neurodegeneration, by administration of either ATROSAB (Antagonistic TNF Receptor One-Specific Antibody), a TNFR1-selective antagonistic IgG1 antibody, or EHD2-scTNFR2, an agonistic TNFR2-selective TNF mutein (i.e., mutated protein). EHD2-scTNFR2 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 CH2 domain of IgE and creates a disulfide bonded dimer that contains hexameric TNF
domains.
Simultaneous injection of NMDA and ATROSAB, or NMDA and EHD2-scTNFR2, 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 at. (2013) PLoS One 8(8):e72156. Similarly, 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 at. (2016) Immun. Inflamm. Dis. 4(2):135-147).

Therefore, improved TNFR1 antagonists and improved TNFR2 agonists are needed that overcome these limitations.
3. Regulatory T Cells (Tregs) and Their Role in the Autoimmune Microenvironment Regulatory T cells (Treg cells or Tregs) 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 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. 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 at.
(2018) Front Immunol. 9:883). For these reasons, and others described below, 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).
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 4-1BB and 0X40, result in the optimal activation and proliferation of Tregs, and in the attenuation of excessive inflammatory responses. Neutralization of TNF (blocking TNFR2) blocks in vivo expansion of Tregs (e.g., Hamano etal. (2011) Eur. I ImmunoL 41:2010-2020).
In comparison to CD4+FoxP3- conventional T cells, CD4+FoxP3+ Tregs constitutively express TNFR2, promoting Treg cell activation, expansion and survival. TNF signaling through TNFR2 (i.e., TNFR2 agonism) promotes the activation and expansion of Tregs, while TNFR2 antagonism results in Treg contraction. For example, 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. 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 etal. (2014) Arthritis & Rheumatology 66(10):2728-2738; Faustman and Davis (2013) Front. Immunol. 4:478; Schmidt etal. (2013) Arthritis & Rheumatism 65(9):2262-2273; Vanamee etal. (2017) Trends in Molecular Medicine 23(11):P1037-P1046; Chen et al. (2013)1 ImmunoL 190(3):1076-1084). In one study, in vitro produced antigen-specific 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). Using Tregs in cellular therapy, while promising, due to manufacturing and other complications, a traditional biologic therapeutic that provides the advantages of Tregs without the complications is needed.
As described and provided herein, TNFR2, and its expression by Tregs, is required for the suppression of inflammatory and autoimmune diseases and RECTIFIED SHEET (RULE 91) ISA/EP

conditions. For example, the mycobacterium bovis bacillus Calmetter-Guerin (BCG) induces transient expansion of Tregs. In a clinical trial, BCG triggered Treg production in patients with type I diabetes, resulting in suppression of disease and temporary restoration of islet cell function, indicating a use of 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):11O. doi: 10.1007/s11892-016-0807-6).
It is described and established herein that modulation of Treg function presents a therapeutic approach for the prevention or treatment of inflammatory and autoimmune diseases and conditions. Tregs, however, 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. Alternative approaches have used TL1A-Ig, a naturally occurring TNF receptor superfamily agonist, or TNFR2 monoclonal antibody agonists, to expand Tregs in vivo and ex vivo, respectively. 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-TNFR1 activity. As described and provided herein, 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).
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.
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. To be useful as a therapeutic, 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 (MDSCs) also express TNFR2, and inhibition of TNFR2 in MDSCs control metastasis, as shown in a murine liver cancer model. Thus, blockade of TNFR2, such as through the .. use of non-aggregating antagonistic antibodies or other therapeutics, as provided herein, presents a useful treatment for certain types of cancers via the inhibition of immunosuppressive Tregs. TNFR2 antagonists, however, only should be administered to patients whose tumors show overexpression of TNFR2 compared to adjacent normal tissue as judged from immunohistochemistry. Thus, such treatment should be accompanied by diagnostics to confirm overexpression (see e.g., Zhang et at.
(2019) Thorac Cancer 10(3):437-444. doi:10.1111/1759-7714.12948; Yang et al. (2017) Oncol Lett. /4(2):2393-2398. doi:10.3892/01.2017.6410; and Yang et al. (2018) Oncol Lett. 16(3):2971-2978. doi:10.3892/01.2018.8998, for exemplary assays).
4. Autoimmune / Inflammatory Diseases Mediated by or involving TNF
Elevated levels or uncontrolled expression of TNF and deregulation of TNF
signaling can cause chronic inflammation, which can result in the development of autoimmune diseases and tissue damage. TNF-a 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. The 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 at. (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.
Exemplary autoimmune and inflammatory diseases and disorders, that can be treated with the constructs provided herein, are discussed below.
a. Arthritis Rheumatoid Arthritis and other types of arthritis Rheumatoid arthritis (RA) 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 (TNFa) 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., Bluml et at. (2012) International Immunology 24(5):275-281; Schmidt et at.
(2013) Arthritis & Rheumatism 65(9):2262-2273; Keffer et at. (1991) EMBO

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 Inflammatory bowel disease (113D) 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.
c. Fibrotic Diseases Constructs herein can be used for treatment of fibrotic diseases. 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. There are no approved therapies for early stage disease, which manifests as nodules that are quiescent for some time and that then become active and progress to cords and flexion deformities of the fingers, resulting in the loss of hand function. Treatment involves surgical excision (fasciotomy) of the diseased tissue or cords, or disruption of the cords using collagenase or needle fasciotomy. The surgical and non-surgical treatments have high rates of recurrence and complications. Therapeutic intervention at the early stages of disease, to prevent progression to cord development and the subsequent flexion contractures of the digits, is advantageous (see, e.g., Nanchahal et al.
(2018) EBioMedicine 33:282-288).
Myoflbroblasts, 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 RECTIFIED SHEET (RULE 91) ISA/EP

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. The use of anti-TNF
therapies, such as adalimumab, however, 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 at. (2018) EBioMedicine 33:282-288). Thus, other therapies are needed.
d. 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 77VFRSF1A 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. The majority of 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).
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. For example, ligand-independent gain-of-function of TNFR1 induces TRAPS pathophysiology, and certain mutations result in the constitutive activity of TNFR1, NF-KB and caspase 1. Traditional anti-TNF
therapies, including etanercept, infliximab, and others, are only partially effective in the treatment of TRAPS (see, e.g., Greco et at. (2015) Arthritis Research &
Therapy 17:93), and thus, other therapies are needed.

e. Other Diseases Mediated by or involving TNF
i. Neurodegenerative Diseases Aging and several neurodegenerative diseases are associated with elevated levels of TNF in the central nervous system (CNS). 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. In animal models of disease, TNF is implicated in chronic glial activation and impaired neuronal viability through its actions on TNFR1. In aged animals, neurologic changes include synaptic dysfunction and Ca2+ 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 Ca2+ channels (L-VSCCs); a similar effect is observed in hippocampal neurons of memory impaired aged rats. Studies in rats have shown that TNF blockade in the cerebellum accelerates learning in a delayed eyeblink task. Selective blockade of TNFR1 signaling, using XPro1595, 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 CA1 neurons. These results indicate that TNF signaling via TNFR1 is implicated in modifying the neurologic phenotype of aged animals, and can result in pathological changes associated with neurological diseases. (See, e.g., Sama et al. (2012) PLoS ONE 7(5):e38170).
a) Alzheimer's Disease 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). In animal models of AD, TNF promotes microglial activation, synaptic dysfunction, neuronal cell death, accumulation of plaques and tangles, and cognitive decline. For example, in a triple transgenic AD
mouse model (3xTg-Ad), with mutations in presenilin 1, amyloid precursor protein (APP) and tau, TNF levels were elevated in entorhinal cortex, coincident with the earliest appearance of pathology (see, e.g., McCoy et at. (2006)1 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. 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 (AP) in the brains of AD patients, which hinders efficient plaque removal by microglia. 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 (f3-CTF), in the hippocampus, cortex and amygdala. Genetic deletion of TNFR1 in 3xTgAD mice also prevents the LPS-induced accumulation of 0-CTF, which is neurotoxic. Neuronal cells bearing familial AD (FAD) mutations accumulate f3-CTF intracellularly, implicating its involvement in the pathogenesis of AD. These results indicate that soluble TNF
is a mediator of the effects of neuroinflammation on early, pre-plaque pathology in 3xTgAD mice, and that targeted inhibition of solTNF in the central nervous system (CNS) can slow the appearance of amyloid-associated pathology, cognitive deficits, and the progressive loss of neurons in AD (see, e.g., McAlpine et at. (2009) Neurobiol. Dis. 34(1):163-177).
b) Parkinson's Disease Parkinson's disease (PD) 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 nigrostriatal 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. 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. XENP345 also attenuated 6-0HDA-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 at. (2006)1 Neurosci. 26(37):9365-9375).
c) Multiple Sclerosis (MS) CNS-specific overexpression of TNF in transgenic mice results in spontaneous demyelination, which is indicative of a role of TNF in multiple sclerosis (MS). A
polymorphism in the gene encoding TNFR1 is linked to an increased susceptibility of developing MS. TNFR1 is necessary for the disease induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and 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).
Endometriosis TNF-a has been implicated in the pathophysiology of endometriosis. TNF-a 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:

2023). Peritoneal fluid TNF-a is produced locally by activated peritoneal macrophages, and TNF-a induces IL-8 secretion by peritoneal mesothelial cells.
The peritoneal fluid concentrations of TNF-a 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.
Cardiovascular Disease TNFa was the first cytokine to be identified in human atherosclerotic plaque;
TNFa 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 blockade of TNFa by TNF binding protein, or IL-1 by an IL-1 receptor antagonist, partially protects apoE knockout mice from atherosclerosis. Atherogenesis primarily is the result of the production of TNFa by myeloid cells. The plaque area in apoE-/- and TNF-/- mice on a high fat diet is half the size of the plaque area in mice that are apoE-/-. Transplantation of bone marrow from apoE4- and TNF-/- mice, into apoE-/- mice, reduced atherosclerotic lesion size by 83%.
Atherosclerotic lesion size also was reduced following treatment of apoE-/-mice with a recombinant soluble p55 (TNFR1) TNF blocker, indicating the role that TNF
plays in atherosclerosis. NF-KB signaling is involved in the production of TNF-a in human atherosclerotic plaques. The peripheral blood levels of TNFa in patients with cardiovascular disease also is correlated with the development of myocardial infarction. Cardiotoxicity primarily is attributed to TNF-induced cardiomyocyte apoptosis. The use of anti-TNF therapies, such as infliximab and etanercept, in clinical trials for the treatment of heart failure failed, and resulted in increased mortality; anti-TNF therapies have thus not been tested for the treatment of RECTIFIED SHEET (RULE 91) ISA/EP

cardiovascular disease (see, e.g., Udalova et at. (2016) Microbial Spectrum 4(4):MCHD-0022-2015; Kalliolias and Ivashkiv (2016) Nat. Rev. Rheumatol.
12(1):49-62). As a result, alternative therapies are required.
iv. Acute Respiratory Distress Syndrome (ARDS) Acute respiratory distress syndrome (ARDS) affects approximately 190,000 patients per year in the U.S. and has mortality rates of up to 40%. There are no effective therapeutics targeting the underlying pathophysiological mechanisms of ARDS. 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-a 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-a 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. In a randomized, placebo-controlled trial of nebulized GSK1995057 in 37 healthy humans challenged with a low dose of inhaled endotoxin, treatment with GSK1995057 attenuated pulmonary neutrophilia, inflammatory cytokine release, and signs of endothelial injury in bronchoalveolar lavage and serum samples. These results indicate the potential for pulmonary delivery of selective TNFR1 antagonists for the prevention and treatment of ARDS (see, e.g., Proudfoot etal. (2018) Thorax 73:723-730).
v. Severe Acute Respiratory Syndrome (SARS) and Subjects infected with severe acute respiratory syndrome coronavirus (SARS-CoV) present with fever and respiratory illness, general malaise and lower respiratory tract symptoms, including cough and shortness of breath, with an overall fatality rate of 10%. TNF signaling promotes pathogenesis of SARS, by inducing excessive inflammation, which causes significant tissue damage. The development of a cytokine release syndrome (CRS) plays a role in severe COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The persistent viral stimulation results, in some subjects in an increase in the levels of circulating cytokines, such as IL-6 and TNFa, which leads to reduced lymphocyte counts and triggers inflammatory organ damage, particularly to the lungs, and blood vessels.
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. The expression of the ACE2 receptor is upregulated by SARS-CoV-2 ihfection and by inflammatory cytokine stimulation. In SARS-CoV
infection, S-proteins induce the TNF-a-converting enzyme (TACE)-dependent shedding of the ACE2 ectodomain, which is a process that is strictly coupled to TNFa production. The loss of ACE2 activity due to shedding is associated with lung injury due to an increased activity of the renin-angiotensin system. 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 TNFa production can thus facilitate viral infection and result in organ damage, such as lung injury.
As discussed, regulatory T cells (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 CD251" and feature inducible levels of intracellular transcription RECTIFIED SHEET (RULE 91) ISA/EP

factor forkhead box P3 (FOXP3). Naturally-occurring Tregs express TNFR2 at a higher density than TNFR1. TNF signaling through TNFR2 promotes Treg activity:

TNF-mediated TNFR2 activates and induces proliferation of Tregs (100) and expression indicates maximally suppressive Tregs Thus, in the case when TNF is .. being overproduced in response to an infection (influenzas, SARS type viruses, endotoxemia) Treg's 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 etal. (2008) Proc. Nail. Acad Sci. USA. /05:7809-7814).
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 /0:93). 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.
TNFa inhibition reduces the severity of virally-induced lung diseases, such as those caused by respiratory syncytial virus (RSV) or influenza virus, in mice.
The depletion of TNF using anti-TNF antibody in these mouse models reduced the pulmonary recruitment of inflammatory cells, reduced the production of proinflanunatory cytokines (e.g., IFNy, 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. I Immunol. 31:2566-2573). These results indicate that 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 RECTIFIED SHEET (RULE 91) ISA/EP

pulmonary dysfunction that resembles SARS pneumonia. Elevated levels of TNFa 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.
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 at. (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.
As a result, 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. The constructs provided herein, thus, are ideal therapeutics for these indications.
D. THERAPIES FOR RHEUMATOID ARTHRITIS AND OTHER
CHRONIC INFLAMMATORY AND AUTOIMMUNE DISEASES AND
DISORDERS
There is no cure for rheumatoid arthritis (RA), but 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, Behcet's disease, inflammatory bowel disease (MD; e.g., Crohn's disease and ulcerative colitis), multiple sclerosis, and lupus, as well as for the treatment of some cancers.

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; Plaquenilg), an anti-malarial agent;
sulfasalazine (Azulfidineg), an anti-inflammatory drug; and leflunomide (Aravag), an immunosuppressant that inhibits dihydroorotate dehydrogenase. Biologic DMARDs include, for example, abatacept (Orenciag), a fusion protein that prevents T cell activation and contains the Fc region of IgG1 fused to the extracellular domain of CTLA-4; anakinra (sold, for example, under the trademark Kineretg), a recombinant human IL-1 receptor antagonist; rituximab (sold under trademarks, including) Rituxang, Truximag, MabTherag), a chimeric monoclonal antibody against CD20, which induces apoptosis in CD20+ cells, such as B cells;
tocilizumab (atlizumab, Actemrag, RoActemrag), a humanized monoclonal antibody against the IL-6 receptor (IL-6R); corticosteroids; tofacitinib (Xeljanzg), a small molecule inhibitor of Janus kinase (JAK), a protein tyrosine kinase involved in mediating cytokine signaling; and TNF-inhibitors/anti-TNF agents, such as, for example, certolizumab pegol (Cimziag), infliximab (Remicadeg), adalimumab (Humirag), golimumab (Simponig), and etanercept (Enbrelg). 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.
The following sections describe existing therapies, and the problems associated with each, to highlight how the therapies provided herein solve the problems.
1. Conventional Synthetic Disease Modifying Anti-Rheumatic Drugs (csDMARDs) Conventional synthetic Disease Modifying Anti-Rheumatic Drugs (csDMARDs) are typically the first line treatment for RA and other autoimmune and chronic inflammatory diseases and disorders. 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 production, reducing levels of IL-6 and IL-8, suppressing cell-mediated immunity, and inhibiting synovial collagenase gene expression. Other conventional synthetic DMARDs act by inhibiting the proliferation of lymphocytes or causing lymphocyte dysfunction. For example, 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).
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. Therapy with methotrexate, leflunomide, and sulfasalazine 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-antagonists that preserve TNFR2 immunosuppressive activity advantageously can avoid the need for these immunosuppressive drugs.
2. Anti-TNF Therapies/TNF Blockers Anti-TNF therapies/TNF-blockers (a type of biological DMARD) 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 IgG1 constant region), and the fully humanized mAbs (IgGls) adalimumab (Humirag) 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 (Enbre1 ), which contains the extracellular receptor region that contains the binding site of human TNFR2 fused to the Fc of human IgG1 . 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 (MD; e.g., Crohn's disease and ulcerative colitis).
Because of the immunosuppressive effects of therapies that target TNF, 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. 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. For example, TNF
blocking agents are associated with the reactivation ofM 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. As described herein, 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. Other anti-rheumatic drugs, such as methotrexate and glucocorticoids, also can induce apoptosis in immune cells (see, e.g., Vigna-Perez et at. (2005) Cl/n. 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).
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 at. 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/).
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 at. (2019) Sci. Rep. 9:17231) of developing invasive aspergillosis (IA), a life-threatening mold (i.e., fungal) infection caused by Aspergillus fumigatus. Treatment with etanercept results in the downregulation of genes involved in immune responses and TNF signaling, including genes involved in NF-KB 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).
Other side effects associated with the use of 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 at. (2016) Proc. Natl. Acad. Sci. USA
113(43):12304-12309; Zalevsky et at. (2007)1 Immunol. 179:1872-1883; Zoran et at. (2019) Sci.
Rep. 9:17231). Thus, 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. As a result, 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 at.
(2014) Arthritis & Rheumatology 66(10):2728-2738). Non-responsiveness also occurs in non-RA patients receiving anti-TNF therapeutics. Depending on the anti-TNF
agent, 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 at.
(2019) MABS 11(4):653-665). Thus, there is a need for therapies with improved therapeutic efficacy and safety.
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. As discussed elsewhere herein, 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. Thus, as described herein, 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 at. (2014) Arthritis & Rheumatology 66(10):2728-2738; Schmidt et at.

(2013) Arthritis & Rheumatism 65(9):2262-2273; Bluml et at. (2012) International Immunology 24(5):275-281; Zalevsky et al. (2007)1 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. For example, in a phase II trial for the treatment of relapsing remitting MS, a TNFR1 receptor-Fc IgG1 fusion protein anti-TNF therapeutic, lenercept (Ro 45-2081), failed, and symptoms were increased/worsened compared to patients receiving a placebo, with neurologic deficits being more severe in lenercept-treated patients. These results indicate that anti-TNF
therapies can aggravate demyelinating diseases. While 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. The blockade of TNFR1 with ATROSAB, a humanized monoclonal antibody that blocks TNFR1, or the activation of TNFR2 with EHD2-scTNFR2, an agonistic TNFR2-selective TNF mutein, results in the protection of cholinergic neurons against cell death, and reverts the neurodegeneration-associated memory impairment in a mouse model of NMDA-induced acute neurodegeneration. This is likely to be immunogenic. ATROSAB is a partial TNFR1 agonist; those of skill in the art would not administer a TNFR1 agonist. The blockade of TNFR1 and TNFR2, however, 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 at. (2016) Proc.
Natl. Acad. Sci. USA 113(43):12304-12309).
Due to the adverse effects associated with the use of anti-TNF agents, the non-responsiveness of some patients, the lack of a sustained response in patients that had an initial response, and the failure to treat and/or exacerbation of neurodegenerative diseases, such as MS, other therapies are needed. Such therapies are provided herein.

E. THERAPEUTICS FOR TARGETING TNFR1/TNFR2 The following section discusses exemplary therapeutics that target TNFR1/TNFR2, and describes some problems and limitations with these therapeutics.
Existing therapeutics can be modified as described in Section F and the Examples, or are used, in whole or in part, or are modified to improve their properties for use, in the constructs that are provided herein.
1. TNFR1-Selective Antagonists As discussed and provided herein, therapy with TNF blockers, such as etanercept, infliximab, adalimumab, and others) abrogates TNF signaling via and TNFR2. While 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. Therapy with 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. , 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.
a. TNFR1 antagonistic Antibodies Among TNFR1 antagonist antibodies, is ATROSAB (Antagonistic TNF
Receptor One-Specific Antibody). ATROSAB, the first TNFR1 blocking antibody, is a full-length IgG1 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 Fe region in ATROSAB is mutated to RECTIFIED SHEET (RULE 91) ISA/EP

eliminate FCyR receptor binding and complement fixation, thereby avoiding unwanted immune system activation (see, e.g., Kalliolias and Ivashkiv (2016) Nat.
Rev. Rheumatol. 12(1):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. For example, 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. The cross-linking of TNFR1 also can occur due to secondary events, such as interactions with FcyRs or anti-drug antibodies (ADAs), which must be avoided to maintain the antagonistic nature of TNFR1 inhibitors. ADAs have been observed in patients treated with infliximab or adalimumab at rates of 50% and 31%, respectively (see, e.g., Richter et al. (2019) mAbs 11(4):653-665; Richter et al., (2019) mAbs 11(1):166-177).
b. Monovalent TNFR1 antagonistic Antibodies/Antibody Fragments 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, however, 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. For example, 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. For example, 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 (antiTNFR1 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. coil) often lack the conserved disulfide bond found in variable heavy domains, which both decreases their melting point and can decrease their ability to refold. 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, which can reduce patient compliance. Because these molecules (see, e.g., Holland et al. (2013) J Clin Immunol 33(7):1192-203) were produced in E coil, and were often not correctly folded, leading to poor solubility and immunogenicity, resulting in their clinical failure (see, e.g., adisinsight.springer.com/drugs/800037882).
The 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. Provided herein are constructs containing small antibody fragments, such as dAbs, with specificity towards TNFR1 and/or TNFR2, that exhibit improved pharmacological, such as pharmacokinetic, properties, including longer serum half-life, increased stability, reduced/slower peripheral clearance, and lower immunogenicity compared to the dAbs of the prior art.
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 Humirag); cancers, such as non-Hodgkin's lymphoma (e.g., rituximab and ibritumomab tiuxetan, sold under the trademarks Rituxang and Zevalin , respectively) and breast and gastric cancers (e.g., trastuzumab, sold under the trademark Herceptinc)); and respiratory syncytial virus infection (e.g., palivizumab, sold under the trademark Synagis ). Manufacturing of complete antibodies has several limitations, such as the reliance on mammalian cell expression.
As a result, 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 VH-CH1 polypeptide, linked to a VL-CL polypeptide via a disulfide bond; an scFv is a fusion protein containing a VH domain and VL 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 (Vii) or of the light chain (VL) 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).
I. Fab- and scFv-Based TNFR1 antagonists As discussed above, the 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 TNF. The parental mouse antibody, H398, possesses stronger inhibitory potential, which is due to the faster dissociation of ATROSAB (i.e., a higher koff value) compared to H398.
This was determined using quartz crystal microbalance (QCM) measurements, in which antigen density on the chip was reduced to favor monovalent interactions; a slower dissociation of monovalently bound H398 from TNFR1, and the resulting longer receptor occupation, contributes to the improved blockade of TNFR1.
Thus, to eliminate the TNFR1 agonistic activity of ATROSAB, and to improve its TNFR1 antagonistic activity, monovalent derivatives of ATROSAB were developed.
To increase the affinity and antagonistic activity of ATROSAB, 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 TNFR1-Fc.
RECTIFIED SHEET (RULE 91) ISA/EP

The scFv of ATROSAB contains the VH domain, corresponding to residues 1-115 of the ATROSAB heavy chain (see, SEQ ID NO:31), linked by a short peptide linker to the VL 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, 555Q, H57E, Y59K, and E62D, with reference to SEQ ID NO:31, exhibited slower receptor dissociation and improved equilibrium binding to human TNFR1-Fc, and improved inhibition of TNF-induced TNFR1 activation. This clone was further subjected to random mutagenesis, generating the clone scFv T12B (see, SEQ ID NO:675), containing the mutations Q1H, Y52V, Y545, 555Q, H57E, Y59K, and E62D in the VH domain (with reference to SEQ ID NO:31), and 596G in the VL domain (with reference to SEQ ID NO:32).
scFv T12B had reduced dissociation from immobilized TNFR1-Fc compared to the scFv of ATROSAB and to scFv IG11, and increased TNFR1 inhibitory 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/d8e7/8b 87d76dce36225 cld497939ef37445 cfa8 a.
pdf).
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 TNFR1-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. Based on scFv 13.7, an IgG and a Fab, with constant regions identical to ATROSAB, 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 TNFR1-Fc, compared to Fab ATR, with an
18.8-fold improved monovalent affinity. Thus, 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 TNFR1-Fc and did not bind to a TNFR2-Fc fusion protein; Fab 13.7 bound to human and rhesus TNFR1-Fc, but not to mouse and rat TNFR1-Fc, showing a similar binding pattern to ATROSAB. In vitro, 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 etal. (2019) ntAbs 11(1):166-177).
Compared to ATROSAB, which has an initial half-life of 0.44 hours, a terminal half-life of 32.1 hours, and an area under the curve (AUC) of 181 ps/m1 x h, Fab 13.7 (with a molecular mass of 47 IcDa). 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 [tg/m1 x h, which were similar to the values obtained for Fab ATR. To extend the half-life, a Fab' fragment, Fab' 13.7, was generated by introducing a free cysteine residue at the C-terminus of the CH1 domain, which was chemically coupled to a branched PEGoidm 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 Fab13.7-MSA.
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 (IgG1ha1f13.7). A
monovalent Fv-Fc molecule also was generated by fusing the VII and VL domains to a hetero-dimerizing knob-into-hole (kih) Fc chain lacking the cysteine residues in the hinge region (Fv13.7-Fck,h). None of the derivatives showed any agonistic activity, and, compared to Fab 13.7, a slightly reduced binding to human TNFR1-Fc was observed for Fab13.7PEG, Fab13.7-MSA and IgG1ha1f13.7; binding of Fv13.7-Fckth was not affected. Inhibition of TNF-mediated TNFR1 activity was reduced by 1.5-3.3 fold compared to Fab 13.7; Fab13.7PEG showed the strongest impairment in function, and Fv13.7-Fckin showed the lowest change in bioactivity.
IgGhaif13.7 showed a similar half-life to Fab 13.7, and an AUC value that was increased by 7.1-fold. Fab13.7PEG, Fab13.7-MSA and Fv13.7-Fckin had extended terminal half-lives, RECTIFIED SHEET (RULE 91) ISA/EP

with values of 14.4 h, 9.7 h, and 10.5 h, respectively, and increased AUC
values.
Thus, the fusion protein Fv13.7-Fchh, 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/d8e7/8b 87d76dce36225 cld497939ef37445 cfa8 a.
pdf).
In another study, to improve the pharmacokinetic properties, such as serum half-life, of Fab 13.7, an IgG-like Fc was incorporated into the molecule, while retaining the Fab-like heterodimerization of the polypeptide chains. To achieve this, the variable domains of the heavy and light chains of Fab 13.7 were fused to the N-termini of newly generated heterodimerizing Fc chains, known as Fc-one/kappa (Fclic). The Fc heterodimerization approach is based on interspersed Ig domains, derived from the heterodimerizing IgG1 constant heavy chain domain, CH1, and the kappa light chain constant domain, CLK, and containing sections of the IgG1 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 CH1 and CH3, and "CH3kappa" (CH3K), which contains amino acid sequence fragments of CD< and CH3. IgG1 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. Addition of the IgG1 hinge region to the N-termini of the domains results in a covalently linked heterodimerizing Fc moiety, known as Fclic. In contrast to other Fc heterodimerization technologies, such as knobs-into-holes, which involve replacement of one or more amino acids at the CH3-CH3 interface, Fc heterodimerization was achieved by exchanging larger amino acid sequence stretches obtained from human antibody sequences. Asymmetric scFv-Fclic fusion proteins were prepared and compared to scFv fusions with Fcs 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 at. (2019) mAbs 11(4):653-665).

The variable domains of the TNFR1-specific Fab 13.7 molecule were fused to the CH2 domain N-termini of the CH31- or CH3x-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-CH3x; VL1C/VIIKC), generating the .. monovalent TNFR1-specific antagonistic antibody-derived molecule (Fv-Fc1x 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 Fclx; the lack of binding to effector molecules of the immune system prevents the activation of due to secondary crosslinking of Atrosimab bound to cells expressing FcyRs.
Atrosimab bound to TNFR1 with high affinity (KD 2.7 nM), inhibited TNF-induced activation of TNFR1 with IC50 values of 16-55 nM in various in vitro assays, and in the presence of anti-human IgG antibodies (i.e., cross-linking antibodies), and displayed improved pharmacokinetic properties. Compared to the parental Fab 13.7 molecule, 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 [tg/m1 x h. The terminal half-life of Atrosimab was extended almost 40-fold compared to that of Fab 13.7, and was extended by 1.3-fold compared to ATROSAB; these values may be inaccurate, however, because the injected doses of Fab 13.7 and ATROSAB were lower, which can affect pharmacokinetic properties (see, e.g., Richter et at.
(2019) mAbs 11(4):653-665).
Domain Antibody (dAb)-Based TNFR1 antagonists Another class of therapeutics, 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 (VH) or of the light chain (VI) 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). Similar to dAbs, are the nanobodies that occur in camelids, which produce antibodies that contain only heavy chains, where the antigen-binding site is a single unpaired variable domain, known as a VHH.
In a dAb, there are three complementarity determining regions (CDRs) on each VH and each VL; thus, each dAb contains three out of the six CDRs from a VH-VL pair in an antibody, which are the highly diversified loop regions that bind to the target antigen.
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 at. (2003) Trends in Biotechnology 21(11):484-490; Schmidt et at.
(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. As with other antibody fragments, 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 at. (2003) Trends in Biotechnology 21(11):484-490; Goodall et al. (2015) PLoS ONE 10(9):e0137065).

a) Anti-TNFR1 dAb-Anti-Albumin dAb Fusion Constructs 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-TNFR1 dAb, fused with an albumin-binding dAb (AlbudAb; to extend serum half-life). DMS5540, which does not bind human TNFR1, was found to inhibit TNFa-mediated cytotoxicity in the mouse fibroblast cell line L929 (which is highly sensitive to TNFa-mediated cytotoxicity). DMS5540 was administered to mice intravenously, followed four hours later with an intravenous bolus injection of TNFa, and serum IL-6 levels were assessed. DMS5540 demonstrated a dose-dependent inhibition of TNFa-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 at.
(2015) PLoS ONE 10(9):e0137065).
In another study, mice with collagen-induced arthritis (CIA) 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 IgG1 Fc domain (mTNFRII-Fc; mTNFR2.Fc), which blocks both receptors (TNFR1 and TNFR2) and inhibits mouse TNF, and disease progression was monitored. 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. 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., IFNy, 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. Additionally, 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. These results indicate that inhibiting TNFR1, but not TNFR2, 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 at.
(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. TNFR2-deficient mice displayed an increase in LPS-induced bone destruction. In vitro, the human equivalent of DMS5540, DMS5541, which contains an anti-human TNFR1 dAb, reduced human osteoclastogenesis in the presence and absence of low-dose TNF more effectively than etanercept. These results indicate an osteo-protective role for TNFR2 signaling.
As a result, 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 at.
Biochem. Biophys. Res. Commun. 464:1145-1150).
DMS5541 (also known as TNFRI-AlbudAb), which contains a noncompetitive human TNFR1-specific dAb fused to AlbudAb, was evaluated for the selective blockade of TNF signaling via TNFR1, in ex vivo cultured human rheumatoid arthritis (RA) synovial membrane mononuclear cells (MNCs), which express TNFR1 and TNFR2, and produce inflammatory cytokines and chemokines spontaneously, in the absence of exogenous stimulation. DMS5541 inhibited the production of the proinflammatory cytokines GM-CSF, IL-10, IL-10 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 TNFa-induced cytotoxicity in human rhabdomyosarcoma KYM-1D4 cells in a dose-dependent manner, similar to TNF blockade with etanercept.
In addition, DMS5541 inhibited the production of soluble TNFR1, but not soluble TNFR2, demonstrating selectivity for TNFR1. These results indicate that the pathway is the dominant inflammatory pathway that is responsible for the TNF
response observed in the ex vivo cultured RA synovial membrane MNC disease model (see, e.g., Schmidt et at. (2013) Arthritis & Rheumatism 65(9):2262-2273).

b) Domain antibody fragments designated GSK1995057 and GSK2862277 The domain antibody fragment designated GSK1995057 (see, SEQ ID NO:55) is a short-acting, fully human domain antibody (dAb) fragment (containing a VH
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. 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-a contributes to increased endothelial permeability, and GSK1995057 prevents this increase, indicating that TNFR1 signaling mediates TNF-induced endothelial permeability (see, e.g., Proudfoot et at. (2018) 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 coil 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. W02008/149148A2).
In monkeys exposed to a single inhaled lipopolysaccharide (LPS) challenge, which is a well-established model that triggers a clinically relevant inflammatory response modeling subclinical tissue injury, 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. In a clinical trial, in which healthy human subjects were pretreated with a single nebulized dose of GSK1995057 and then exposed to a low dose of inhaled LPS, the pretreated subjects experienced less systemic inflammation, as well as less neutrophilic lung inflammation and signs of endothelial injury in response to LPS challenge, in comparison to subjects who received a placebo. Despite these results, translation into the clinic is not likely. In the trial GSK1995057 was administered prior to the LPS challenge, but patients with ARDS, generally require treatment after the initial injury (see, e.g., Proudfoot et al.
(2018) Thorax 73:723-730), not before.
Another difficulty is the detrimental effects of anti-drug antibodies (ADAs) on anti-TNFR1 agents, which were observed in a clinical Phase I study of GSK1995057, in which cytokine release infusion reactions, at doses of 2-10 g/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. Specifically, the ADAs were human anti-VH (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).
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 VH dAbs, including the dAb GSK1995057.
To counter this, 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. In serum samples from healthy subjects that screened positive for HAVH autoantibodies that bind to GSK1995057, the frequency of pre-existing autoantibodies decreased from 51%
for GSK1995057-specific HAVH autoantibodies, to 7% for GSK2862277-specific autoantibodies. Human in vitro systems and animal in vivo experiments showed that GSK2862277 does not induce TNFR1 activation, even in the presence of GSK2862277-specific autoantibodies, and that the pharmacology and biophysical properties of GSK2862277, including target affinity, in vitro potency and in vivo pharmacokinetics and pharmacodynamics, is comparable to those of the parent dAb (GSK1995057).
A Phase I clinical trial to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of single and repeat doses of inhaled (i.h.) and intravenous (i.v.) GSK2862277 found that GSK2862277 was generally well tolerated when RECTIFIED SHEET (RULE 91) ISA/EP

administered via inhalation or intravenously. One subject, however, had a mild infusion reaction with cytokine release after repeated IV dosing; this subject had high serum levels of pre-existing antibodies to GSK2862277, and the serum antibodies from this subject were shown to activate TNFR1 signaling in an in vitro assay.
The interaction between GSK2862277 and the autoantibodies result in antibody-mediated, GSK2862277-dependent cross-linking of cellular TNFR1, agonizing the receptor and leading to cytokine release. Thus, despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, adverse effects were still associated with the presence of a new pre-existing antibody response, that was specific to the modified dAb framework. These results highlight challenges in developing biological antagonists against TNFR1 (see, e.g., Cordy et al. (2015) Cl/n. Exp. Immunol. 182:139-148).
Thus, there remains a need for improved TNFR1 antagonists.
Nanobodies (Nbs) Similar to dAbs, nanobodies (Nbs) 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.
iv. Anti-TNFR1 Nanobody-Anti-Albumin Nanobody Fusion Constructs TROS (TNF Receptor One-Silencer; also called Nb Alb-70-96) is a trivalent high-affinity nanobody-based selective inhibitor of human TNFR1 that competes with TNF for binding to TNFR1. To generate TROS, two anti-human TNFR1 nanobodies (Nb 70 and Nb 96; see, SEQ ID NOs: 683 and 684, respectively), which had been generated from a VHH library that was constructed by immunization of alpacas with recombinant human soluble TNFR1, were linked and, via (Gly4Ser)3 linkers, were linked to an anti-albumin nanobody (Nb Alb) to increase serum half-life to produce the trivalent TROS. The serum half-life of the resulting TROS is ¨24 hours;
the serum half-life of monovalent Nbs is only ¨1.5 hours. Treatment with TROS delays disease onset in mouse experimental autoimmune encephalomyelitis (EAE; a model of MS), and prevents established disease; the therapeutic effects are due to the diversion of TNF to signal through TNFR2, and the effects of such signaling. 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 at. (2015)1 Biol. Chem.
290(7):4022-4037; Steeland et al. (2017) Sci. Reports 7:13646).
c. Dominant-Negative Inhibitors of TNF (DN-TNFs)/TNF
Muteins Another class of 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-KB 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.
As selective inhibitors of soluble TNF, 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, 586Q, 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 at. (2007)1 Immunol. 179:1872-1883).
For example, the TNF mutations R32W and 586T, with reference to SEQ ID
NO:2, result in a several hundred-fold loss in affinity towards TNFR2, but do not affect binding to TNFR1. The R32W/586T double mutant abrogates all binding to TNFR2, with no loss in binding to TNFR1. The mutations L295, L29G, L29Y, R31E, R3 1N, R32Y, R32W, 586T, L295/R32W, L295/586T, R32W/586T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, and E146R, with reference to SEQ ID NO:2, also impart selectivity towards 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 at. (1993)1 Biol. Chem.
268(35):26350-26357; U.S. Patent No. 5,422,104).
A modified TNF, designated XPro1595 (INmuneBio; see, SEQ ID NO:701), is a PEGylated, soluble DN-TNF mutein that preferentially inhibits TNFR1 signaling, and contains the mutations V1M, R31C, C69V, Y87H, C101A and A1456R, with reference to SEQ ID NO:2 (see, e.g.,U U.S. Publication No. 2015/0239951).
XPro1595 decreases neuroinflammation and is being investigated in the treatment of Alzheimer's disease (see, e.g., clinical trial identifier No. NCT03943264).
XPro1595 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, XPro1595 reduced amyloid, improved cognition, rescued neuron communication, and also, normalized innate and adaptive immune responses.
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.
When treated with XPro1595, aged rats exhibit improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased levels of the GluR1 type glutamate receptors, and lower L-type voltage sensitive Ca2+ channel (L-VSCC) activity in hippocampal CA1 neurons, indicating that functional changes associated with brain aging can occur from selective alterations in TNF signaling. In animal models of Parkinson's disease and aging, XPro1595 suppresses neuroinflammation and the activation of microglia.
In EAE (a model of MS), XPro1595 ameliorates disease, improves remyelination and reduces CNS lesions and neuroinflammation. XPro1595 also ameliorates inflammatory arthritis, and decreases susceptibility to infection in treated animals. In comparison to etanercept, which had no therapeutic effect, treatment with XPro1595 delayed the onset of EAE and ameliorated symptoms more efficiently. XPro1595 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 at. (2018) Front. Immunol. 9:784; Sama et at. (2012) PLoS ONE
7(5):e38170). Since XPro1595 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 (see, SEQ ID NO:702) 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)1 Neurosci. 26(37):9365-9375;
McAlpine et at. (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 A845, V85T, 586T, 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). To increase the in vivo half-life of RlantTNF, a PEGylated version, PEG-RlantTNF, 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 Th17 cell activation and inflammatory T-cell infiltration in the spinal cord. PEG-RlantTNF also inhibits NF-KB, 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. When the effects on antiviral immunity of PEG-RlantTNF and etanercept were compared using a recombinant adenovirus vector, PEG-RlantTMF 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 at.
(2018)Front. Immunol. 9:784; Shibata et at. (2008) J Biol. Chem. 283(2):998-1007;
Kitagaki et at. (2012) J Atheroscler. Thromb. 19(1):36-46; Fischer et at.
(2015) Antibodies 4:48-70; Horiuchi et al. (2010) Rheumatology (Oxford) 49:1215-1228).
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. In contrast to inhibitors of solTNF, such as DN-TNFs, TNFR1 antagonists can block the binding of lymphotoxin-a (LT-a), another member of the TNF superfamily, to TNFR1. 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 at.
(2015) Antibodies 4:48-70).
2. TNFR2-Selective Agonists 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. The Treg-stimulatory effect of the TNF-TNFR2 signaling pathway can be leveraged for the treatment of several human diseases and disorders, including autoimmune and chronic inflammatory diseases, through agonism, and cancer, through antagonism (see, e.g.,Zou et at. (2018) Front. Immunol. 9:594).

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. 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. Thus, 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.
a. TNFR2 agonistic Antibodies Human TNFR2-selective agonist antibodies include the commercially available MR2-1 (a monoclonal mouse IgG1 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. 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 at. (2018) Front. Immunol. 9:594).
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+CD1271' 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. (2018) Front. Immunol. 9:594).
b. TNFR2-Selective TNF Muteins and Fusions Thereof As described herein, 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 U.S. Patent No. 9,081,017). Other mutations in TNF that impart selectivity for TNFR2 include, but are not limited to, for example, K65W, D143E, D143W, D143V, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, and D143V/A1455, with reference to SEQ ID NO:2 (see, e.g., U.S. Patent Publication No. 2020/0102362).
TNF ligand trimerization is essential for signaling via TNFRs. At low concentrations, such as in serum, the trimers dissociate, resulting in their degradation.
To generate functionally active, receptor-specific TNF muteins, it is necessary to create stable trimers. TNFO7 is a soluble TNF (sTNF or solTNF) mutein, containing the mutations 595C/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. The S95C/G148C mutations result in the formation of an intermolecular Cys-Cys covalent bond; a stable trimer is thus formed as a result of covalent internal disulfide cross-linking of sTNF at a strategic location between TNF monomers. TNFO7 acts as a TNFR2 agonist despite lacking TNFR2-selective mutations. TNFO7 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. (2018) Front.
Immunol.
9:594).
Several TNFR2 agonists, containing fusions of single-chain TNFR2-selective TNF mutein trimers, with multimerization domains, have been generated. As described herein, the primary ligand for TNFR2 is membrane-bound TNF (memTNF;
also referred to herein as transmembrane TNF or tmTNF). The addition of 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. Commonly used dimerization domains include EHD2, which is derived from the heavy chain CH2 domain of IgE
and MHD2, which is derived from the heavy chain CH2 domain of IgM.
Dimerization domains also can include Fc domains, such as those derived from IgG1 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.
STAR2 (also known as 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. Residues 116-120), and the 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 trimerization 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. Pretreatment of allogeneic hematopoietic stem cells with STAR2 prior to transplantation in mice prolonged the survival and decreased the severity of GvHD in a TNFR-2 and Treg-dependent manner. 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 etal. (2016) J Exp. Med. 213(9):1881-1900; Zou etal. (2018)Front. Immunol.
9:594).
TNC-scTNFR2 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 (scTNFR2; SEQ ID NO:803), contains three TNF domains connected by two short peptide linkers (GGGGS). The TNFR2-selective TNF
molecule, scTNFR2, 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 scTNFR2, generating TNC-scTNFR2. TNC-scTNFR2 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 (memTNF) activity, induces the clustering of TNFR2 and the formation of TNFR2 signaling complexes, efficiently activating TNFR2. TNC-scTNFR2 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-scTNFR2 rescued neurons after induction of cell death by 6-0HDA. These results indicate that TNC-scTNFR2 can ameliorate neurodegenerative processes (see, e.g., Fischer et at. (2011) PLoS ONE 6(11):e27621).
EHD2-scTNFR2 (see, SEQ ID NO:810) is an agonistic TNFR2-selective TNF
mutein fusion protein that contains a covalently stabilized human TNFR2-selective single-chain TNF trimer (scTNFR2; 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 CH2 domain of IgE, and creates a disulfide bonded dimer that contains hexameric TNF domains. Each TNF subunit within scTNFR2 contains residues 4-157 of SEQ ID NO:2. EHD2 is fused to the N-terminal end of the trivalent human single-chain scTNFR2 via a peptide linker (GGGSGGGSGGGSGGGSGGGSGGSEFLA; SEQ ID NO:809), and the three TNF domains of scTNFR2 are connected via two GGGGS peptide linkers. EHD2-scTNFR2 exhibits neuroprotective properties in a mouse model of NMDA-induced acute neurodegeneration (see, e.g., Dong et at. (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 (scTNFR2) 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 (scTNFR2-Fc). The Fc can be an IgG4 or IgG1 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 (scTNFR2) by a third short peptide linker. The three linkers can all be the same or can be different, and can include GS linkers, such as (GGGGS),, residues 116-121 of SEQ ID NO:707, and/or other combinations of Gly and Ser), where n = 1-5, or can include all or a portion, at least 10, 15, or 20 contiguous residues, of the stalk region of TNF-a (GPQREEFPRDLSLISPLAQAVRSSSRTPSDK (SEQ ID NO:812), corresponding to residues 57-87 of SEQ ID NO:1). Dimerization enhances signaling by the TNFR2 agonist, and also improves the half-life of the fusion protein. Alternative dimerization domains that can be used in the fusion proteins include Fc fusion proteins derived from other dimerizing molecules, such as the IgE heavy chain domain 2 (EHD2;
see, SEQ ID NO:808) and IgM heavy chain domain 2 (MHD2; see, SEQ ID NO:811) (see, e.g., International Application Publication No. WO 2019/226750).
3. Anti-TNFR2 Antagonistic Antibodies and Small Molecule Inhibitors 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. TNFR2 antagonists also induce the expansion of T effector cells, including cytotoxic CD8+ T cells, via the inhibition of Treg expansion and activity. As a result, 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 at. (2017) Sci.
Signal.
10:eaaf8608).
As discussed herein, expression of 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 NO:4 (see, e.g., U.S. Patent Publication No. 2019/0144556).
In general, 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 (e.g., less than a 10-fold difference in affinity), are not antagonistic TNFR2 antibodies.
.. Antagonistic TNFR2 antibodies include TNFRAB1 (see, SEQ ID NOs:1213 and 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, for example, 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. 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). These data indicate that the CDR-H3 sequence of an antagonistic antibody largely dictates the antigen-binding properties, and that the CDR-H3 motif is a modular domain that can be substituted into anti-TNFR2 antibodies that do not exhibit antagonistic activity, in order to impart such antibodies or antigen-binding fragments thereof with TNFR2 dominant antagonistic features. For example, replacement of the CDR-H3 sequence of a neutral anti-TNFR2 antibody (i.e., an antibody that is neither antagonistic nor agonistic), with the CDR-H3 of an antagonistic TNFR2 antibody, such as the CDR-H3 sequences of TNFRAB1, TNFRAB2 or TNFR2A3, for example, 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 (see, e.g., U.S. Patent Publication No.
2019/0144556).
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:1213; or the CDR-L3 sequences set forth in any of SEQ ID NOs: 1221, 1222, and 1241-1244, or the CDR-L3 sequence of TNFRAB1, corresponding to residues 88-96 of SEQ ID NO:1213. 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 at., 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)1 Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. I Immunol. 24:827-836). In this way, the variable domain framework residues and CDRs can be identified by sequence alignment. One can substitute, for example, the CDR-H3 of the consensus human antibody with the CDR-H3 of an antagonistic TNFR2 antibody, such as the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, to produce a humanized TNFR2 antagonist antibody. 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.

Other 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 at. (2018) 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.
As a result, TNF-induced activation of NF-KB pathways in Tregs is inhibited, and conversion of transmembrane TNFR2 (tmTNFR2) to soluble TNFR2 (sTNFR2) is suppressed. 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. These results indicate the therapeutic potential for TNFR2 antagonists in the treatment of tumors, by targeting tumor-infiltrating Tregs as well as tumor cells (see, e.g., Zou et al. (2018) Front. Immunol. 9:594; Torrey et al. (2017) Sci.
Signal.
10: eaaf8608).
In addition to anti-TNFR2 antagonistic mAbs, small molecules can inhibit TNFR2. For example, 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-KB, 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. It has been shown that 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. In patients with multiple myeloma, however, 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. (2018) Front. Immunol. 9:594).
Another small molecule inhibitor of TNFR2 is 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 IFNy and IL-2 production by effector T cells results in a therapeutic effect in these patients (see, e.g., Zou et at. (2018) .. 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. In a mouse model of mesothelioma, cyclophosphamide treatment depleted TNFR2hi Tregs. The combination of cyclophosphamide with etanercept 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 at. (2018) 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. (2018) Front. Immunol. 9:594).
F. SELECTIVE TARGETING OF THE TNFR1 AND/OR TNFR2 AXIS
As described herein, existing 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, however, 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. Other side effects associated with the use of 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.
Provided herein are 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 axis. These therapeutic approaches include, but are not limited to treatments of autoimmune, chronic inflammatory, neurodegenerative, and demyelinating, diseases, disorders and conditions, and cancer, which also has an inflammatory component. As described herein, concomitant or sequential selective agonism of TNFR2 with 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-KB 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.
1. Selective Blockade of TNFR1 with TNFR1 antagonists The use of multivalent agents, such as antibodies against TNFR1, however, is not feasible. 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 at. (1994)1 Immol. 153(5):1963-72). They 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 at.
(2019) Sci Signal. 12(592):eaav5637).
As a result, antibodies and other multivalent agents that bind to TNFR1 likely are not suitable for use as antagonists, because they can cause super-clustering leading to activation of TNFR signaling. 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 As described herein, selective activation of TNFR2 can be achieved using 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 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.
In contrast to the antagonism of TNFR1, to agonize TNFR2, dimeric and trimeric molecules are used that mimic the action of membrane-bound TNF, which is the primary ligand that activates TNFR2. As such, TNFR2 agonists include TNFR2-selective TNF muteins and antibody fragments. Exemplary are TNF mutein and antibody fragments that fused with multimerization domains, particularly dimerization or trimerization domains, as discussed below. For extending half-lives of these molecules they can be associated with or coupled to polyethylene glycol with or without cleavable linkers (see, e.g., Santi et at. (2012) Proc. Natl. Acad.
Sci. U.S.A.
109:6211-6216), or fused or bound to 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 residues in length), ELPylation (see, e.g., Floss et at. (2010)1 Trends Biotechnol.28(1):37-45), Hapylation (glycine homopolymer), fusion to human serum albumin, fusion to GLK, fusion to CTP, GLP fusion, fusion to the constant fragment (Fc) domain of a human immunoglobulin (IgG), fusion to transferrin, fusion to non-structured polypeptides such as XTEN (also referred to as rPEG, genetic fusion of non-exact repeat peptide sequence, containing A, E, G, P, S, and T, see, e.g., Schellenberger et al. (2009) Nat BiotechnoL 27(12):1186-90), and other such modifications and fusions that increase size, increase hydrodynamic radius, alter charge, or target to receptors for recycling rather than clearance, and combinations of such modifications. Particular examples of extenders of half-life are discussed and exemplified in detail below.
3. TNFR1 antagonist constructs, TNFR2 agonist constructs; Multi-Specific, Including Bi-Specific, TNFR1 Antagonist and TNFR2 Agonist Constructs Thus, provided herein are constructs for inhibiting TNRFR1 signaling/activity .. and/or for agonizing TNFR2. Included among the constructs provided herein are 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.
Among the constructs provided herein are TNFR1 antagonist constructs.
These include fusion protein constructs, such as TNFR1 antagonist-Fe fusion constructs. As described herein, and exemplified in the Examples, 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.
Also provided are selective TNFR2 agonist constructs, such as TNFR2-Fc fusion constructs that improve the therapeutic efficacy of prior TNFR2 agonists. For example, as shown herein, the half-life of the Fe fusion constructs increases the half-life of prior TNFR1 antagonists or TNFR2 agonists, which, for example, reduces the RECTIFIED SHEET (RULE 91) ISA/EP

frequency of dosing, improves patient compliance, and improves the therapeutic index. Also provided are selective TNFR2 agonist constructs, such as TNFR2-Fc fusion constructs that improve the therapeutic efficacy of prior TNFR2 agonists. For example, as shown herein, 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. (2018) 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):531-536).
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).¨linkerp¨ (activity modifier)q, formula la, or (activity modifier)q¨linkerp¨(TNFR1 inhibitor),, 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. The activity modifier is not a human serum albumin antibody or an unmodified Fc. Also provided are the TNFR2 agonists of formula 3: (TNFR2 agonist).¨linkerp¨ (activity modifier)q, where n, p and q, the linker, and the activity modifier, are as set forth for formula 1.
Also provided are multi-specific, including bi-specific, constructs that contain an TNFR1 antagonist (a TNFR1 inhibitor) and an TNFR2 agonist, linked directly or via a linker. 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 antagonists and TNFR2 agonists.
The structure of the multi-specific, such as, bi-specific, molecules/constructs provided herein is represented by the following formula (Formula 2):
(TNFR1 inhibitor).¨ (activity modifier)ri ¨ Linker (L). ¨ (activity modifier),2¨ (TNFR2 agonist)., where n= 1, 2, or 3, p= 1, 2, or 3, rl and r2 each independently = 0, 1, 2, and q= 0, 1 or 2.
As with formulae 1, the order of components can vary. 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 Fe region, or a modified Fe 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.
Also provided are constructs that contain (formulae 5):
(TNFR2 agonist )n¨linkerp¨ (activity modifier)q, formula 5a, or (activity modifier)q¨linkerp¨(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.
4. Components of the TNFR1 antagonist constructs, TNFR2 agonist constructs, and Multi-Specific, Including Bi-Specific, TNFR1 Antagonist/TNFR2 agonist constructs Description of and examples of the constructs, and each component of the constructs provided herein are described in the sections below. Exemplary forms of each construct are depicted and described by formulae 1 and 2, above, and 3 and 4, below.
a. TNFR1 inhibitor moiety (TNFR1 antagonist) The TNFR1 inhibitor moiety in formula 1, above, and in the multi-specific molecules/constructs (formula 2, above) provided herein is any molecule, including a RECTIFIED SHEET (RULE 91) ISA/EP

polypeptide or small molecule, that inhibits TNFR1 signaling. This includes a inhibitor that selectively inhibits TNFR1 signaling, without inhibiting TNFR2 signaling.
In order to avoid receptor clustering, which agonizes TNFR1, the TNFR1 antagonist construct generally is monomeric/monovalent. TheTNFR1 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. Among 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. In those instances, 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.
For example, as provided herein, 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 (VII) or light chain (VI) domain, dAbs for use herein include, for example, dAbs designated DOM1h-574-208 (SEQ ID NO:54) (from DMS5541; see, SEQ ID NO:38), GSK1995057 (see, SEQ ID NO:55) and G5K2862277 (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. Publication Nos.: 2006/0083747, 2010/0034831, and 2012/0107330; and International Application Publication Nos.: WO 2004/058820, WO 2004/081026, WO 2005/035572, WO 2006/038027, WO 2007/049017, WO
2008/149144, WO 2008/149148, WO 2010/094720, WO 2011/051217, WO
2011/006914, WO 2012/172070, WO 2012/104322, and WO 2015/104322, and other related family member applications and patents; see, also Enever et al., (2015) Protein Engineering, Design & Selection 28(3):59-66, which provides sequences and discussion of various dAbs). Provided are Vhh dAbs that contain a heavy chain.
These RECTIFIED SHEET (RULE 91) ISA/EP

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-TNFR1 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 at. (2018) PLoS Comput Biol.14(3):e1005983), which describes a method for de-immunizing proteins).
Thus, for example, 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.
Other TNFR1 antagonists include, for example, antigen-binding antibody fragments. For example, 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), hsFy (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.
For example, 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 VH (residues 1-115 of SEQ
ID NO:31) and/or VL (residues 1-113 of SEQ ID NO:32) of ATROSAB, or a VH or VL containing at least 85%, 90%, 95%, or more, sequence identity to the VH or VL of ATROSAB. For example, 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. For example, the scFv contains the VH domain, corresponding to residues 1-115 of the ATROSAB heavy chain (see, SEQ ID NO:31), 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 VI, domain, corresponding to residues 1-113 of the ATROSAB light chain (see, SEQ ID NO:32). 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 13.7, containing the sequence set forth in SEQ ID NO:676, or variants containing at least 90% sequence identity to the sequences of scFv IG11, scFv Ti 2B, and scFv 13.7. 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. These 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 XPro1595 (see, SEQ ID NO:701). XPro1595 contains the mutations VIM, R31C, C69V, Y87H, C101A and A1456R, with reference to SEQ ID NO:2. Other exemplary TNFR1-selective antagonistic TNF muteins are derived from XENP345 (see, SEQ ID NO:702), which contains the mutations 197T/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 A845, V85T, S86T, Y87H, Q88N and T89Q, with reference to SEQ ID NO:2. TNFR1 inhibitors to be RECTIFIED SHEET (RULE 91) ISA/EP

used in the TNFR1 antagonists also include small molecule inhibitors that can be chemically conjugated to a linker.
As described herein, see e.g., the Examples, 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. TNF binds to TNFR1 with low pM affinity (Ka 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.

antagonist constructs provided herein, include those that specifically bind to 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). In certain embodiments, 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 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).
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. For example, the TNFR1 antagonist constructs can specifically bind to human TNFR1 or a TNFR1-derived peptide, with an affinity that is greater than the affinity for another RECTIFIED SHEET (RULE 91) ISA/EP

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.
Among the TNFR1 antagonist constructs provided herein are those that exhibit high kon and low koff values upon interaction with TNFR1, consistent with high-affinity receptor binding. For example, the TNFR1 antagonist constructs provided herein can exhibit koo values in the presence of TNFR1 of greater than or equal to, or greater than, about 104 M"1s"1 (e.g., greater than or equal to 1.0 x 104 M"1s"1, 1.5 x 104 M"1s"1, 2.0 x 104 M"ls"1,2.5 x 104 m-is-i, 3.0 x 104 M-is-i, 3.5 x 104 M's, 4.0 x 104 1\4"
is-1, 4.5 x 104 M's', 5.0 x 104 M"Is"1, 5.5 x 104 M4s"1, 6.0 x 104 M"Is"1, 6.5 x 104 M"1s"1, 7.0 x 104 M"1s"1, 7.5 x 104 m4s-1, 8.0 x 104 M"ls"i, 8.5 x 104. m ¨is-1, 9.0 x 104 M"1s"1, 9.5 x 104 m-is-i, 1.0 x 105 M"1s"1, 1.5 x 105 M's', 2.0 x 105 M"1s"1, 2.5 x 105 m-is-i, 3.0 x 105 M-1s-1, 3.5 x 105 M4s-1, 4.0 x 105 M"Is4, 4.5 x 105 M"1s"1, 5.0 x 105 M"1s"1, 5.5 x 105 M"ls"1, 6.0 x 105 Ms', 6.5 x 105 WO, 7.0 x 105 M-ls"1, 7.5 x 105 M"1s"1, 8.0 x WO, 8.5 x 105 WO, 9.0 x 105 WO, 9.5 x 105 M"1s"1, 1.0 x 106 M"1s1). For example, the TNFR1 antagonists provided herein can exhibit koff values, when complexed to TNFR1, of less than or equal to, or less than about 10 s4 (e.g., less than or less than about 1.0 x 10 s"1, 9.5 x 10 s"1, 9.0 x 104 s"1, 8.5 x le s-1, 8.0 x 10 s"1, 7.5 x 104 s"1, 7.0 x 104 s"1, 6.5 x 104 s"1, 6.0 x 104 s"1, 5.5 x 104 s"1, 5.0 x 1040, 4.5 x 10 s"1, 4.0 x 104s', 3.5 x 10 s"1, 3.0 x 104 s"1, 2.5 x 104 s"1, 2.0 x 104 s-1, 1.5 x 104 s"1, 1.0 x 104 s"1, 9.5 x 10"5 s"1, 9.0 x 10 s"1, 8.5 x 10"5 s"1, 8.0 x 10 s"1, 7.5 x 10 s"1, 7.0 x 10 s"1, 6.5 x 10"5 s"1, 6.0 x 10"5 s"1, 5.5 x 10"5 s"1, 5.0 x 10"5 s"1, 4.5 x 10"5 s"1, 4.0 x 10"5 s"1, 3.5 x 10"5 s1, 3.0 x 10"5 s"1, 2.5 x 10"5 s"1, 2.0 x 10 s-1, 1.5 x 10-5 s"1, or 1.0 x 10"5 s"1).
RECTIFIED SHEET (RULE 91) ISA/EP

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. Alternatively, the N-terminus of the 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, For example, 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. For example, in some embodiments, the linker is a .. poly(ethylene glycol) (PEG) molecule, or a branched PEG molecule, such as those whose molecular mass is at or about 30 kDa or more.
b. TNFR2 Agonist Constructs and TNFR2 Antagonist Constructs 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) 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).
RECTIFIED SHEET (RULE 91) ISA/EP

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.
As discussed above, also are provided are 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.
Development of 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 RECTIFIED SHEET (RULE 91) ISA/EP

epitopes from distinct regions of the TNFR2 polypeptide in a conformation-dependent manner. For example, 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). In general, 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 (KQEGCRLCAPLRKCRPGFGV; SEQ ID NO:848), and/or residues 110-147 of SEQ
ID NO:4 (CTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGF; SEQ ID
NO:849), and/or an epitope containing at least five continuous or discontinuous residues from positions 106-155 of SEQ ID NO:4 (ETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTE;
SEQ ID NO:850), and/or residues 137-144 of SEQ ID NO:4 (CAPLRKCR; SEQ ID
NO:851), and/or residues 141-149 of SEQ ID NO:4 (RKCRPGFGV; SEQ ID
NO:852).
In another aspect, 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 RECTIFIED SHEET (RULE 91) ISA/EP

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 1VIR2-1, using ELISA. Using this assay, 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 (e.g., peptides having the sequence of any one of SEQ ID NOs: 853-1194, and particularly, those that contain the KCSPG motif, as in SEQ ID NOs: 905, 921, 927, 970, and 1085) 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. WO
2017/040312). To generate TNFR2 agonists, 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) are immobilized on a solid support or in the phage. 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. To isolate the binding phage, 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.
As provided herein, 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. For example, the VH and VL 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 (VH) or light chain (VL) domain of MR2-1 or MAB2261, or a VH or VL with at least or at least about 85%, 90%, 95%, or more, sequence identity to the VH or VL or MR2-1 or MAB2261, provided the resulting TNFR2 retains TNFR2 agonist activity. 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), hsFAT
(helix-stabilized Fv), minibody, diabody, anti-idiotypic (anti-Id) antibody, free light chains, or antigen-binding fragments of any of the above. 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-CH2-scFv, scFv fusions (C term, or N term), Fab-fusions (HC C-term, or LC C-term), RECTIFIED SHEET (RULE 91) ISA/EP

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.
In some embodiments, 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. In some embodiments, 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). Exemplary of such embodiments, are 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/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, and D143V/A145S, and combinations thereof, such as a combination of D143V/A145S with S95C/G148C, with reference to SEQ ID NO:2.
For example, TNF variants with the mutations D143N/A145R (SEQ ID NO:781) 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. As described herein, the primary ligand for TNFR2 is membrane-bound TNF (memTNF; also referred to herein as RECTIFIED SHEET (RULE 91) ISA/EP

transmembrane TNF or tmTNF). The addition of 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, activate TNFR2 signaling.
Dimerization domains include, for example, EHD2 (SEQ ID NO:808), discussed above. EHD2 is derived from the heavy chain CH2 domain of IgE and MEID2 (SEQ
ID NO:811), which is derived from the heavy chain CH2 domain of IgM.
Dimerization domains also include Fc domains, such as those derived from IgG1 (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.
As provided herein, 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. 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.
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-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula 4) or TNFmut-L1-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.

In particular embodiments, the multimerization 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 IgG1 Fc, or an IgG4 Fc. Where the dimerization domain is an IgG1 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 IgG1 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/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A145I/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, and D143V/A1455, with reference to SEQ ID NO:2. 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. In particular, the linkers can contain a short peptide linker, such as a GS linker. For example, the linker can contain (GGGGS)., where n = 1-5 (SEQ ID NO:1471). The linkers also can contain all or a portion (at least 10, 15, or 20 contiguous residues) of the stalk region of TNF-a, 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. For example, 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)., 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), 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 provided herein, 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). In certain cases, the TNFR2 agonists specifically bind to TNFR2 with a KID
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 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).
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. 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%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100%), as measured, for example, by FACS analysis, relative to a subject or sample containing a population of cells not treated with the TNFR2 agonist.
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. For example, 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. 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), relative to a subject not treated with the agonist. Additionally or alternatively, 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%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100%), in a subject, as measured, for example, by FACS analysis, relative to a subject not treated with the TNFR2 agonist.
For example, 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.
The 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. For example, 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, 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 agonist for another TNFR superfamily member, such as TNFR1.
The TNFR2 agonists provided herein include those that exhibit high k0 and low koff values upon interaction with TNFR2, consistent with high-affinity receptor binding. For example, the TNFR2 agonists provided herein can exhibit k0 values in the presence of TNFR2 of greater than or equal to, or greater than about 104 Avs-1 (e.g., greater than or greater than about 1.0 x 104 m-ls-1, 1.5 x 104 M-1-s1, 2.0 x 104 M-1-s1, 2.5 x 104 M's', 3.0 x 104M-ls-1, 3.5 x 104 M's', 4.0 x 104 Avs-1, 4.5 x 104 M-1-s-1, 5.0 x 104 m-ls-1, 5.5 x 104 Avs-1, 6.0x 104 M's', 6.5 x 104 M's', 7.0 x 104 M's', 7.5 x 104 Avs-1, 8.0 x 104 M's', 8.5 x 104 M's', 9.0 x 104 Avs-1, 9.5 x 104 M"
1.0 x 105 M-1-s-1-, 1.5 x 105 M's', 2.0 x 105 M-1-s-1-, 2.5 x 105 M-1-s-1-, 3.0 x 105 Avs-1, 3.5 x 105 M's', 4.0 x 105 M's', 4.5 x 105 M-1-s-1-, 5.0 x 105 M's', 5.5 x 105 M's', 6.0 x 105 M-1-s-1-, 6.5 x 105 M-1-s-1-, 7.0 x 105 M-1-s1, 7.5 x 105 M-1-s-1-, 8.0 x 105 M-1-s1, 8.5 x 105 M-1s-1, 9.0 x 105 M's', 9.5 x i0 M's', or 1.0 x 106 M's'). For example, the agonists provided herein can exhibit koff 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 10-3s-1, 9.5 x 10-4 s-1, 9.0 x 10' s-1, 8.5 x 10' s-1, 8.0 x 10' s-1, 7.5 x 10' s-1, 7.0 x 10' s-1, 6.5 x 10' s-1, 6.0 x 104s-1, 5.5 x 104 s-1, 5.0x 104s', 4.5 x 10-4 s-1, 4.0x 104s', 3.5 x 10' s-1, 3.0x 10' s-1, 2.5 x 10' s-1, 2.0 x 10' s-1, 1.5 x 10' s-1, 1.0 x 10' s-1, 9.5 x 10-5s-1, 9.0 x 10-5 s-1, 8.5 x 10-5 s-1, 8.0 x 10-5 s-1, 7.5 x 10-5 s-1, 7.0 x 10-5 s-1, 6.5 x 10-5 s-1, 6.0 x 10-5 s-1, 5.5 x 10-5 s-1, 5.0 x 10-5 s-1, 4.5 x 10-5 s-1, 4.0 x 10-5 s-1, 3.5 x 10-5 s-1, 3.0 x 10-5 s-1, 2.5 x 10-5 s-1, 2.0 x 10-5 s-1, 1.5 x 10-5 s-1, or 1.0 x 10-5 s1).
As provided herein, 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. For example, the N-terminus of 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.
Alternatively, the C-terminus of the TNFR2 agonist can be fused with the N-terminus of the TNFR1 antagonist. Where the TNFR2 agonist has the structure set forth in Formula 3, the N-terminus of the multimerization domain is linked to the C-terminus of the TNFR1 antagonist, and where 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-TNFR1 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. In some embodiments, the linker is a poly(ethylene glycol) (PEG) molecule, or a branched PEG molecule, of 30 kDa or more. As discussed above, where the TNFR2 agonist has the structure set forth in Formula 3 or 4, if the multimerization domain is an Fc, then it is the same Fc that is used to link the TNFR1 antagonist to the TNFR2 agonist.
c. Linkers 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).
Included are polypeptide linkers and also chemical linkers for chemical conjugation. 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 inununoglobulin (Ig). In embodiments herein, for example, 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.
In other embodiments, 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. As discussed below and elsewhere herein, in embodiments where the 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.
RECTIFIED SHEET (RULE 91) ISA/EP

i. Peptide Linkers 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.
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.
/5:871-879).
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. The incorporation of 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.
RECTIFIED SHEET (RULE 91) ISA/EP

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).. By adjusting the copy number "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.
To confer protease resistance and to increase the flexibility of the fusion protein, 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. For example, polypeptide linkers include, but are not limited to, (GlySer)., where n= 1-10; (GlySer2); (Gly4Ser)., where n= 1-10; (Gly3Ser)., where n= 1-5; (SerGly4)., where n= 1-5; (GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS (see SEQ ID NOs: 816-827 for the GS linkers). 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. Another 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.
Several other types of flexible linkers, including KESGSVSSEQLAQFRSLD
(SEQ ID NO:829), and EGKSSGSGSESKST (SEQ ID NO:830). The Gly and Ser residues in the linker provide flexibility, and the Glu and Lys improve the solubility.

b) Rigid linkers 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 (SEQ ID
NO:831) 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. An example of a rigid linker is: A(EAAAK)nA (SEQ ID NO:832), wherein n = 2 ¨5. This linker displays an a-helical conformation, which was stabilized .. by the Glu¨Lys+ salt bridges within segments. 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. 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-.
Those of skill in the art can select from known linkers or design linkers.
Desirable properties and requisites therefor are known. The following discussion summarizes some exemplary linkers (see, Chen et al. (2013) Adv. Drug. Deliv.
Rev.
65:1357-1369), which provides details of flexible and rigid and cleavable linkers and that can be used). 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.
RECTIFIED SHEET (RULE 91) ISA/EP

In Formula 2, above, 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.
In certain embodiments, 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. For example, the N- or C-terminal extensions from an Fc can be used as a linker. The C-terminal extension from human IgG, ELQLEESSAEAQDGELDG
(SEQ ID NO:833) or a sequence having at least 98% or 99% sequence identity thereto, or a variant containing the sequence ELQLEESSAEAQGG (SEQ ID
NO:834) or a sequence having at least 98% or 99% sequence identity thereto, also 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. In certain embodiments, the second Fc subunit is fused with a second TNFR2 agonist, creating a bivalent antibody-like structure. In other embodiments, only one Fc subunit is included (an Fc monomer).
Chemical Linkers In some embodiments, the linker is a chemical linker. These include 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.
RECTIFIED SHEET (RULE 91) ISA/EP

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 etal., (2010) Toxins 3:848-883). Exemplary chemical cross-linking linkers include, but are not limited to, SMCC (Succinimidy1-4-(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. STAB is a short, NHS-ester and iodoacetyl cross-linker for amine-to-sulfhydryl conjugation. Other exemplary 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 (succinimidy1-(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, besides maleimide, 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. For example, 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)1 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.
WO
02/088172, WO 03/026577, WO 03/043583, and WO 04/032828. For example, linker RECTIFIED SHEET (RULE 91) ISA/EP

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 at.

(1998) Bioconj. Chem. 9:72-86). Chelating linker labelling reagents, such as DOTA-NHS (1,4,7,10-tetraazacyclododecane-1,4,7,10-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 at. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215;
Sun et at. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King et at.
(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 U.S. Patent Publication No.
2005/0276812).
Another example of a chemical linker (also can be an activity modifier for use in constructs herein) are 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. Activity Modifiers Among the components of 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.
As provided herein, the constructs can include, as an activity modifier, the Fc region of a human immunoglobulin, such as an IgG, for example, an IgG1 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). In particular, the Fc is derived from an IgG1 or IgG4 antibody. For example, the linker can include an IgG1 kappa Fc region, such as the IgG1 Fc derived from trastuzumab, containing the CH2 and CH3 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 (Opdivog), containing the and CH3 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). In some embodiments herein, for example, where the construct is a bi-specific molecule is used to treat inflammatory and autoimmune diseases and conditions, immune effector functions are eliminated or reduced. Where the therapeutic is used in the treatment of a tumor or cancer, immune effector functions can be enhanced to improve the anti-tumor immune response and therapeutic efficacy. Additionally, or alternatively, the Fc region is modified to enhance FcRn recycling, to increase the in vivo serum stability and half-life of the molecules provided herein.

For purposes here, the Fe 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. Small antibody fragments, however, 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.
Several methods can be used to increase the retention and in vivo half-life of small antibody fragments, such as dAbs. For example, as provided herein, 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 Fe region of an IgG, such as IgG1 or IgG4. The Fe can be a monomer or a dimer.
.. Fusion of small antibody fragments, such as dAbs, to the Fe 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 Fe receptor (FcRn) expressed on endothelial cells, which protects antibodies from lysosomal degradation and prolongs their in vivo half-life. The addition of an Fe, however, 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.
As described herein, the Fe 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. For example, the Fe region is modified to take advantage of/enhance neonatal FcR recycling to increase the in vivo half-life, and/or is mutated to eliminate Fe-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). Additionally, in embodiments where 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 Fe dimer, the dimer is mutated to introduce knobs-in-holes to prevent homodimerization.
Numerous modifications to Fe portions (or regions) are known to those of skill in the art (see, e.g., Li etal., (2014) Expert Opin Ther Targets /8:335-350).
i. Modifications to the Fc portions a) Knobs-in-Holes Bispecific antibodies (bsAbs) 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. For example, bsAbs can include an Fe 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 CH3 domains of antibody (IgG) heavy chains are engineered for heterodimerization, to allow for the construction of Fe-containing bi-functional therapeutic molecules that will not self-associate.
The knobs-in-holes approach involves asymmetrically mutating interfacial residues in the CH3 domains of the two parental heavy chains in a complementary RECTIFIED SHEET (RULE 91) ISA/EP

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 CH3 domains, and "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 CH3 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. Residue T366 on the first domain is within hydrogen-bonding distance of residue Y407 on the second or partner CH3 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 Fe 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 IgG1 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.
Table 6:
IgG1 Fc Modifications that Introduce Knobs-into-Holes Modifications by Modifications Modifications Modification Sequential by EU by Kabat Type Numbering Numbering Numbering (SEQ ID NO:9) Knob S354C S375C S237C
Knob T366Y T389Y T249Y
Knob T366W T389W T249W
Knob T394W T422W 1277W
Hole Y349C Y370C Y232C
Hole T366S T389S T249S
Hole L368A L391A L251A
Hole F405A F436A F288A
RECTIFIED SHEET (RULE 91) ISA/EP

IgG1 Fc Modifications that Introduce Knobs-into-Holes Modifications by Modifications Modifications Modification Sequential by EU by Kabat Type Numbering Numbering Numbering (SEQ ID NO:9) Hole Y407T Y438T Y290T
Hole Y407A Y438A Y290A
Hole Y407V Y438V Y290V
Ligand Trap Constructs Fcs modified to have "knobs-in-holes" as described above also can be employed with other bi-specific molecules to produce heterodimers. For example, U.S. Patent Publication No. 2010/0055093, and Jin et at. (2009) Mol. Med.
15:11-20, 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 HER1/ErbB1 (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 (IgG1) (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 at. (2009)Mol. Med. 15:11-20). RB200 binds HER1/ErbB1 ligands (EGF, TGF-a, HB-EGF, AR, BTC, EPR and EPG) and HER3/ErbB3 ligands (NRG1-a and NRG1-03) with high affinity. RB242 inhibits EGF-stimulated and NRG1 -0 I -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. The EGF family has four members: EGF receptor (EGFR)/HER1/ErbB1, HER2/ErbB2, HER3/ErbB3, and 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), 13-cellulin (BTC), epiregulin (EPR), epigen (EPG) and neuregulin (NRG). Within the EGFRs there are four ECDs;
domains I and III are ligand-binding domains, and domains II and IV mediate binding to each other and to other members of this receptor family. Ligand binding induces the formation of homo- or heterodimers between the receptors. For example, TGF-a and EGF bind to EGFR/HER1/ErbB1, whereas NRG4 binds to HER4/ErbB4.
Depending on the dimer formed, transphosphorylation of intracellular regions occurs, leading to the activation of numerous downstream signaling pathways, which results in cell proliferation, survival and differentiation (see e.g., Jin et at.
(2009)Mol. Med.
15:11-20).
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). In many cancer types, mutations or amplification of one the family members is associated with worsened survival in cancer patients. In autoimmune disease, 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).
In a complementary manner, 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. Thus, the TNF and the EGFR pathways cooperate in the progression of lupus and rheumatoid arthritis, and other autoimmune diseases.
Among the constructs provided herein are 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. Examples of targeted therapeutics that act via the EGFR family are (listed with generic name and exemplary trademark providing source) cetuximab (Erbituxg), panitumumab (Vectibixg), trastuzumab (Hercepting), and pertuzumab (Perjetag). Small molecule inhibitors also target the intracellular tyrosine kinase activity of the EGFR family. Examples of small molecules include lapatinib (Tykerbg), erlotinib (Iressag), and neratinib (Nerlynxg). 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. Similarly, an antibody vs. a single growth factor (e.g., TGF-a, EGF, HB-EGF, and others) inhibits only that growth factor, and the tumor cell will compensate by upregulating other growth factors. 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 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.
b) Modifications that Enhance Neonatal Fc Receptor (FcRn) Recycling There are numerous approaches to increasing the short serum half-life of small polypeptide or protein therapeutics. 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 binds to the Fc portion (within the CH2-CH3 domains) of pinocytosed IgGs in the acidic (¨ pH 6) endosome in a 2:1 FcRn:IgG
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, 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 CH2 and CH3 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. Alternatively, studies have shown that short FcRn-binding peptides (FcRnBPs) allow for the interaction of small proteins with FcRn, obviating the need for fusion to a high molecular weight Fc domain. For example, fusion with an FcRnBP increases the molecular weight by approximately kDa, in comparison to fusions with Fc or albumin, which increase the molecular weight by approximately 50-70 kDa (see, e.g., Datta-Mannan et at. (2019) Biotechnol.

1 14:1800007; Sockolosky et at. (2012) Proc. Natl. Acad. Sci. USA
109(40):16095-16100).
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. 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. This results from increased avidity, with constructs containing four linear FcRnBPs fused to the N- and C-termini of the heavy and light chains of the Fab showing the greatest improvement in pharmacokinetics in cynomolgus monkeys relative to the parental Fab. For example, the half-life improved from 3.7 hours for the parental Fab, to between 15-60 hours for the various FcRnBP-Fab constructs (see, e.g., Datta-Mannan et al. (2019) Biotechnol. 1 14:1800007). While these results indicate an improvement in serum half-life, it is still much lower than the half-life for an IgG, which is about 2-3 weeks. The use of FcRnBPs also does not reduce renal clearance, as they do not significantly increase the molecular weight of the therapeutics.
As discussed above, 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. To improve the pharmacokinetics and overall pharmacology, 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 CH2 and CH3 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-IgG1 complex. For example, the replacements M252Y/5254T/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 IgG1 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 et al. (2002)1 Immunol. 169:5171-5180).
The triple substitution M252Y/S254T/T256E, when introduced into the CH2 domain of 1VIEDI-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. When introduced into the Fc portion of MEDI-522, a humanized, affinity-optimized mAb directed against the human avf33 integrin complex, the replacements M252Y/S254T/T256E (YTE) reduced its ADCC activity and its binding to human FcyRIIIA (F158 allotype). The ADCC
activity of MEDI-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 IgG1 (see, e.g., Dall'Acqua et al. (2006)1 Biol. Chem. 281(33):23514-23524).
Residues at positions 250, 314 and 428 (by EU numbering) of the human IgG
heavy chain, which are conserved among all four human IgG subtypes, also are located near the Fc-FcRn interface. The mutations T250Q, M428L and T250Q/M428L, when introduced into the Fc of a human IgG2 mAb, resulted in an increase in binding to FcRn at pH 6.0 of ¨3-, 7- and 28-fold, respectively, with no binding observed at pH 7.5. When the pharmacokinetics of the mutants were evaluated in rhesus monkeys, it was found that the mean clearance, i.e., the volume of serum antibody cleared per unit of time, was ¨1.8-fold lower for the M428L
mutant, and ¨2.8-fold lower for the T250Q/M428L mutant, while 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. Since these residues are conserved among IgG subtypes, the mutations M428L and T250Q/M428L are expected to have similar effects in human IgGl, IgG3 and IgG4 antibodies (see, e.g., Hinton et at.
(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 IgG1 in rhesus monkeys (see, e.g., Saxena et at. (2016) Front.
Immunol. 7:580).
The mutation N434A (by EU numbering), when introduced into the human anti-HER2 IgG1 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. In contrast, the mutation N434W, which resulted in ¨80-fold increased binding to FcRn at pH 6, exhibited a clearance rate similar to wild-type; this mutant also exhibited significant binding to FcRn at pH 7.4, indicating that maintaining pH-dependent binding of Fc mutants to FcRn is critical for improving the in vivo pharmacokinetics (Yeung et at. (2009)1 Immunol. 182:7663-7671). The N434A
mutation also counters the poor FcRn affinity that can result from the introduction of mutations that increase binding to FcyRs; N434A is typically added to the mutations S298A/E333A/K333A to create a variant with enhanced FcyR 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. 0. (2019) Front. Immunol. 10:1296).
The replacements M428L/N4345 (by EU numbering), when introduced into the humanized anti-VEGF IgG1 antibody bevacizumab (Avasting), resulted in an 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/N4345 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 half-life extension of these anti-tumor antibodies correlated with enhanced tumor reduction in vivo in a mouse model, indicating that the in vivo therapeutic efficacy of the antibodies is increased when the pharmacokinetics, such as clearance rate, are improved. Other mutations engineered into the bevacizumab Fc include (by EU
numbering): N4345, with ¨3-fold improvement in FcRn binding and ¨2.8 fold increase in serum half-life in mice; V259I1V308F, with -6-fold improvement in FcRn binding, and -3-fold and -2-fo1d increases in serum half-life in mice and cynomolgus monkeys, respectively; M252Y/S254T/T256E, with -7-fold improvement in FcRn binding, and -4-fold and 2.5-fold increases in serum half-life in mice and cynomolgus monkeys, respectively; and V259IN308F/M428L, with -20-fold improvement in FcRn binding, and -4-5-fold and 2.6-fold increases in serum half-life in mice and cynomolgus monkeys, respectively (Zalevsky et al. (2010) Nat. Biotechnol 28(2):157-159).
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.
In some embodiments, 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 CH2 and CH3 domains of IgG Fc, which are responsible for binding to FcRn. These include, but are not limited to, the residues T250, L251, M252, 1253, S254, T256, V259, T307, V308, L309, H310, L314, Q311, A378, E380, S383, M428, H433, N434, H435 and Y436, by EU numbering. 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, V25911V308F, V2591/V308F/M428L, E294del/T307P/N434Y, T256N/A378V/S383N/N434Y, and combinations thereof, by EU numbering. Table 7, below, shows the corresponding mutations by Kabat numbering and sequential numbering, with reference to the sequence of the IgG1 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.
RECTIFIED SHEET (RULE 91) ISA/EP

Table 7:
IgG1 Fc Modifications that Enhance FcRn Binding Modifications by EU Modifications by Kabat Modifications by Sequential Numbering Numbering Numbering (SEQ ID NO:9) E294de1/T307P/N434Y E311del/T326P/N465Y El 77del/T190P/N317Y

c) Enhancement of or Reduction/Elimination of Fc Immune Effector Functions There are four human IgG subclasses that differ in effector functions, circulating half-life and stability. IgG1 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 Fe effector functions, and a very long, rigid hinge region. IgG4 is deficient in Fe 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. Thus, Fe 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 IgG1 subclass, and can interact with the humoral and cellular components of the immune system. For example, antibodies engage the humoral immune response via interaction with complement protein Cl q, 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 Fe gamma receptors (FcyRs). The FcyRs include the FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) classes that differ in their cell surface expression and Fe binding affinities. The five activating FcyRs include the high affinity FcyRI that can bind monovalent antibodies, and the lower affinity FcyRIIa, FcyRIIc, FcyRIIIa and FcyRIIIb, which require avidity-based interactions.
.. FcyRIIb is the only inhibitory receptor. Upon binding of an Fe to an activating receptor, intracellular signaling pathways, modulated through the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), result in effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC; also called antibody-dependent cellular cytotoxicity) and antibody-dependent cell-mediated phagocytosis (ADCP; also called antibody-dependent cellular phagocytosis), as well as inflammation due to the induction of cytokine secretion. Signaling via the inhibitory FcyRIIb, which is modulated through the phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITEVIs), recruits phosphatases that counter-balance the activating signaling pathways (see, e.g., Wang et al.
(2018) Protein Cell 9(1):63-73).
The hinge and proximal CH2 amino acid sequence (lower hinge-upper CH2 domain region), and glycosylation of the conserved N297 residue (by EU
numbering) in the C112 domain Asn-X-Ser/Thr glycosylation motif of the Fe region, mediate the interactions of antibodies with FcyRs and complement protein Cl q. Antibody/Fe engineering has been used to modify the immune effector functions of antibodies by altering their binding to Clq and various Fcy receptors. The CDC, ADCC and ADCP
activities of therapeutic mAbs can thus be increased or decreased, depending on the application. For example, the efficacy of anti-cancer mAbs depends in part on their induction of FciR effector functions. The effector function includes the activation of natural killer (NK) cells via FcyRIIIa and the subsequent ADCC activity and release of inflammatory cytokines, the induction of macrophage-mediated ADCP via interactions with multiple FcyRs, and the recruitment and activation of other immune cells, such as neutrophils, the primary receptor for NK cell-mediated ADCC.
FcyRIIIa has two polymorphic variants: one with V158, which has a higher affinity for IgGl;
and one with F158, with a lower affinity for IgGl. Cancer patients with the high affinity V158 polymorphism can have better outcomes following treatment with Cetuximab, trastuzumab and rituximab, compared to patients with the low affinity F158 polymorphism. Results such as these highlight the role that FcyR-mediated immune effector functions play in therapies, and indicate that engineering antibodies and related molecules to have increased affinity to FcyRs can enhance the therapeutic efficacy (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73).
Residues in the lower hinge and proximal CH2 regions of IgGs have been = determined to be critical for binding to FcyRs. Residues that are within 5 angstroms from the FcyR:Fc interface for FcyRI, FcyRIIa, FcyRIIb and FcyRIIIb, 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 N208-1215, with reference to SEQ ID NO:9) (see, e.g., Wang et at (2018) Protein Cell 9(1):63-73).
Modifications of Fe that enhance or decrease ADCC activity and/or enhance affinity/binding to receptors are known to those of skill in the art. For example, Fe modifications that increase the IgG1 affinity for and binding to FcyRIIIa, and/or RECTIFIED SHEET (RULE 91) ISA/EP

enhance ADCC function, include the replacements (by EU numbering):
F243L/R292P/Y300L/V3051/P396L, L235V/F243L/R292P/Y300L/P396L, F243L/R292P/Y300L, S23 9D, 1332E, S239D/I332E, S239D/A330L/1332E, 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/G236W/S298A in one heavy chain and S239D/A330L/1332E in the opposing heavy chain. Additionally, the mutations A327Q/P329A (interact with FcyRI), D265A/S267A/H268A/D270A/K326A/S337A (interact with FcyRIIa), G236A
(interacts with FcyRIIa), and T256A/K290A/S298A/E333A/K334A (interact with FcyRIIIa), result in high affinity interactions with FcyRs.
Fc modifications that increase binding to FcyRIIa and FcyRIIIa, and enhance ADCC and ADCP, include (by EU numbering) G236A/I332E, G236A/S239D/I332E
(also increases binding to FcyRI), and G236A/S239D/A330L/1332E (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et at (2016) Front. Immunol.
7:580; and Saunders, K. 0. (2019) Front. Immunol. 10:1296).
Glyco-engineering of IgGs, which contain a conserved N-linked glycosylation site at residue N297 in the CH2 domain, can enhance Fc effector function.
Glycosylation of N297 is essential for maintaining Fc conformation and mediating its interactions with FcyRs (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. IgGs found circulating in human sera generally are fucosylated, but recombinant IgG
production can alter the glycan composition by expressing the antibody in plant cells, knocking in or out specific glycosidases, or in vitro enzymatic digestion of the glycosylated IgG;
because both heavy chains are glycosylated, a single IgG molecule can have glycan heterogeneity. The glycan directly affects FcyR binding. For example, the N297 glycan on the Fc can clash with glycans on the FcyRIII protein, resulting in poor engagement of effector cells that mediate ADCC. Fc regions containing different glycans at N297 adopt different hinge region conformations, which can affect the Fc's ability to interact with FcyRs. Expression of f3(1,4)-N-acetylglucosaminyltransferase III when expressing IgG generates an antibody that is glycosylated at position with a biantennary glycan; this antibody has increased binding to FcyRIIIa and enhanced ADCC activity. It has been demonstrated that fucose deficient (afucosylated/non-fucosylated) IgG1 s exhibit up to 50-fold increased binding to FcyRIIIa and enhanced ADCC activity. Two glyco-engineered (afucosylated) mAbs, obinutuzumab (anti-CD20) and mogamulizumab (anti-CCR4) have been approved for clinical use, indicating the potential for glyco-engineering for enhanced effector function, and its translation into clinically approved therapeutics (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al. (2016) Front. Immunol.
7:580; and Saunders, K. 0. (2019) Front. Immunol. 10:1296).
The Fc also can be modified to bind with a wider range of Fc receptors. Fc receptors for isotypes other than gamma (i.e., IgA, IgM and IgE) exist on certain leukocytes, and by modifying an Fc region to engage with multiple Fc receptors, an antibody with expanded abilities to engage effector cells is created.
Neutrophils, which are the most abundant leukocytes in the body, engage the Fc of IgA
antibodies via the FcaRI receptor. For example, to engage FcyRs and FcaRI, single domains of IgA2 were added to the end of the IgG1 constant region, creating a four domain constant region, CH1g-CH2g-CH3g-CH3a. The CH1 domain of IgG1 was replaced with the alpha 1 constant region domain, generating a constant region (CH1a-CH2g-CH3g-CH3a) that is closer in structure to the alpha constant region. These four-domain, cross-isotype IgGA chimeric antibodies bound to J chain similarly to natural IgA2, had reduced transport by polymeric Ig receptor, had a 3-5-fold decrease in FcyRI affinity, and the short serum half-life of IgA2 instead of the protracted serum circulation of IgGl. The four-domain, cross-isotype IgGA chimeric antibodies, however, had the ability to mediate complement-dependent lysis of sheep red blood cells and were more pH-resistant than IgGl. Another cross-isotype Fc was created by fusing the gamma 1 and alpha constant regions together to create a tandem G1-A
Fc region, in which the hinge, CH2 and CH3 domains of IgA2 were fused to the C-terminus of IgGl. This tandem cross-isotype IgG/IgA fusion showed similar expression levels, antigen binding and thermostability as IgGl, and, in vitro, bound to FcaRI and FcyRI, FcyRII, FcyRIIIa and FcRn, with affinities similar to wild-type IgA
and IgG, respectively. The binding to various FcRs resulted in ADCC activity with polymorphonuclear cells and NK cells; Clq binding, however, was reduced 3-fold compared to IgG1 . The tandem IgG/IgA had an in vivo half-life similar to that of IgG1 in BALB/c mice. An alternative cross-isotype antibody was created by replacing the CH3 domain and CH2 al loop residues 245-258 (by EU numbering, corresponding to the sequence PKPKDTLMISRTPE; (residues 128-141 of SEQ ID NO:9)) of the IgG1 constant region, with the structurally analogous regions of the IgA
constant region. This chimeric Fc was able to bind FcyRI, FcyRIIa and FcaRI, and antibodies containing the chimeric Fc mediated ADCC with polymorphonuclear cells and ADCP

with macrophages, and activated complement, but lacked binding to FcRn, which regulates antibody half-life; thus, further optimization is required for effective in vivo use (see, e.g., Saunders, K. 0. (2019) Front. Immunol. 10:1296).
Another approach to enhance FcyR binding is the multimerization of IgG, which has shown promise in the treatment of autoimmune diseases. The IgG
multimers are generated, for example, by adding heterologous multimerization domains such as isoleucine zippers, or by adding another hinge region at the N-terminus of the natural hinge, or by adding another hinge region at the C-terminus of the CH3 domain. IgG hexamers are created by appending the IgM tailpiece to the C-terminus of the IgG1 Fc and creating a cysteine bond at position 309; this multimeric IgG bound strongly to FcyRI, FcyRIIa and FcyRIIIa, and weakly to FcyRIlb and FcyRIIlb. The various multimeric IgGs have increased binding to FcyRI, FcyRIlb and FcyRIII, compared to monomeric IgG, and have shown promise in preclinical models of arthritis, neuropathy, and autoimmune myasthenia gravis. This multimeric IgG
design is being further optimized to fine-tune which immune receptors, including FcRn, can bind to the multimer (see, e.g., Saunders, K. 0. (2019) Front.
Immunol.
10:1296).
Residues in the Fc region of IgG that are involved in the interaction with and binding to Clq (and hence, CDC) include (by EU numbering) S267, D270, K322, K326, P329, P331 and E333. Fc modifications that have been shown to enhance CDC
by increasing Clq binding include, for example, K326A, E333A, K326A/E333A, K326W, K326W/E3335, K326M/E3335, C220D/D221C, H268F/5324T, 5267E, H268F, 5324T, 5267E/H268F/5324T, and G236A/I332E/5267E/H268F/5324T (all by EU numbering). In the upper hinge region of the IgG1 Fc, substituting Trp, in various combinations, at positions 222, 223 and 224 (i.e., K222W, T223W, and H224W, by EU numbering), increased Clq binding and CDC activity relative to wild-type IgG1 without affecting FcyRIIIa binding and ADCC activity. Specifically, the mutations included K222W/T223W, K222W/T223W/H224W and D221W/K222W.
The mutations C220D/D221C and C220D/D221C/K222W/T223W, also increased Clq binding and CDC activity (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73;
Saxen et al. (2016)Front. Immunol. 7:580; Saunders, K. 0. (2019)Front.
Immunol.
10:1296; and Dall'Acqua et al. (2006)1 Immunol. 177:1129-1138).
IgG3 has the best in vitro binding to Clq; combining the CH1 and hinge regions of IgG1 with the CH2 and CH3 regions of IgG3 (to retain ADCC activity from IgG1 and CDC activity from IgG3), creating IgG1/IgG3 cross-subtype antibodies, also increases Clq binding and enhances CDC activity. Another IgG1/IgG3 cross-subtype antibody with increased Clq binding and enhanced CDC activity includes the CH1, hinge and CH3 of IgGl, and the CH2 of IgG3; these modifications allow for increased Cql binding since Clq binds the CH2 domain, as well as easy purification, since protein A binds the CH3 domain. Additionally, the modifications E345R/E430G/5440Y, which result in the formation of IgG hexamers with K322 oriented in a position to favorably interact with the hexameric Clq headpiece, enhanced CDC activity. The mutation E345R alone also results in IgG hexamer formation, with increased Clq binding and enhanced CDC activity (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al. (2016)Front. Immunol. 7:580;

Saunders, K. 0. (2019)Front. Immunol. 10:1296).
Glycoengineering also can be used to improve complement binding; the N297 glycan within the CH2 domain of the Fc can be modified to improve CDC
activity.
For example, an overabundance of galactosylation in the IgG1 Fc increases Clq binding and CDC activity compared to the unmodified glycoform of IgGl, and also improves thermostability. Thus, galactosylating the Fc can be used to generate a stable biologic with enhanced CDC activity (see, e.g., Saunders (2019)Front.
Immunol. 10:1296).
Table 8, below, summarizes the Fc modifications that increase binding to FcyRs or Clq, and thus, enhance immune effector functions, including ADCC, ADCP
and CDC, and provides the corresponding modifications by Kabat numbering and by sequential numbering, with reference to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO:9. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fc portions of the constructs provided herein. Other modifications, known in the art to confer enhanced or increased immune effector functions, also are contemplated for use herein.
Table 8:
IgG1 Fc Modifications that Enhance Immune Effector Functions Modifications by EU Modifications by Kabat Modifications by Sequential Numboing Effects Numboing Numboing (SEQ ID NO:9) Increase binding to S239D S252D S122D FcyRIIIa;
enhance ADCC
Increase binding to I332E I351E I215E FcyRIIIa;
enhance ADCC
Increase binding to S239D/I332E S252D/I351E S122D/ I215E FcyRIIIa;
enhance ADCC
Increase binding to FcyRIIIa; enhance ADCC
Increase binding to S298A/E333A/K334A S317A/E352A/K353A S181A/E216A/K217A FcyRIIIa;
enhance ADCC
F243L/R292P/Y300LN305 F256L/R309P/Y319LN324 F126L/R175P/Y183LN188 Increase binding to FcyRIIIa and FcyRIIa;

enhance ADCC

Increase binding to L/P396L L/P424L L/P279L FcyRIIIa;
enhance ADCC
Increase binding to FcyRIIIa; enhance ADCC
L234Y/G236W/S298A (1st L247Y/G249W/S317A (1st L117Y/G119A/S181A (1st Increase binding to heavy chain) and heavy chain) and heavy chain) and FcyRIIIa; enhance S239D/A330L/I332E (2" S252D/A349L/I351E (2" S122D/A213L/I215E
(2"
ADCC
heavy chain) heavy chain) heavy chain) 9M/H268D/D270E/S298A 2M/H281D/D283E/S317A 2M/H151D/D153E/S 181A Increase binding to (Pt heavy chain) and (Pt heavy chain) and (15t heavy chain) and FcyRIIIa; enhance 34E (21x1 heavy chain) 53E (21x1 heavy chain) 17E (2" heavy chain) A327Q/P329A A346Q/P348A A210Q/P212A Increase binding to FcyRI

Increase binding to FcyRIIa T256A/K290A/S298A/E33 T269A/K307A/S317A/E35 T139A/K173A/S181A/E21 Increase binding to 3A/K334A 2A/K353A 6A/K217A FcyRIIIa Increase binding to G236A G249A G119A FcyRIIa;
enhances ADCP
Increase binding to G236A/I332E G249A/I351E Gil 9A/I215E FcyRIIa and FcyRIIIa;
enhance ADCC and ADCP

Increase binding to FcyRI, FcyRIIa and FcyRIIIa; enhance ADCC and ADCP
Increase binding to FcyRIIa and FcyRIIIa;
enhance ADCC and ADCP
Increases binding to Biantennary glycan at N297 Biantennary glycan at N314 Biantennary glycan at N180 FcyRIIIa; enhances ADCC
Increases binding to Afucosylated glycan at Afucosylated glycan at Afucosylated glycan at FcyRIIIa; enhances ADCC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC and preserve ADCC activity Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC and preserve ADCC activity Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC and preserve ADCC activity Increase binding to Clq;
23W 36W 06W enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
Increase binding to Clq;

enhance CDC
G236A/I332E/5267E/H268 G249A/I351E/5280E/H281 Gil 9A/I215E/S150E/H151 Increase binding to C lq;
F/5324T F/5343T F/5207T enhance CDC
Increase binding to Clq;

enhance CDC; IgG1 hexamer formation Increase binding to Clq;

enhance CDC; IgG1 hexamer formation Therapeutic antibodies also can be engineered to reduce or eliminate immune effector functions. For purposes herein, in some embodiments, it of interest, for example, to reduce or eliminate ADCC activity. Constructs herein that include Fc generally are modified to reduce or eliminate ADCC activity.
It is of interest to reduce or eliminate immune effector functions, for example, where: the therapeutic antibodies are antagonistic in order to prevent receptor-ligand interactions and signaling; the antibodies are receptor agonists to crosslink receptors and induce signaling; the antibodies are drug delivery vehicles that deliver a drug to antigen-expressing target cells; and, where the reduction or elimination of effector functions prevents target cell death or unwanted cytokine secretion. Reduced effector function also prevents antibody-drug conjugates from interacting with FcyRs, which reduces off-target cytotoxicity. The importance of reducing or eliminating effector functions became evident following adverse events associated with the administration of the first approved mAb, muromonab, which was designed to prevent T cell activation in transplant patients receiving a donor kidney, lung or heart.
Patients administered muromonab experienced a dangerous induction of pro-inflammatory cytokines (i.e., a cytokine storm); this was due, in part, to the interaction of muromonab with FcyRs (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73;
and Saunders, K. 0. (2019) Front. Immunol. 10:1296).
There are many known mutations that reduce or eliminate receptor function.
For example, replacements L235E and F234A/L235A in human IgG4, and L235E and L234A/L235A (all by EU numbering) in human IgG1 reduce FcyR and C lq binding, and reduce effector functions, such as inflammatory cytokine release.
Inflammatory cytokine release from therapeutic antibodies, can result in adverse effects.
The replacements S228P/L235E, when introduced into IgG4, also reduce binding to FcyRs; the S228P mutation improves stability of IgG4. The mutations S228P/F234A/L235A in the IgG4 Fc decrease binding to FcyRI, ha and Ma, and reduce ADCC and CDC. The triple mutant L234E/L235F/P331S in IgG1 Fc decreases binding to FcyRI, FcyRII, FcyRIII and C 1 q, and reduces CDC, the mutations L234A/L235A/P329G in the IgG1 Fc eliminate FcyRI, FcyRII, FcyRIII and C lq binding, and reduce ADCP. The mutations L234F/L235E/P331S also reduce binding to FcyRs and Clq, and reduce effector functions of the IgG1 Fc. The mutations G237A and E318A in the IgG1 Fc each decrease binding to FcyRII and reduce ADCP; the mutations D265A and E233P decrease binding to FcyRI, FcyRII
RECTIFIED SHEET (RULE 91) ISA/EP

and FcyRIII, and decrease ADCC and ADCP, and the mutations G236R/L328R
decrease binding to all FcyRs and reduce ADCC. Crystal structure data revealed that conformational changes at residue P329, which packs between two conserved tryptophan residues that occur in all FcyRs, form a "proline sandwich," can be .. detrimental to the interaction with FcyRs, and that modifications at residue D270 can negatively impact interactions with Clq (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, K. 0. (2019) Front. Immunol. 10:1296; International Application Publication No. WO 2019/226750).
Induction of the complement cascade is associated with antibody injection site adverse reactions, and eliminating Clq binding to Fe, which is the initial even in the activation of CDC. Modification of Fe region eliminate Clq binding can be used to eliminate CDC in constructs containing Fe regions. Many of the mutations that eliminate FcyR binding also eliminate C 1 q binding, as shown above. For example, the mutation A330L disrupts Clq binding and reduces CDC, and also eliminates FcyRIIb binding. The mutations D270A, P329A, K322A and P331A also result in reduced C lq binding and reduced CDC activity (see, e.g., Saunders, K. 0. (2019) Front.
Immunol. 10:1296).
Glyco-engineering can be used to ablate FcyR and Clq binding. As discussed elsewhere herein, the glycan at residue N297 is a complex biantennary glycan.
Modification of this glycan to a high mannose glycan (i.e., high mannose glycosylation) reduces the affinity of IgG1 Fe for Clq and reduces CDC
activity.
Mutations in the Fe that reduce or eliminate Clq and FcyRI binding also can result in an increase in galactosylation and sialylation of the N297 glycan; such mutations include F241A, V264A and D265A, for example. The mutations N297A, N297Q, N297D and N297G, by EU numbering, remove the glycosylation site at N297 and reduce effector functions, such as CDC and ADCC, by abrogating Fe interactions with Clq and FcyRs, respectively. The combination N297G/D265A almost completely abrogates binding to FcyRs and Clq. An IgG3 Fe lacking glycosylation (the aglycone Fe) has reduced binding to FcyRI and Clq. (see, e.g., Wang etal.
(2018) Protein Cell 9(1):63-73; Saunders, K. 0. (2019) Front. Immunol.
10:1296).
To reduce or eliminate Fe effector functions, large portions of Fe regions from different subclasses, that lack opposing functions, can be exchanged to generate cross-RECTIFIED SHEET (RULE 91) ISA/EP

subclass Fe regions. For example, IgG2 has poor FcyR binding but binds Clq, and IgG4 lacks Clq binding but reacts with FcyRs; thus, combinations of IgG2 and IgG4 CH domains that are devoid of both Clq and FcyR binding, can be constructed.
In general, in IgG1/IgG4 chimeras, the hinge and CH1 domain is from IgG2, and the and CH3 domains are from IgG4. Since IgG1 and IgG3 recruit complement more effectively than IgG2 and IgG4, and because IgG2 and IgG4 are limited in their ability to induce ADCC, a cross-subclass approach can reduce effector function. For example, the anti-05 mAb eculizumab, contains IgG2 residues 118-260 (by EU
numbering; corresponding to residues 114-273 by Kabat numbering, and residues 139 with reference to SEQ ID NO:11), and IgG4 residues 261-447 (by EU
numbering; corresponding to residues 274-478 by Kabat numbering, and residues 141-327 with reference to SEQ ID NO:15), and has limited or undetectable effector function. Similarly, an IgG2 variant (IgG2m4) with the point mutations H268Q1V309L/A330S/P331S from IgG4 (by EU numbering; corresponding to .. H281Q1V328L/A349S/P350S by Kabat numbering, and H147QN188L/A209S/P210S with reference to SEQ ID NO:11) lacks binding to all FcyRs and Clq and exhibits reduced effector functions. A variant (called IgG2a) containing the IgG2 to IgG4 cross-subclass mutations V309L/A330S/P331S (by EU
numbering; corresponding to V328L/A349S/P350S by Kabat numbering, and V188L/A209S/P210S with reference to SEQ ID NO:11), and the non-germline mutations V234A/G237A/P238S/H268A (by EU numbering; corresponding to V247A/G250A/P251S/11281A by Kabat numbering, and V114A/G116A/P117S/H147A with reference to SEQ ID NO:11), eliminates binding to FcyRs and Clq and exhibit undetectable CDC, ADCC and ADCP activities. The IgG1/IgG4 cross-subclass variant IgGla, which includes the mutations L234A/L235A/G237A/P238S/H268A/A330S/P331S, lacks binding to FcyRI and Ma, and has very weak binding to FcyRIIa and IIb at high concentrations of antibody, resulting in reduced ADCC and CDC activities (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, K. 0. (2019) Front. Immunol. 10:1296).
Tables 9 and 10, below, summarize some IgG1 and IgG4 Fc modifications that reduce or eliminate binding to FcyRs and/or Cl q, and thus, reduce or eliminate immune effector functions, including ADCC, ADCP and CDC, which can be introduced RECTIFIED SHEET (RULE 91) ISA/EP

into the Fe regions in constructs herein. The tables provide the corresponding modifications by Kabat numbering and by sequential numbering, with reference to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO:9, or the IgG4 heavy chain constant domain set forth in SEQ ID NO:15. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fe portions of the constructs provided herein. Other modifications, known in the art to reduce or eliminate immune effector functions, also are contemplated for use herein.
Table 9:
IgG1 Fc Modifications that Reduce or Eliminate Immune Effector Functions Modifications by Modifications by Modifications by Sequential Effects EU Numbering Kabat Numbering Numbering (SEQ
ID NO:9) Reduces FcyR binding; reduces ADCC
Reduce FcyR and Clq binding; reduced ADCC, ADCP and CDC

L117E/L118F/P214S Reduce FcyR and Clq binding; reduce CDC
Reduce FcyR and Clq binding; reduce effector functions Eliminate FcyR and Clq binding;

reduce ADCP and CDC

Reduced binding to FcyR1, ha, Ilb and Ina; reduced ADCC and CDC

Reduced binding to FcyRs; reduced ADCC
Reduces binding to FcyRII; reduced ADCP
Reduces binding to FcyRII; reduced ADCP
Reduces binding to FcyRI, II, III;

reduced ADCC and ADCP
Reduces binding to FcyRI, II, III;

reduced ADCC and ADCP
Remove glycosylation site; decrease interaction with FcyRs; reduce effector functions (CDC, ADCC, ADCP) Remove glycosylation site; decrease interaction with FcyRs; reduce effector functions (CDC, ADCC, ADCP) Remove glycosylation site; decrease interaction with FcyRs; reduce effector functions (CDC, ADCC, ADCP) Remove glycosylation site; decrease interaction with FcyRs; reduce effector functions (CDC, ADCC, ADCP) Reduces binding to FcyRs and Clq;

reduces effector functions Reduced Clq binding; reduced CDC

Reduced Clq binding; reduced CDC

Reduced Clq binding; reduced CDC

Reduced Clq binding; reduced CDC

Reduced Clq binding; reduced CDC

Reduced Clq binding; reduced CDC

Reduced Clq binding; reduced CDC
Table 10:
IgG4 Fc Modifications that Reduce or Eliminate Immune Effector Functions Modifications by Modifications by Modifications by Sequential Effects EU Numbering Kabat Numbering Numbering (SEQ
ID NO:15) Reduces FcyR binding; reduces ADCC

Reduce FcyR and Clq binding; reduced ADCC, ADCP and CDC

Reduce FcyR binding; reduced effector functions Reduced binding to FcyRI, Ha and Ina;
reduced ADCC and CDC
Other Modifications of Fc portions The Fc portion also can be modified to increase binding to inhibitory FcyRs, which results in the suppression of the immune response. Therapeutic antibodies with .. immunosuppressive Fc modifications are advantageous for the treatment of inflammatory diseases. These mutations can be incorporated into the Fc portions of constructs herein that are intended for treatment of diseases and conditions with an inflammatory component or etiology or involvement. For example, the immunosuppressive version of an anti-CD19 antibody (XmAb5871; Xencor), .. containing the mutations S267E/L328F (by EU numbering), binds inhibitory FcyRIIb with ¨430-fold increased affinity, and depletes CD19+ B-cells in patients with systemic lupus erythematosus (SLE). The same mutations, when introduced into a humanized anti-IgE antibody (XmAb7195; Xencor), prevent the binding of IgE to its high-affinity receptor (FccRI) that is present on basophils and mast cells, increases affinity for FcyRIIb by ¨430-fold, and is used for the treatment of allergies, including allergic asthma. The anti-CD3 antibody TRX4 (Tolerx), containing the aglycosylating Fc mutation N297A (by EU numbering), suppresses pathogenic T-cells and restores normal Treg cell activity in type-1 diabetes (autoimmune) patients (see, e.g., Saxena et al. (2016)Front. Immunol. 7:580).
An additional example is the monomeric IgG1 Fc (mFc), containing the mutations L351S/T366R/L368H/P395K (by EU numbering), which binds FcRn and exhibits similar in vivo half-life to dimeric Fc, and selectively binds FcyRI
with high affinity, but does not bind FcyRIIIa, abrogating Fc-mediated cytotoxicity, including ADCC and CDC. FcyRI is expressed on inflammation-related cells, such as inflammatory macrophages. Targeting this receptor can be used for the treatment of chronic inflammatory diseases, such as arthritis, multiple sclerosis and cancer. The variant mFc, when fused to the P seudomonas exotoxin A fragment (PE38), kills FcyRt macrophage-like U937 cells. Neither the variant mFc, nor the fusion protein, exhibits any cytotoxicity (ADCC or CDC) in vitro (see, e.g., Ying et at.
(2014) mAbs 6(5):1201-1210).
Modifications that increase binding to, or that confer selective binding to, inhibitory FcyRIIb, and/or FcyRI but not FcyRIIIa, can be engineered into the IgG Fc regions in the TNFR1 antagonists and TNFR1 antagonist/TNFR2 agonist constructs provided herein. These modifications include, but are not limited to, one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F, L351S/T366R/L368H/P395K, and combinations thereof, by EU numbering. Table 11, below, shows the corresponding replacements by Kabat numbering, and by sequential numbering, with reference to the sequence of the IgG heavy chain constant domain set forth in SEQ ID NO:9.
Table 11:
IgG1 Fc Modifications that Increase Binding to Inhibitory FcyRIIb Modifications by Modifications by EU Modifications by Kabat Sequential Numbering Numbering Numbering (SEQ ID NO:9) iii. Human Serum Albumin (HSA) A problem with the dAbs as previously provided (see, e.g., International PCT
application No. 2008/149144) was that their serum half-life was insufficient for their use as therapeutics. They were linked to anti-HSA antibodies to bind to HSA;
the half-life was insufficient. Herein, the dAbs or Vhh antibodies are linked to HSA. HSA
has 33 cysteines; Cys34 is the only cysteine with a free sulfhydryl group that does not participate in a disulfide linkage. HSA can be linked via its N or C terminal to a dAb, directly or via a linker, such as a Gly-Ser linker, to extend the serum half-life of the dAb. It also can be linked via the free cysteine. Example 6 exemplifies a construct that contains a dAb linked via a Gly-Ser linker to the N-terminus of HSA.
e. Multi-specific TNFR1 antagonist / TNFR2 agonist Constructs To selectively inhibit TNFR1 signaling, while enhancing the beneficial effects of TNFR2 signaling, multi-specific, such as bispecific, constructs, containing an TNFR1 antagonist and a TNFR2 agonist, are provided (see, e.g., Formula 2 above).
These multi-specific constructs can include linkers and activity modifiers to confer advantageous properties, as needed, as discussed above.
The TNFR1 inhibitor and TNFR2 agonist portions of constructs provided herein can be polypeptides or small molecules or combinations thereof; they can be linked directly in any order, or indirectly via a linker, such as a Gly-Ser linker, including any described herein, and/or a hinge region, or they can be linked via a chemical linker. The construct can contain an activity modifier, such as an Fc region or modified Fc, and/or other activity modifier, such as a polypeptide, such as HSA, that extends half-life, and can be polymer, such as PEG or polymeric moiety.
The C-terminus of a human TNFR1 antagonist, such as the TNFR1 antagonist set forth in any of SEQ ID NOs: 54-703, or an TNFR1 antagonist with about or at least about 95% sequence identity to the TNFR1 antagonist set forth in any of SEQ ID
NOs: 54-703, is fused with the N-terminus of a first IgG1 Fc, such as the IgG1 Fc derived from trastuzumab. The order can be reversed.
The Fc region contains the CH2 and CH3 domains of the trastuzumab heavy chain (see, e.g., residues 234-450 of SEQ ID NO:26). In some embodiments, the linker between the TNFR1 antagonist and the first Fc subunit contains all or a portion of the hinge sequence of an antibody, such as trastuzumab (SCDKTH;
corresponding to residues 222-227 of SEQ ID NO:26). To confer protease resistance and increase flexibility of the fusion protein, the SCDKTH hinge sequence or the protease cleavage site or both can be replaced with a Gly-Ser short peptide linker, such as, for example, GSGS, GGGGS, or GGGGSGGGGSGGGGS, and others described herein and/or known in the art. In other embodiments, only a GS linker is included. In another embodiment, the linker contains a PEG or a branched PEG, with a molecular weight of 30 kDa or more.

In some embodiments, the Fe subunits (also referred to as regions or domains) can be multimerized. The first Fe subunit is attached to a second Fe subunit via disulfide bonds. For bi-specific constructs, the C-terminus of the second Fe subunit is connected to the N-terminus of a TNFR2 agonist, such as, for example, the agonist of any of SEQ ID NOs: 765-801, 803, and 810, or a TNFR2 agonist with about or at least about 95% sequence identity to the TNFR2 agonist of any of SEQ ID
NOs: 765-801, 803, and 810, or to a small molecule TNFR2 agonist. The second Fe subunit and the TNFR2 agonist are connected via the linker, such as the SCDKTH

hinge sequence of trastuzumab, alone, or in combination with a short GS
linker, as described above. In other embodiments, only a GS linker is included. In alternative embodiments, the single chain FIT fragment (scFv), or the Fab region, or other antigen-binding fragment, of a TNFR2 agonistic monoclonal antibody can be used;
the scFy or Fab are dimerized by N-terminal fusion with the C-terminus of the Fe. As provided herein, the antigen-binding fragment can be derived from the TNFR2 agonistic mAbs 1VIR2-1 and MAB226.
The Fe subunit can be modified to alter its activity. For example, a dimer is modified to prevent homodimerization, and/or to eliminate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC), and/or to enhance neonatal FcR (FcRn) recycling to increase the in vivo half-life and stability of the recombinant construct, as described below.
In embodiments in which the constructs are for treatment of inflammatory diseases, the Fe portion is modified to have reduced or eliminated effector functions.
In embodiments, for example, where construct is for the treatment of cancer, the Fe dimer is modified to enhance immune effector functions, such as ADCC, ADCP
and/or CDC. The particular Fe modifications depend upon the intended disease target.
In some embodiments, the Fe subunit(s) can contain an IgG4 Fe region, such as the IgG4 Fe derived from nivolumab (Opdivog), containing the CH2 and CH3 domains of the nivolumab heavy chain (see, e.g., residues 224-440 SEQ ID
NO:29).
A short peptide linker, containing all or a portion, sufficient to provide flexibility, of the hinge sequence of nivolumab, ESKYGPPCPPCP (see, e.g., residues 212-223 of SEQ ID NO:29), can be included between the nivolumab Fe region and the TNFR1 antagonist and/or the TNFR2 agonist. Optionally, or alternatively, a GS linker also can be included.
In exemplary embodiments, since TNFR2 can require receptor aggregation/clustering for signaling, a bivalent antibody-like structure can be generated to achieve superior agonism. In this embodiment, the C-terminus of the first and second Fc subunits each is fused to the N-terminus of an TNFR2 agonist, as described above. The Fe dimer is modified to prevent homodimerization, to eliminate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and to enhance neonatal FcR recycling to increase the in vivo half-life of the recombinant construct, as described elsewhere herein.
PEGylation for Linking Components of the Multi-Specific Constructs, PEG-centered Multi-Specific Construct, such as Bi-Specific, TNFR1 Antagonist/TNFR2 Agonist Constructs PEGylation, which refers to the covalent attachment of the biocompatible and biologically inert polymer poly(ethylene glycol) (PEG) to molecules, such as proteins, peptides, drugs and other molecules, is another modifier of the activity of a construct.
It can increase the aqueous solubility of molecules, increase the molecular weight of the molecule, prolong the in vivo circulation time, decrease peripheral clearance rates, minimize non-specific uptake, and target tumors via the enhanced permeability and retention (EPR) effect. PEGylation of therapeutic, including protein therapeutics, can mask undesirable antigenic surface markers to protect therapeutics from the action of antibodies and antigen processing cells, and reducing degradation by proteolytic enzymes and other inactivating processes. PEGylation also increases the molecular weight of the protein therapeutic, prolonging the in vivo half-life and reducing peripheral clearance, and allowing for less frequent administrations.
The chemical conjugation of therapeutic molecules to polymers such as PEG
can form stable ester or amide bonds, as well as disulfide bonds. Conjugation of PEG
to a molecule of interest, such as the TNFR1 antagonists and TNFR2 agonists provided herein, can be achieved, for example, by using coupling agents, such as, for example, dicyclohexylcarbodiimide (DCC), 1-ethy1-3-(3-dimethylaminopropyl)carbodiimide (EDC), HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid RECTIFIED SHEET (RULE 91) ISA/EP

hexafluorophosphate), or others known in the art, or by using N-hydroxysuccinimide (NHS) esters, such as PEG NHS esters. Other methods include the use of PEG
maleimides, which react with sulthydryl groups on the protein or peptide; PEG
pentafluorophenyl (PFP) esters, which react with primary and secondary amines;
thiol PEG, which reacts with thiols on the side chains of cysteine residues; and click chemistry techniques. PEG azides, propargyl PEG, aminooxy PEG, hydroxy PEG, amino PEG, PEG acid, biotin PEG, PEG tosylate, and PEGs with other functional groups also are commercially available and can be used for conjugation to peptides and other therapeutic molecules.
A common method to prepare PEG-protein conjugates is by coupling -NH2 groups on the protein and monomethoxy PEG (mPEG) with an electrophilic functional group; this approach results in the formation of polymer chains that are covalently linked to a globular protein at the core. This property is exploited herein to provided PEG-centered constructs in which PEG, or chemically similar or suitable moieties, display a plurality of binding or interacting moieties to one or a plurality of different targets. In order to increase drug (binding moiety) loading, multi-arm or branched chain PEGs (or similar moieties or branched chain moieties) can be used.
Alternatively, the drug can be conjugated to small PEG dendrons (see, e.g., the PEG
conjugation protocols on the BROADPHARM website, available at broadpharm.com/web/protocols.php; see, also, Banerjee et al. (2012) Journal of Drug Delivery, Article ID 103973). In order to attach a plurality, such as two, different therapeutic moieties, a heteromultifunctional, such as heterobifunctional, PEG
moiety, with different reactive groups, such as at each end, can be used. The PEG
moiety can have two, three, or more different reactive groups. Such molecules can be used to deliver two or more different ligands, targeting two different receptors on the same cell or different cells, such as TNFR1 and TNFR2, as described herein, or to deliver two targeting agents that bind to different sites on the same receptor, or to cluster a receptor, for example, to activate or inhibit the receptor, or to cross-link two different receptors, for example, to inhibit receptor activity. Homobifunctional PEG
molecules, with identical reactive groups at each of the ends, can be used to cluster identical receptors on the same cell, or, if the PEG chain length permits, on different cells.
Such constructs can be used to trap circulating soluble receptors, or ligands, such as RECTIFIED SHEET (RULE 91) ISA/EP

TNF. Figures 3 herein provide exemplary constructs that employ PEG moieties to display drugs (binding reactive moieties).
To increase reactivity and flexibility, enhance ligand-protein binding, and reduce steric hindrance, the constructs can include a linker molecule or plurality thereof as described herein as a spacer molecule. Such spacers include, for example, amino acid spacers, such as alanine, glycine, and small peptides. Any of the linkers described herein, including GS linkers and other flexible linkers, as well as rigid linkers, can be used to conjugate reactive moieties, such as TNFR1 inhibitor moieties, as described herein and/or TNFR2 agonists described herein to a multifunctional PEG
molecule. These constructs also can include the activity modifiers, such as Fc regions.
The use of branched PEG moieties, or multi-arm PEG moieties as described herein (see, e.g., Figure 5) , with or without linkers, is not limited to use in constructs containing a TNFR1 inhibitor moiety or TNFR2 agonist, or combinations of both, but can be used to present other inhibitor and/or agonist moieties of any receptor(s) of interest and/or to also produce immunotoxins and other toxic conjugates.
Methods for synthesis of a multitude of PEG moieties and variations thereof are known (see, e.g., US Patent Publication No. 2010/0221213; Han et at., (2014) Sci Rep 4:4387.
For example, in some embodiments that contain TNFR1 inhibitor and TNFR2 agonist moieties, the construct includes a bifunctional PEG moiety, and also includes linkers between the PEG moiety and each of the TNFR1 antagonist and the TNFR2 agonist. The multi-specific construct, contains a branched PEG polymer to which the linker is attached and to which one or both of the TNFR1 inhibitor moiety and TNFR2 moiety is/are attached. A suitable PEG moiety can have a molecular weight of kDa or more, for example, 30-40 kDa or more. An exemplary branched PEG
25 molecule can be, for example, a 3-arm, heterobifunctional PEG molecule that contains one arm, with one type of reactive group (RG1; e.g., -NH2), that is linked to a TNFR1 inhibitor moiety, and two arms, with a different type of reactive group (RG2;
e.g., -COOH), that each are linked to a TNFR2 agonist. Such 3-arm heterobifunctional branched PEG molecules are commercially available (e.g., from BROADPHARMg).
30 The first PEG arm can be linked to the N-terminus or C-terminus of the inhibitor moiety, and the other two arms can be linked to the N-terminus or C-terminus of the TNFR2 agonists or to the TNFR2 agonists, if they are small molecules. In some embodiments, the constructs also can include an optional linker, as described herein. Such linker can be included between the PEG arms and the TNFR1 inhibitor moiety and/or TNFR2 agonist(s). Such PEG-centered bi-specific constructs provide monovalency for the TNFR1 antagonist activity, which prevents TNFR1 receptor clustering that leads to unwanted agonism, and provides bivalency for TNFR2 receptor clustering, which enhances TNFR2 signaling. An exemplary structure for the PEG-centered bi-specific TNFR1 antagonist/TNFR2 agonist constructs described herein, among those depicted in Figures 3.
In another embodiment, the linker between the TNFR1 antagonist and the .. TNFR2 agonist contains a branched PEG with a molecular weight of 30 kDa or more.
The branched PEG molecule contains one branch that it linked to the N-terminus of the TNFR1 antagonist, and two branches that are each linked to an TNFR2 agonist, providing bivalency for TNFR2 receptor clustering, which enhances TNFR2 signaling.
Figures 3A-D depict various configurations of multi-specific constructs in which PEG moieties link functional moieties. PEGylation moieties and PEGylation procedures are discussed in more detail in section H, below. Methods for preparing various PEG linkers and configurations are well known to those of skill in the art (see, e.g., creativepegworks.com/pegylation literature.php; and .. broadpharm.com/web/protocols.php). In Figures 3, each n can be independently 1-10, such as 1-7, 1-5, and 1-3, 1, or 2. In figures 3A, each n is generally 1 -3, depending upon the particular ligands that are displayed. In the others of Figures 3, n generally is 1 to 5. N can be 1 in all the embodiments. Those of skill in the art will recognize other routine changes in the PEG moieties that serve as the central linkers; similar moieties .. can be used in place of PEG.
In Figure 3A:
RI is H or lower alkyl (Cl to C5, or Cl or C2), such as CH3, n is generally 1 to 5, such as 1 or 2. The figures depict ligands or epitopes that bind to multiple targets (i.e., epitopes on a receptor, such as targets (the circles) 1 a,b,c represent different .. epitopes on the same receptor. Target 2 can be an epitope on a different receptor. In Figures 3B and 3C, the circles are ligands to target epitopes or receptors, n is generally 1-5, typically n is 1 or 2, generally 1. FIG 3D depicts a homobifunctional RECTIFIED SHEET (RULE 91) ISA/EP

construct; n typically is 1-3, generally lor 2, such as 1. The activity modifier, such as an Fc or others as described herein or known to those of skill in the art, is optional. In all of these constructs, the PEG portion is generally 'inactive' except for providing the activity modifying activity, such as half-life extension and/or sterically connecting the .. operative pieces that bind to intended targets (peptides, small molecules, aptamers, and others).
Figure 3A depicts an exemplary bivalent construct in which PEG is a central portion. One circle 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 a different moiety, such as a polysaccharide or receptor ligand. The bivalent structure provides for clustering of targets for receptor activation. In some embodiments provided herein, the targets include TNFR1 and TNFR2, and the circles represent the TNFR1 inhibitors and TNFR2 agonists as described throughout the disclosure herein. Figure 3B
depicts a monovalent single ligand, such as CD3+, which can prevent cytokine release syndrome, linked via the PEG moieties to the agonist, antagonist, or binding protein, which is bivalent for receptor clustering. Figure 3C depicts a heterobifunctional PEG
(or other such carrier) for crosslinking two different cell targeting agents, or two agents, such as trastuzumab and pertuzumab, that bind to different sites on the same receptor or two receptors. The constructs of Figures 3B and 3C 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.
These constructs also can serve as prodrugs that be directed to or accumulate in hypoxic regions with lower pH where a linked moiety can be released chemically by protonation. For example, for a tumor, this can be a toxin, or can be a TNF
inactivator (i.e., aptamer or peptide) that is released locally.
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(s), 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.
Other structures, where X and Y refer to reactive groups, such as binding moieties, molecules that interact with a target, also are contemplated (see, Figure 4):
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f. Additional Activity Modifiers ¨ Fusion Proteins that Include Portions or Entire Polypeptides that Increase Serum Half-Life Properties of the constructs can be altered by adding full-length polypeptides or portions thereof that increase serum half-life, but that do not substantially or do not alter the interaction of a construct with TNFR1 and/or TNFR2. This includes albumination and other such modifications (see discussion above regarding half-life extenders; reviewed in, Strohl (2015) BioDrugs 29(4):215-239, see also, Tan et at.
(2018) Current Pharmaceutical Design 24:4932-4946).
5. Prediction and Removal of Immunogenicity in Protein Therapeutics Many protein therapeutics, including those that contain human germline sequences, such as recombinant human cytokines and human antibodies, are immunogenic, and induce host immune responses against the therapeutics. As described herein, the constructs provided herein, including the TNFR1 antagonist molecules and TNFR2 agonist, and multi-specific constructs, described and provided herein, can be modified, if needed, such as by amino acid replacement, to remove or eliminate epitopes that are immunogenic or with which pre-existing antibodies interact.
The constructs are subjected to the prediction, identification and removal of immunogenic B-cell and/or T-cell epitopes, thus decreasing or eliminating any potential immunogenicity, and increasing the safety, tolerability and efficacy of the therapeutic molecules. The molecules are tested in in vitro assays and in vivo animal models to determine immunogenicity before and after the removal of immunogenic sequences.
As discussed in more detail below, protein therapeutics can contain immunogenic B-cell and/or T-cell epitopes. When the immune system recognizes a protein therapeutic as a foreign agent, a coordinated, undesirable immune response towards the therapeutic is induced. The response can result in clinical complications including, for example, rapid drug clearance, reduced drug functionality and efficacy, delayed infusion-like allergic reactions, anaphylaxis, and in some cases, life-threatening autoimmunity. Immune responses against protein therapeutics occur via two different mechanisms; a classical immune response and by breaking tolerance.
The immunological discrimination between self and non-self proteins determines the mechanism of the immune response, and proteins recognized as foreign induce a classical immune response, characterized by the formation of antibodies within days to weeks after administration, often after a single injection of the protein therapeutic.
This response is long-lasting and difficult to reverse once memory B-cells have formed. Subsequent exposure to the protein induces a secondary response, characterized primarily by significant amounts of IgG that negatively impacts therapy.
Therapeutic proteins that induce classical immune responses include replacement therapies, such as rhGAA (recombinant human acid alpha-glucosidase) and FVIII, as well as monoclonal antibody (mAb) therapeutics, where the complementarity-determining region (CDR) is highly immunogenic and leads to the generation of anti-idiotypic alloantibodies due to a lack of central tolerance to the CDR region.

Therapeutic proteins that are homologous to endogenous proteins typically do not result in an immune response due to established immune tolerance, however, they can become immunogenic by breaking B-cell tolerance after repeated administrations, such as the case with IFN-y, INF-f3 and erythropoietin (EPO) (see, e.g., Baker et at.

(2010) Selffonself1(4):314-322; Choi et at. (2017) Methods Mol. Biol. 1529:375-398; Dingman et al. (2019)1 Pharm. Sci. 108(5):1637-1654).
Factors that influence the immunogenicity of a protein therapeutic, include, for example, the duration of treatment, and the route and frequency of administration;
subcutaneous administration of protein therapeutics is more immunogenic than intravenous administration, and prolonged, frequently administered therapies are more immunogenic. Patient-related factors, such as the immune status of the patient and polymorphisms of the MHC (or HLA in humans) molecules, also affect protein immunogenicity. For example, MHC molecules are highly polymorphic, and several different alleles for MHC II exist, including different subunits, such as DP, DM, DOA, DOB, DQ and DR; these receptor subtypes differ in binding affinity for epitopes, and thus, differences in MHC subtypes between patients can affect the immune response against a protein therapeutic. Patient immune status also can affect immunogenicity, as autoimmune patients respond more strongly than immunocompromised patients to protein therapeutics. Other factors affecting immunogenicity include properties of the protein product, including, for example, the presence of immunogenic epitopes recognized by MHC II, the formation of aggregates in the final product, the oxidation of proteins, aggregates in formulations, and post translational modifications, such as glycosylation. Recombinant proteins can be produced in several different cell types, including, for example, bacterial cells, such as E. coli, and mammalian cells, such as CHO cells. Proteins expressed in bacteria are not subjected to post translational modifications such as glycosylation, but proteins produced in mammalian cells are, which can lead to different immunogenicity. For example, the interferon sold under the trademark Betaseron , .. an interferon 13-1b, is produced in E. coli cells and is not glycosylated, while the later developed product sold under the trademark Avonex (an interferon (3-1a), is produced in CHO cells with recombinant DNA technology. Betaseron has a much higher immunogenicity than Avonex interferon, at 35% vs. 5%, respectively.
This difference can be attributed in part to the differences in glycosylation patterns, that can lead to aggregation (see, e.g., Dingman et al. (2019)1 Pharm. Sci.
108(5):1637-1654).

An effect following administration of a protein therapeutic is the development of high-affinity anti-therapeutic antibodies, which are also known as anti-drug antibodies, or ADAs. The generation of ADAs involves the stimulation of adaptive and non-adaptive immune responses, that primarily are polyclonal, and that can have neutralizing and non-neutralizing effects on protein therapeutics. ADAs can contain multiple isotypes (e.g., IgM, IgE and IgG) as well as sub-classes (e.g., IgG1-4) of heavy chain constant regions, and contain variable regions that bind with high affinity to the protein therapeutic, and thus, have undergone somatic hypermutation of variable region genes. The immune responses are induced by the recognition of immunogenic peptide fragments, such as B-cell and T-cell epitopes, in the protein therapeutic. Thus, many protein therapeutics require de-immunization, while retaining the desired therapeutic activity, before they can be applied to the clinic (see, e.g., Baker et al. (2010) Self/Nonself 1(4):314-322; Choi et al. (2017) Methods MoL
Biol.
1529:375-398).
The formation of anti-drug antibodies (ADAs) against protein therapeutics is mediated by antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, and by B and T lymphocytes. MHC class II-restricted T-cell epitopes present in the sequences of protein therapeutics can result in the development of humoral responses against protein therapeutics. For example, DCs, stimulated via pattern recognition receptors (PRRs), stimulate T-cells and induce the generation of a T-cell-dependent high-affinity ADA response. In the first step in a T-cell-dependent antibody response, APCs phagocytose the protein therapeutic, process the antigens into peptide epitopes, and present the epitopes to nave T cells by coupling them with major histocompatibility complex (MHC) class II molecules on the APC cell surface.
To fully activate a T-cell, which is required to activate B-cells, a T-cell receptor (TCR) must interact with the MHC II-epitope complex, and this must be accompanied by additional signals from costimulatory molecules, such as CD80 and CD86, which are provided by the APC. Naïve B-cells are activated by the interaction between IgM
and IgD receptors on the B-cell surface and their cognate antigens. Antigen specific T-cells then secrete cytokines that stimulate B-cell proliferation and maturation to plasma cells, which results in the engagement of CD40 and CD40 ligands, providing a further signal that leads to antibody production by B-cell clonal expansion and RECTIFIED SHEET (RULE 91) ISA/EP

differentiation into antibody-secreting plasma cells and memory B-cells.
Memory B-cells remain dormant until subsequent exposure to the therapeutic protein, while plasma cells secrete antibodies that recognize specific epitopes on the therapeutic protein that are presented by APC MHC receptors. Many protein therapeutics, including recombinant human proteins, contain potent T-cell epitopes. For example, the immunogenicity of IFNI3lb was ameliorated after the mapping and removal of a single immunodominant (but not a sub-dominant) T-cell epitope via amino acid mutation.
Immunogenicity also can occur in a T-cell-independent process, whereby the antigen engages B-cells directly. High molecular weight aggregates of a protein therapeutic can induce T-cell dependent and independent anti-drug antibody responses by stimulating DCs or by cross-linking B-cell receptors. For example, T-independent stimulation of B-cells, generating an ADA response, can occur if the protein therapeutic forms a multimeric structure that can cross-link the B-cell receptor (BCR) sufficiently effectively to obviate the need for co-stimulation from T-cells.
There is a correlation between enhanced immunogenicity and aggregated or multimeric proteins. For example, aggregated, but not monomeric, recombinant human interferon (lIFN)ct results in the generation of IFNa-specific antibodies in human IFNa transgenic mice. The formation of aggregates depends on drug solubility and production processes (see, e.g., Baker et al. (2010) Serslonself1(4):314-322;
Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654; De Groot, A. S. and Moise (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340).
ADA responses against protein therapeutics can be in the form of neutralizing or binding antibodies. Neutralizing antibodies recognize regions within the protein therapeutic that are necessary for biological activity, and eliminate its activity directly. The humoral response is directed against B-cell epitopes within the protein therapeutic, which results in the ability to neutralize the protein therapeutic. For example, human anti-mouse (HAMA) or human anti-human (HAHA) responses, directed against the idiotype of antibody therapeutics, are neutralizing and can be generated against humanized and fully human antibodies. For example, in 30% of hemophilia A patients, neutralizing ADAs develop against administered recombinant FVIII, abrogating its hemostatic efficacy, and in 89-100% of Pompe disease patients RECTIFIED SHEET (RULE 91) ISA/EP

receiving rhGAA, anti-rhGAA neutralizing antibodies destroy the therapeutic efficacy. Binding antibodies alter the pharmacokinetic properties of the protein therapeutic, and indirectly impact its efficacy by reducing systemic exposure, for example, by promoting rapid clearance of the protein. For example, long-term use of adalimumab results in the development of ADAs in ¨28% of patients, resulting in lower adalimumab concentrations and poorer clinical outcomes (see, e.g., Baker et al.
(2010) Self/Nonself 1 (4):314-322; Dingman et al. (2019)1 Pharm. Sci.
108(5):1637-1654).
ADA levels can be assessed and monitored before, during, and after treatment.
Various assays are available. These assays include bridging immunoassays, which involve labeling the drug, and detecting ADAs that form a bridge between two labeled drug molecules. Bridging assays can be used for all antibody classes and with any type of sample.
Ligand-binding assays (LBAs), which are used to detect binding to a target, include surface plasmon resonance (SPR), electrochemiluminescence, and biolayer interferometry, and also can be used to detect ADAs. Protein specific assays, such as the Bethesda Assay, which has been used to measure the concentration of neutralizing anti-F VIII antibodies, can be used.
Anti-PEG antibodies also can be measured in an assay where biotin-PEG is conjugated to magnetic beads, and the amount of anti-PEG antibodies bound to the beads is measured using a sensor that detects changes in the size of the complex. Drug-tolerant assays overcome the limitations caused by the presence of drug in the sample, and improve the quantification of ADAs. These include, for example, pH shift idiotype antigen-binding assays, acid dissociation assays, temperature shift assays, and electrochemiluminescence assays. Enzyme-linked immunosorbent assays (ELISAs) can be used to detect ADAs; the protein therapeutic is coated on a plate and incubated with samples to measure bound ADA. ELISAs for the detection of ADAs, can be limited because of the lack of standards for the ADAs. Other methods include Immune-PCR, an extension of bridging assays, in which the complex is labeled with biotin that is detected using an anti-biotin antibody conjugated to DNA. The DNA then is amplified using PCR and quantified to assess ADA levels. Immuno-LC/MS can be used to detect ADAs in plasma samples; the samples must be enriched by tagging the drug with biotin, or by spiking excess drug RECTIFIED SHEET (RULE 91) ISA/EP

into the sample to saturate ADA binding (see, e.g., Dingman et at. (2019)1 Pharm.
Sci. 108(5):1637-1654).
The prediction of and removal of immunogenic epitopes from protein therapeutics (i.e., de-immunization) can increase the efficacy and safety of the therapeutics, and prevent adverse effects that could lead to drug failure in clinical trials. For example, the depletion of T-cell epitopes from protein therapeutics by de-immunization has been successful in the progression of protein therapeutics, particularly antibodies, into clinical trials. These results indicate the importance of T-cell epitopes in the generation of ADA responses, and that de-immunization provides .. safer, less immunogenic therapeutics (see, e.g., Baker et at. (2010) Selffonself 1(4):314-322). These methods can be used to detect or identify or predict immunogenicity of the constructs provided herein, and can be used to identify amino acid mutations to eliminate or reduce immunogenicity or immune responses in subjects. Provided are constructs that have been modified to decrease or eliminate .. immunogenicity. De-immunization of protein therapeutics involves the identification of highly immunogenic B-cell and/or T-cell epitopes, and deletion of the identified epitopes by mutagenic substitution of key amino acid residues. As discussed below, preclinical prediction and assessment of immunogenic regions within a protein therapeutic sequence includes the use of in silico tools that focus on epitope mapping, .. in vitro methods, such as epitope mapping, MHC/HLA affinity assays and T-cell proliferation assays, and in vivo testing in animal models. To increase the efficiency of protein therapeutic de-immunization, computational epitope predictive tools are used. In silico tools include databases and algorithms to rapidly predict immunogenic sequences in peptide libraries. The results then can be confirmed, and the specific .. immunogenic effects of the epitopes on B-cells or T-cells can further be evaluated using in vitro assays. The effects on the immune response to the protein therapeutics can be evaluated using in vivo assays in animal models, such as transgenic mice, and non-human primates.
Once an immunogenic epitope is identified, the amino acid sequence of the .. therapeutic can be modified to remove the epitope. Methods for removal include random or site-directed mutagenesis to remove the immunogenic sequence (i.e., to de-immunize the epitope). Following mutagenesis, the immunogenic sequence is re-evaluated to confirm that it is no longer immunogenic. There are in silico tools to streamline this process; for example, programs are available that sequentially replace each amino acid in the immunogenic sequence, with one of the other 19 naturally occurring amino acids (particularly alanine), and then re-evaluate the immunogenicity of the sequence. In this way, non-immunogenic sequences can efficiently be narrowed down to the most promising candidates prior to peptide synthesis and in vitro and/or in vivo re-evaluation of immunogenicity. The prediction and mutagenic deletion of immunogenic epitopes, however, is not necessarily sufficient for protein de-immunization, as the protein must retain its folded, stable and active structure in order to retain its therapeutic efficacy. Thus, epitope-deleting mutations must be selected that are compatible with the protein's structure and function.
The methods and approaches discussed below, are used to predict, identify, and eliminate epitopes from the constructs provided herein.
a. B-cell and T-Cell Epitopes The interaction between antigen and antibody is important for the induction of a humoral immune response against an invading pathogen. A specific antibody recognizes a particular antigen at discrete regions that are known as antigenic determinants, or B-cell epitopes. B-cell epitopes contain clusters of amino acids that are solvent-exposed and surface accessible, and that are recognized and bound by secreted antibodies, or by B-cell receptors (BCRs), which contain membrane-bound immunoglobulins that induce a cellular or humoral immune response.
The identification of B-cell epitopes is part of the development of antibody and other protein-based therapeutics. B-cell epitopes are classified, based on spatial structure, as continuous (also known as linear) epitopes, that contain sequential residues, and discontinuous (also known as conformational) epitopes, which are nonlinear and conformational. Discontinuous B-cell epitopes contain groups of solvent-exposed amino acid residues that are not fully sequential, but that are brought together in close proximity when the protein/antigen is folded into its three-dimensional conformation. Approximately 90% of B-cell epitopes are conformational.
Linear B-cell epitopes can be recognized by antibodies following antigen denaturation, but conformational epitopes are no longer recognized if the antigen is denatured. The minimal amino acid sequence, or contact residue span, that is required for proper folding of a discontinuous B-cell epitope ranges from approximately 20 to 400 residues in native proteins. The majority of identified linear B-cell epitopes are thought to be components of conformational B-cell epitopes, and it has been shown that over 70% of discontinuous B-cell epitopes are contained of 1-5 linear segments, each of 1-6 amino acids in length (see, e.g., Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830; Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160).
T-cell epitopes are linear and bind to major histocompatibility complex (MHC) molecules via the interaction of the amino acid side chains with binding pockets in the MHC epitope-binding groove. The presence or absence of specific side chains determines if, and how tightly, an epitope binds to MHC (see, e.g., De Groot, A. S. and Moise, L. (2007) Curr. Op/n. Drug Discov. Devel. 10(3):332-340). T-cells have T-cell receptors (TCRs) that recognize antigens that are displayed on the surfaces of antigen presenting cells (APCs) and bound to MHC molecules. T-cell epitopes are presented by MHC class I (MHC I) and II (MHC II) molecules, that are recognized by CD8+ and CD4+ T-cells, respectively; thus, there are CD8+ and CD4+
T-cell epitopes. CD8+ T-cells form into cytotoxic T lymphocytes (CTLs) after CD8+
T-cell epitope recognition, while primed CD4+ T cells form into helper T (Th) cells, which amplify the immune response, or into regulatory T (Treg) cells, which are immunosuppressive (see, e.g., Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160).
b. In Silico Epitope Prediction Methods Experimental studies and in silico analyses indicate that the majority of epitopes span 15-25 residues and an area of 600-1000 A2, organized in loops, and that the epitope sequence is enriched with tyrosine, tryptophan, charged, and polar amino acids with exposed side chains, and with specific amino acid pairs. However, it has been demonstrated that the differences between epitope and non-epitope residues are not significant, and amino acid composition alone is insufficient for differentiating between epitopes and non-epitopes. The combination of epitope mapping technologies and bioinformatics has led to the development of immunoinformatics, which involves the use of computational methods in immunology for the identification of structures of antibody, B-cell, T-cell and allergen, the prediction of MHC binding, the modelling of epitopes, and the analysis of immune networks (see, e.g., Potocnakova et at. (2016) Journal of Immunology Research, Article ID
6760830).
i. In Silico Prediction of B-Cell Epitopes B-cell epitope prediction identifies immunogenic epitopes so that they can be replaced/de-immunized, for example, for therapeutic protein production.
Databases of known B-cell epitopes have been developed, and include multifaceted databases such as the Immune Epitope Database (IEDB) and IEDB-3D (available at iedb.org) and AntiJen (available at ddg-pharmfac.net/antijen/AntiJen/antijenhomepage.htm); B-cell oriented databases such as BciPep (available at imtech.res.in/raghava/bcipep/info.html), Epitome (available at rostlab.org/services/epitome/) and the Structural Database of Allergenic Proteins (SDAP; available at fermi.utmb.edu/); and single pathogenic organism oriented databases, such as the HIV Molecular Immunology Database (available at hiv.lanl.gov/content/immunology/index.html), FLAVIdB (available at cvc.dfci.harvard.edu/flavi/), and the Influenza Sequence and Epitope Database (ISED;
available at influenza.cdc.go.kr). Other B-cell epitope databases include the Conformational Epitope Database (CED; available at immunenet.cn/ced/), the Protein Data Bank (PDB; available at rcsb.org), and the Structural Epitope Database (SEDB;
available at sedb.bicpu.edu.in) (see, e.g., Potocnakova et at. (2016) Journal of Immunology Research, Article ID 6760830).
Several algorithms for predicting B-cell epitopes from their sequence or structure are available. The algorithms have been developed, which initially relied on the identification of linear epitopes through propensity scale, but have been improved through the development of methods based on machine learning, such as the Hidden Markov Model (HMM), recurrent neural network (RNN), and support vector machine (SVM). In sit/co B-cell epitope prediction tools include those that predict continuous/linear B-cell epitopes, and those than predict discontinuous/conformational B-cell epitopes. Prediction of discontinuous B-cell epitopes requires information on amino acid statistics, spatial information and surface exposure. Web available tools for continuous/linear B-cell epitope prediction include, for example, ABCPred (available at crdd.osdd.net/raghava/abcpred/), APCPred (available at omictools.com/apcpred-tool), BCPREDs (BCPred and FBCPred, available at ailab.ist.psu.edu/bcpred/), BepiPred (available at cbs.dtu.dk/services/BepiPred/), LBtope (available at crdd.osdd.net/raghava/lbtope/), BcePred (available at crdd.osdd.net/raghava/bcepred/), EPMLR (available at bioinfo.tsinghua.edu.cn/epitope/EPMLR/), BEST (B-cell Epitope Prediction using Support Vector Machine Tool; available at biomine.cs.vcu.edu/datasets/BEST/), COBEpro (available at scratch.proteomics.ics.uci.edu/), PEOPLE (available at iedb.org/), and SVMTrip (available at sysbio.unl.edu/SVMTriP/). Web available tools for discontinuous/conformational B-cell epitope prediction include, for example, CEP
(available at bioinfo.ernet.in/cep.htm), DiscoTope (available at cbs.dtu.dk/services/DiscoTope-2.0/), BEpro (formerly known as PEPITO;
available at pepito.proteomics.ics.uci.edu/), ElliPro (available at tools.immuneepitope.org/ellipro/), SEPPA (Improved Spatial Epitope Prediction of Protein Antigens server; available at badd.tongji.edu.cn/seppa/), EPITOPIA
(available at epitopia.tau.acill), CBTOPE (available at crdd.osdd.net/raghava/cbtope/), EPCES
(available at sysbio.unl.edu/EPCES/), EPSVR (Antigenic Epitopes Prediction with Support Vector Regression server; available at sysbio.unl.edu/EPSVR/), EPMeta (available at sysbio.unl.edu/EPMeta/), PEASE (Predicting Epitopes using Antibody Sequence; available at ofranlab.org/PEASE), EpiPred (available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/EpiPred.php), 3DEX (3D-Epitope-Explorer; not available online), PEPOP (available at pepop.sys2diag.cnrs.fr/), PEPOP
2.0 (available at sys2diag.cnrs.fr/index.php?page=pepop), and EpiSearch (available at curie.utmb.edu/episearch.html) (see, e.g., Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830; Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160; Sun et al. (2013) Comput. Math Method M., Article ID 943636).
Sequence-based and binding site prediction methods also can be used to predict B-cell epitopes. Sequence-based prediction tools rely on the primary sequence of an antigen, and employ propensity scales to measure the probability of each residue being part of an epitope. Sequence-based prediction tools include BEST, which predicts conformational B-cell epitopes. Binding site prediction tools, which aim to identify the binding sites for conformational B-cell epitopes on antibodies, include, for example, ProMate, ConSurf, PINUP and PIER (see, e.g., Sun et at. (2013) Comput. Math Method M., Article ID 943636).
Conformational B-cell epitopes in a protein or antigen with a known 3D-structure can be identified using mimotope-based epitope prediction methods.
Mimotopes are peptides selected from randomized peptide libraries for their ability to bind to an antibody raised against a native antigen. Mimotope-based methods require the input of antibody affinity-selected peptides (i.e., mimotopes), and the 3D-structure of the selected antigen. Epitope prediction methods based on mimotopes derived from phage display experiments are available, and map mimotopes to the overlapping .. location patches on the antigen surface using statistical features of mimotopes, or use mimotope mapping back to the antigen sequence through alignment, which can indicate B-cell epitope location. To identify affinity-selected peptides, or mimotopes, random peptides are displayed on the surface of filamentous phages, and peptides that bind to a monoclonal antibody with a certain degree of affinity are screened, eluted and amplified. This selection process is repeated for a total of 3-5 times, narrowing down the peptides to those with the highest affinity. Mimotopes and epitopes can combine the same paratope of a monoclonal antibody and cause an immune response, and thus have similar functionality. The selected mimotopes share high sequential similarity, indicating that certain key binding motifs and physicochemical preferences exist during the interaction with the antibody. Thus, mapping mimotopes back to the source antigen can help find the true epitope more accurately. Mimotopes have similar physicochemical properties and spatial organization, but rarely show sequence similarity to the native antigen. Databases that provide information on mimotopes include, for example, ASPD (available at mgs.bionet.nsc.ru/mgs/gnw/aspd), RELIC
Peptides (available upon request), PepBank (available at pepbank.mgh.harvard.edu), and MimoDB (available at immunet.cn/mimodb). In sit/co mimotope-based prediction tools are essential for mapping mimotopes back to the surface of the source antigen, in order to locate the best alignment sequences and predict possible epitope regions.
In sit/co B-cell epitope prediction tools based on mimotope analysis include, for example, MIMOX (available at immunet.cn/mimox/), MimoPro (available at informatics.nenu.edu.cn/MimoPro), Pep-3D-Search (available at kyc.nenu.edu.cn/Pep3DSearch), MIIVIOP/MimCons (available upon request), MIMOP/MimAlign (available upon request), LocaPep (available at atenea.monstes.upm.es/#soft), Epi Search (available at curie.utmb.edu/episearch.html), Pepitope/PepSurf (available at pepitope.tau.ac.il/sources.html), PepMapper (available at informatics.nenu.edu.cn/PepMapper/), FINDMAP (not available online), EPIMAP
.. (not available online), MEPS (available at capsur.it/meps), 3DEX (3D-Epitope-Explorer; not available online), Mapitope (available at pepitope.tau.ac.i1), and SiteLight (not available online) (see, e.g., Potocnakova et at. (2016) Journal of Immunology Research, Article ID 6760830; Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160; Sun et at. (2013) Comput. Math Method M., Article ID 943636).
In Silico Prediction of T-Cell Epitopes Linear T-cell epitopes bind to MHC via the interaction of their amino acid side chains with binding pockets in the MHC epitope-binding groove, and the presence or absence of specific side chains determines if, and how tightly, a T-cell epitope binds to MHC. For in sit/co predictions, there are databases that provide libraries of existing epitopes, such as, for example, IEDB, Epitome, and SEDB, which provide information on two dimensional T-cell epitopes. Other in sit/co T-cell epitope databases include CED, AntiJen, Bcipep, and the HLA Epitope Registry. There are several webpages and programs available, that can be used to analyze sequences and predict immunogenic epitopes on protein therapeutic candidates. For example, a number of MHC-binding motif-based tools that scan protein sequences for potential T-cell epitopes are available. These T-cell epitope mapping tools include, for example, EpiMatrix, IEDB, SYFPEITHI, MHC Thread, MHCPred, MHCPred 2.0, EpiJen, NetMHC, NetCTL, nHLAPred, SVMHC, and Bimas (see, e.g., De Groot, A.
S. and Moise, L. (2007) Curr. Op/n. Drug Discov. Devel. 10(3):332-340). For example, the MHCPred algorithm provides information on the MHC binding potential of an amino acid sequence to various alleles, and EpiMatrix/JanusMatrix predicts allele specific binding of protein therapeutics to MHC class II receptors, and can assess binding at the T-cell receptor interface. Other algorithms and programs for .. epitope prediction include, for example, ProPred, MMBPred, and Protean 3D.
Epitope prediction should be combined with in vitro methods and activity assessment, to ensure that any modifications to remove immunogenic sequences retain the therapeutic protein's activity (see, e.g., Dingman et at. (2019)1 Pharm. Sci.
108(5): 1637-1654).
Peptide-MHC Class II Binding Prediction Structure-based methods for identifying T-cell epitopes rely on modeling the peptide-MHC structure, and evaluating the interaction using molecular dynamic simulations, for example. Structure-based methods are computationally intensive and have lower predictive performance than data-driven methods. Structure-based T-cell epitope prediction tools include EpiDOCK (available at epidock.ddg-pharmfac.net).
Data-driven methods for peptide-MHC binding predictions are based on peptide sequences that are known to bind to MHC molecules; the peptide sequences are available in epitope databases, such as IEDB, EPIMHC, AntiJen, and others described herein and known in the art. Peptide-MHC binding predictions can be based on sequence motifs (SMs), which include frequently occurring amino acids at particular positions (anchor residues) that are known to bind MHC molecules. Motif matrices (MM) evaluate the contribution of every residue, including non-anchor residues, to the binding of MHC molecules, but do not account for binding affinities.
Quantitative affinity matrices (QAMs) predict peptide-MHC binding as well as binding affinities.
Quantitative structure-activity relationship (QSAR) additive models predict the binding affinity of peptides to MHC as the sum of amino acid contributions at each position, plus the contribution of adjacent side chain interactions. Machine learning (ML), which is the most popular and robust approach, uses algorithms that are trained on data sets consisting of peptides that either bind or do not bind MHC
molecules, and examples of ML-based discrimination models include those based on artificial neural networks (ANNs), support vector machines (SVMs), decision trees (DTs), and Hidden Markov Models (HMMs) (see, e.g., Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160).
Models to predict the immunogenicity of protein therapeutics include in sit/co peptide-MHC class II algorithms, which predict, with reasonable accuracy, the ability of a peptide sequence to bind to MHC class II. Such algorithms allow for the rapid screening of libraries of sequences. In silico and in vitro MHC class II
binding analyses, however, lead to high levels of false positives, in which the identified immunogenic peptides fail to stimulate T-cell responses in vitro and in vivo.
Such analysis does not take into account other factors that influence the formation of epitopes, such as protein processing, recognition by T-cell receptors (TCRs) and T-cell tolerance to peptides. To address this, in vitro T-cell assays are used.
The combination of in silico analysis and in vitro assays is very useful for the identification of epitopes, and for the design of peptide variants with epitope-depleted protein sequences that have a reduced capacity for MHC binding (see, e.g., Baker et at. (2010) Selffonself1(4):314-322).
The prediction of T-cell epitopes via peptide-MHC binding models also is complicated by MEW polymorphism; in humans MEW molecules are known as human leukocyte antigens (HLAs), and there are hundreds of allelic variants of class I
and class II HLA molecules that bind different peptides and require specific models for predicting peptide-MHC binding (see, e.g., Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160). T-cell epitope prediction tools based on peptide-MHC binding models include, for example, EpiDOCK, MotifScan, Rankpep, SYFPEITHI, MAPPP, PREDIVAC, PEPVAC, EPISOPT, Vaxign, IVIRCPred, EpiTOP, BEVIAS, TEPITOPE, Propred, Propred-1, EpiJen, IEDB-MHCI, IL4pred, MULTIPRED2, IVIEIC2PRED, NetMHC, NetMHCII, NetIVIRCpan, NetMCHIIpan, nHLApred, SVMEIC, SVRMHC, NetCTL, and WAPP
(see, e.g., Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160).
EpiSweep is a suite of protein design algorithms that integrates computational predictions of immunogenic T-cell epitopes with sequence-based or structure-based assessment of the effects of epitope-deleting mutations on protein stability, structure and function, allowing for the selection of combinations of mutations that optimize the protein therapeutic for low immunogenicity and high activity and stability (see, e.g., Choi et al. (2017) Methods Mol. Biol. 1529:375-398, for a step-by-step guide to the application of the EpiSweep suite of deimmunization algorithms).
c. In Vitro Epitope Prediction Methods In vitro methods can be used to determine cellular mechanisms of the immune response, to identify immunogenic epitopes, and to assess MHC affinity, T-cell proliferation, and immunogenic effects of the whole protein therapeutic. For example, epitope mapping identifies immunogenic epitopes by analyzing peptide fragments individually. The peptide fragments are exposed to immune cells, and immunogenicity is determined by measuring cytokines and surface markers that are indicative of an inflammatory immune response. Epitope mapping of a full protein is labor intensive, and in silico programs are used in conjunction with mapping to identify regions that may be immunogenic and narrow down epitope candidates. In vitro epitope prediction methods include, for example, structural epitope mapping methods, such as X-ray crystallography, nuclear magnetic resonance and electron microscopy methods, and functional epitope mapping, such as antigen fragmentation/antigen binding assays, competitive binding assays, modification testing/mutagenesis, display technologies, such as phage display and yeast display, and mimotope analysis.
i. In Vitro B-cell Epitope Prediction Methods Experimental methods for identifying B-cell epitopes include, for example, solving the 3D structure of antigen-antibody complexes, screening peptide libraries for antibody binding, or performing function assays where the antigen is mutated and the effects on antigen-antibody interaction are analyzed. Antibody-producing B-cells recognize structural epitopes, which are ¨16-22 residues in size, and contain amino acids that come into contact with the antibody, and functional epitopes, which are ¨3-5 residues in size and affect the affinity between the protein and the antibody. The most accurate method to identify structural epitopes is by X-ray crystallography of antigen-antibody complexes, which identifies sequences that bind to the antibody, can be used to locate the exact position of an epitope within the protein structure, can identify both continuous and discontinuous epitopes, and provides information on the strength of binding. Structural epitope mapping identifies residues in direct contact with an antibody, but does not always provide information on which residues contribute to the binding strength. The FTProd program, which is freely available, can be used as a computational alternative to X-ray crystallography. Nuclear magnetic resonance (NMR) can be used to identify structural epitopes without the need to generate crystals, but its use is limited to small proteins and peptides that are <25 I(Da in size. NMR provides data about the structure, dynamics, and binding energy of antigen-antibody complexes, and is performed in solution, obviating the need for generating crystals. Two additional methods for epitope mapping with moderate resolution include saturation transfer difference NMR, and antibody inhibition of RECTIFIED SHEET (RULE 91) ISA/EP

hydrogen-deuterium exchange in the antigen. Electron microscopy also can be used for epitope mapping, but it is a low resolution structural method that is typically used for larger antigens, such as whole viral particles or viral capsids. Electron microscopy cannot detect contact residues, but can be used to confirm the epitope's surface accessibility. Cryoelectron microscopy is an alternative method in which rapidly frozen antigen-antibody complexes are observed in physiological buffers, obviating the need for stains and fixatives (see, e.g., Dingman et at. (2019)1 Pharm.
Sci.
108(5):1637-1654; Potocnakova et at. (2016) Journal of Immunology Research, Article ID 6760830).
Functional epitopes can be identified by a variety of methods, including antigen fragmentation, competitive binding, and modification testing. For example, functional B-cell epitope mapping methods generally include screening of antigen-derived proteolytic fragments or peptides for antibody binding, and testing the antigen-antibody reactivity of mutant proteins that have been subjected to site-directed or random mutagenesis. Functional epitope mapping tools, thus, are used to identify and characterize residues within epitopes that are important for antibody binding. The majority of functional methods detect the binding of antibody to antigen fragments, synthetic peptides, or recombinant antigens, such as mutated variants, antigens arrayed by in situ cell-free translation, and/or expressed using selectable systems such as phage display. For example, antigen fragmentation and binding assays involve the immobilization of peptides on a solid support, and the use of Western blot, dot blot, and/or ELISA to determine whether an epitope fragment binds an antibody; binding indicates that the peptide fragment may be immunogenic.
Competitive binding assays, which provide low-resolution mapping, provide information on the number of potentially immunogenic epitopes on a protein, by assessing whether multiple antibodies can bind to epitopes on the protein at the same time, or if they compete with each other for binding to the same epitope (see, e.g., Dingman et al. (2019) J Pharm. Sci. 108(5):1637-1654; Potocnakova et al.
(2016) Journal of Immunology Research, Article ID 6760830).
Modification testing, or mutagenesis, is an epitope mapping method in which individual residues (referred to as hot-spots) in a functional epitope are substituted, and the effects of the modifications on binding of the antibody to the immunogenic sequence are assessed. Hot-spots, which most frequently include Tyr, Arg and Trp residues, are energetically important residues that contain a fraction of the complete protein-protein interface area. Mutation of individual residues allows for the identification of detrimental residues which can be replaced, provided that the protein retains its structure and activity. For epitope mapping via mutagenesis, a peptide library is generated by random or site-directed mutagenesis; the combination of mutagenesis with display techniques allows for the screening of large numbers of mutated proteins. Saturation mutagenesis is another method for epitope mapping, in which an amino acid residue at a particular position within the epitope is replaced with all 20 naturally occurring amino acids, and loss of antibody binding is monitored. A disadvantage to this method is that the loss of immunoreactivity can be due to the disruption of antigenic structure, rendering the interpretation of results difficult. Most of the contacts made between an epitope and antibody occur via amino acid side chains, and alanine scanning mutagenesis can be used to define the contributions that each residue side chain makes to antibody binding. This is performed by sequential alanine substitution of each non-alanine residue, one at a time, which truncates sides chains to the (3-carbon without adding flexibility to the protein backbone. This method identifies critical residues whose side chains make the highest energetic contributions to the paratope-epitope interaction.
Computational alanine scanning also can be used to rapidly determine the effect of alanine mutation on a binding free energy in protein-protein complexes by using a simple free energy function. Combinatorial mutagenesis is based on the combinatorial randomization of a discrete antigenic region, and the grouping of mutated residues (primary sequence proximity), to maximize the chances of identifying combined effects mediated by neighboring residues; this allows for the identification of residues that are not critical for binding, but that contribute to the formation of the epitope, or that form multiple interactions with the paratope that individually are weak. Shotgun mutagenesis is a high throughput method, based on large-scale mutagenesis, in which each clone has a defined amino acid mutation (e.g., alanine substitution), and involves direct cellular testing for mAb reactivity of natively folded proteins. Shotgun mutagenesis allows for the identification of both linear and conformational epitopes with mapping rates of RECTIFIED SHEET (RULE 91) ISA/EP

over 20 epitopes/month (see, e.g., Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830).
Other techniques for epitope mapping include display technologies and mimotope analysis, which are inexpensive, flexible and fast. Display technologies, such as phage display and yeast display, are based on testing the binding capacity of a variety of peptides displayed on the display platforms (e.g., tethering peptides to ribosomes-mRNA complex, or to the surface of phage, bacteria, mammalian, insect or yeast cells) to the mAb of interest through the affinity selection method of biopanning (see, e.g., Potocnakova et at. (2016) Journal of Immunology Research, Article ID
6760830).
In Vitro T-cell Epitope Prediction Methods In vitro methods, such as MHC or HLA binding assays and T-cell assays, can be used to predict T-cell epitopes and evaluate T-cell responses to a protein therapeutic antigen. The synthesis of hundreds or thousands of overlapping peptides for in vitro assays is a limiting factor that can be overcome by the use of in silico epitope prediction tools that can accurately model the MHC:epitope interface and predict immunogenic peptide sequences (see, e.g., De Groot, A. S. and Moise, L.
(2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340) MHC/HLA Binding Assays T-cell epitope prediction identifies the shortest peptide sequences within an antigen that stimulate CD8+ or CD4+ T cells, which, thus, are immunogenic. The immunogenicity of T-cell epitopes depends on antigen processing, peptide binding to WIC molecules, and recognition by a cognate TCR; WIC-peptide binding is the most selective process and a primary basis for predicting T-cell epitopes. MHC
binding assays can be used to detect high affinity peptides, and often are applied in conjunction with epitope mapping to identify regions of the protein that are likely to be immunogenic. In vitro WIC class II binding assays include cell-based binding assays and soluble HLA binding assays. High throughput MHC binding assays involve incubating various doses of peptides of interest with control peptides and soluble WIC proteins to assess binding affinity; high affinity peptides bind more strongly to MHC and the epitopes are more likely to be recognized by T-cells.
For example, MHC II:epitope binding can be evaluated by measuring the ability of exogenously added peptides to bind to the surface of lymphoblastoid cell line B-cells expressing MHC class II alleles, and competition-based HLA assays can be adapted for high throughput screening. MHC binding assays to identify potentially immunogenic epitopes are commercially available, for example, from ProImmune (see, e.g., Dingman et al. (2019)1 Pharm. Sci. 108(5):1637-1654; De Groot, A.
S.
and Moise, L. (2007) Curr. Op/n. Drug Discov. Devel. 10(3):332-340; Sanchez-Trincado et at. (2017) Journal of Immunology Research, Article ID 2680160).
In Vitro T-Cell Assays The presence of T-cell epitopes in a protein therapeutic can be detected by assessing T-cell responses in vitro in T-cell assays. T-cells proliferate and release cytokines upon stimulation by an immunogenic protein. T-cell epitopes induce the secretion of cytokines, such as IL-2, IL-4, IL-5 and IFNy by effector T-cells, and induce the secretion of the cytokines TGF0 and TNFa, and chemokines, such as MIP1a/10, by regulatory T-cells (Tregs). The proliferation of T-cells in response to immunogenic peptides/epitopes can be measured by radiolabeling with thymidine or by labeling with fluorescent dyes, such as carboxyfluorescein succinimidyl ester (CFSE). ELISA or ELISpot methods, as well as flow cytometry, can be used to measure the levels of cytokines, such as IL-2 and IFN-y, that are secreted by T-cells, to determine immunogenicity. ELISpot methods are highly sensitive and can detect individual T-cells directly from splenocytes or peripheral blood, as well as measure the number of antigen-specific T-cells that secrete specific cytokines.
ELISpot assays for measuring IL-2 and IL-4, for example, are commercially available. Flow cytometry also can be used to measure T-cell responses, whereby T-cells that respond to a particular epitope can be directly labeled using tetramers (MHC class II:epitope complexes). T-cell proliferation and cytokine release assays can be combined with T-cell phenotyping to classify the type of T-cell response that occurs. The number and phenotype of T-cells responding to an antigen can be determined using methods such as flow cytometry, by identifying cell surface markers, such as CD25 for effector T-cells, and FoxP3 for Tregs, and/or by identifying intracellular cytokine expression.
Thus, identification of T-cell epitopes can be coupled with phenotypic studies, to evaluate if the immune response will be inflammatory or suppressive.
Peripheral blood mononuclear cell (PBMC) assays, which use PBMC preparations include several types of immune cells (e.g., CD4+ and CD8+ T-cells), better mimic in vivo immune systems, and can be used to assess the immunogenicity of a protein, and the potential immune response, without testing in humans. The PBMCs are stimulated with whole therapeutic proteins, or with peptides derived from therapeutic proteins, in in vitro cultures. Innate immune screening, using innate cell systems, such as PBMC
preparations lacking CD8+ reactive T cells, or innate lymphoid cells (ILCs), can be used to distinguish the innate and adaptive immune responses to immunogenic proteins. To be useful, in vitro T-cell assays should test peptides against PBMCs from large cohorts of donors with a broad spectrum of MHC class II allotypes. In vitro T-cell assays can provide information on the number and potency of T-cell epitopes, which can be used to determine the risk of immunogenicity during preclinical development, and to guide the removal of such epitopes by targeted amino acid substitutions (see, e.g., Baker et al. (2010) Self7Nonself1(4):314-322;
Dingman et al.
(2019)1 Pharm. Sci. 108(5):1637-1654; De Groot, A. S. and Moise, L. (2007) Curr.
Opin. Drug Discov. Devel. 10(3):332-340).
d. In Vivo Epitope Prediction Methods In vivo assessments of immunogenicity of protein therapeutics in humans use animal models, such as mice. In general, any human or humanized protein therapeutic can be immunogenic when administered to a non-human animal. Animal models, .. however, are useful for the prediction of immunogenicity, the comparison of the relative immunogenicity between products, drug formulations or administration routes, the determination of the immunogenicity of aggregates, and the elucidation of immune mechanisms. Adoptive transfer and T-cell proliferation studies in animal models can be used to determine the role of T-cells and B-cells in protein immunogenicity. Immunogenicity of human protein therapeutics cam be difficult to assess in animals, because animal MHC receptors do not directly mimic human HLA
receptors, and because HLA and MHC genes are highly polymorphic, with high inter-subject variability in HLA/MHC expression. To overcome these limitations, HLA
transgenic mice have been generated that mimic a human subject, and that can be tolerized to a particular protein; the mouse will tolerate the protein therapeutic being assessed, and any immunogenicity that develops is due to the breaking of self-tolerance, and not due to a classical immune response to foreign antigens. In vivo methods to determine the immunogenicity of a protein therapeutic include the exposure of HLA-transgenic mice to the whole protein or to epitope peptides.
Several transgenic mouse strains, expressing common HLA gene products, such as HLA-A, HLA-B and HLA-DR molecules, have been generated, and can be used to measure T-S cell responses, as well as antibodies induced by exposure to the protein therapeutic, by ELISA and neutralizing antibody assays. B-cell epitopes in a protein therapeutic also can be identified by immunizing HLA transgenic mice with the protein (see, e.g., see, e.g., Dingman et al. (2019)1 Pharm. Sci. 108(5):1637-1654; De Groot, A.
S. and Moise, L. (2007) Curr. Op/n. Drug Discov. Devel. 10(3):332-340).
NOD scid gamma (NSG) mice, which are highly immunocompromised and lack most immune cells as well as complement and cytokine signaling, can be transfected to investigate the human immune system in an in vivo model. For example, CD34+ humanized NSG mouse models are engrafted with cord blood-derived hematopoietic stem cells to develop a functional immune system with normal T-cell and inflammatory function. Animal models also include non-human primates, such as rhesus monkeys and chimpanzees, which are more useful in predicting protein immunogenicity, because their proteins exhibit a higher degree of homology with human proteins, and because their immune mechanisms are similar to those of humans (see, e.g., Dingman et al. (2019) J Pharm. Sci. 108(5):1637-1654).
e. Removal of Predicted B-cell and T-cell Epitopes (De-immunization) As described herein, the prediction and removal of immunogenic epitopes from protein therapeutics (i.e., de-immunization) can increase the efficacy and safety of the constructs provided herein, and reduce the likelihood or prevent adverse effects.
For example, the removal of identified epitopes, such as B-cell epitopes, can prevent the formation of ADAs, which reduce the efficacy of administered protein therapeutics by neutralizing the therapeutics and/or by inducing their rapid elimination from the body.
De-immunization of protein therapeutics involves the identification of highly immunogenic B-cell and/or T-cell epitopes, and deletion of the identified epitopes by mutagenic substitution of key amino acid residues. As discussed above, prediction and assessment of immunogenic regions within a protein therapeutic sequence includes the use of various in silico, in vitro, and in vivo methods. Upon the identification of an immunogenic epitope, the amino acid sequence of the epitope is modified by random or site-directed mutagenesis, to remove the immunogenic sequence and de-immunize the epitope. For example, most of the contacts made between an epitope and antibody occur via amino acid side chains, and alanine scanning mutagenesis can be used to define the contributions that each residue side chain makes to antibody binding. This is performed by sequential alanine substitution of each non-alanine residue, one at a time, to identify critical residues whose side chains make the highest energetic contributions to the paratope-epitope interaction.
The prediction and mutagenic deletion of immunogenic epitopes, however, is not sufficient for protein de-immunization, as the protein must retain its folded, stable and active structure in order to retain its therapeutic efficacy; epitope-deleting mutations that are compatible with the protein's structure and function must be selected.
There are in silico tools to increase the efficiency of this process. For example, programs are available that sequentially replace each amino acid in the immunogenic sequence with one of the other 19 naturally occurring amino acids, and then re-evaluate the immunogenicity of the new sequences. For example, OptiMatrix is a tool that iteratively substitutes all 20 amino acids in any given position of a peptide sequence, and then re-analyzes the predicted immunogenicity of the modified sequence (see, e.g., De Groot, A. S. and Moise, L. (2007) Curr. Opin. Drug Discov.
Devel. 10(3):332-340). EpiSweep is a suite of protein design algorithms that integrates computational predictions of immunogenic T-cell epitopes with sequence-based or structure-based assessment of the effects of epitope-deleting mutations on protein stability, structure and function, allowing for the selection of combinations of mutations that optimize the protein therapeutic for low immunogenicity and high activity and stability (see, e.g., Choi et al. (2017) Methods Mol. Biol.
1529:375-398, for a step-by-step guide to the application of the EpiSweep suite of deimmunization algorithms). Computational alanine scanning also can be used to rapidly determine the effect of alanine mutation on a binding free energy in protein-protein complexes by using a simple free energy function (see, e.g., Potocnakova et at. (2016) Journal of Immunology Research, Article ID 6760830).

G. PAN-GROWTH FACTOR TRAP CONSTRUCTS
1. Receptor Tyrosine Kinases (RTKs) Receptor tyrosine kinases (RTKs) are high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. RTKs are involved in .. many signal transduction pathways, and play a role in a variety of cellular processes, including cell division, proliferation, differentiation, migration and metabolism. RTKs can be activated by ligands that bind specifically to their cognate receptors.
Such activation, in turn, activates events in a signal transduction pathway, such as by triggering autocrine or paracrine cellular signaling pathways, for example, activation of second messengers, which results in specific biological effects.
Approximately 20 different classes of RTKs have been identified, which include, for example, the epidermal growth factor receptor (EGFR) family (class I, also known as the ErbB
family); the insulin receptor family (class II); the platelet-derived growth factor receptor (PDGFR) family (class III); the vascular endothelial growth factor receptor .. (VEGFR) family (class IV); the fibroblast growth factor receptor (FGFR) family (class V); the hepatocyte growth factor receptor (HGFR) family (class VIII);
and the Eph receptor family (Ephs, after erythropoietin-producing human hepatocellular receptors; class IX), among others.
RTKs are associated with regulating pathways involved in angiogenesis, including physiologic and tumor blood vessel formation, and are implicated in the regulation of cell proliferation, migration and survival. RTKs have been implicated in a number of diseases, including autoimmune diseases and cancers, such as breast and colorectal cancers, gastric carcinomas, gliomas and mesodermal-derived tumors.

Dysregulation of RTKs has been associated with several cancers. For example, breast cancer has been associated with amplified expression of p185-HER2. RTKs also have been associated with ocular diseases, including diabetic retinopathies and macular degeneration. Additionally, members of the epidermal growth factor receptor (EGFR) family, as well as EGF-like growth factors (ligands), have been shown to be overexpressed in synovial fibroblasts and macrophages in patients with rheumatoid arthritis (RA).

a. Human Epidermal Growth Factor Receptor (HER) Family Among the RTKs associated with disease is the class I human EGFR (HER;
also referred to as the ErbB) family of receptors, which includes RERVEGFR
(ErbB1), HER2 (ErbB2/Neu), HER3 (ErbB3) and HER4 (ErbB4). HER1, HER3 and HER4 collectively bind over 11 canonical ligands, including epidermal growth factor (EGF), transforming growth factor (TGF)-a, heparin-binding (HB)-EGF, amphiregulin, 13-cellulin (BTC), epiregulin, epigen, and neuregulin (NRG)1-4.

does not bind any of these ligands, but acts as a signal amplifier by heterodimerization with other HER family members, such as HER3 and HER4 (see, e.g., Jin et at.
(2009) Mol. Med. 15(1-2):11-20). HER1, HER2 and HER4 are active as tyrosine kinases, whereas HER3 is inactive as a kinase (despite having a kinase domain), and signals via the phosphatidylinositol 3-kinase pathway.
All members of the HER family have an extracellular ligand-binding domain, a single transmembrane domain, and a cytoplasmic tyrosine-kinase-containing domain. The extracellular region of each HER family member contains four subdomains, Li, CR1, L2 and CR2, where "L" refers to a leucine-rich repeat domain and "CR" refers to a cysteine-rich region/domain (also known as a furin-like repeat domain); the four subdomains also are referred to as domains I-TV, respectively.
Domains I and III are ligand-binding domains, and domains II and IV mediate binding to each other and to other members of the receptor family. Domain II contains sequences required for dimerization, known as the dimerization arm, and domain IV
contains sequences which allow for domain II/IV tethering, with the exception of HER2, which does not undergo a tethered conformation. In the absence of ligands, EGFR, HER3 and HER4 subdomains II and IV form an intramolecular auto-inhibitory tether. Upon ligand binding, the subdomains undergo conformational changes, allowing subdomains I and III to form a high-affinity ligand-binding pocket.
It has been shown that mutagenic disruption of the tether formed by subdomains II
and IV, or C-terminal deletion of subdomain IV, increases ligand-binding affinity by up to 15-fold (see, e.g., Jin et al. (2009)Mol. Med. 15(1-2):11-20).
HER family members are expressed in various tissues of epithelial, mesenchymal and neuronal origin. Under normal physiological conditions, activation of the HERs is controlled by the spatial and temporal expression of their ligands, which are members of the EGF family of growth factors. Ligand binding induces the formation of receptor homodimers and multiple combinations of heterodimers, leading to the activation of the intrinsic kinase domain, self-phosphorylation of specific tyrosine residues in the cytoplasmic tail, the recruitment and phosphorylation of several intracellular proteins, and coupling to multiple downstream signaling cascades. The activated signaling pathways include the Ras-Raf-mitogen-activated protein kinase mitogenic pathway, the phosphatidylinositol 3-kinase-AKT cell survival pathway, and the stress-activated protein kinase C and Jak/Stat pathways.
The induced signaling pathways result in a variety of cellular responses, including, for example, cell migration, invasion, proliferation, survival, and differentiation (see, e.g., Sarup et al. (2008)Mol. Cancer Ther. 7(10):3223-3236).
b. Diseases Associated with the Human Epidermal Growth Factor Receptor (HER) Family and their Ligands Dysregulation of members of the HER family, as well as their ligands, by overexpression or due to mutations, has been shown to play a role in cancer and other diseases. For example, HER1 and HER2 have been implicated in the development and pathology of many human cancers, and alterations in these receptors have been associated with more aggressive disease and with poor clinical outcome. TGF-a overexpression has been associated with prostate, pancreatic, lung, ovarian, and colon cancers, while NRG1 overexpression has been associated with mammary adenocarcinomas. HER1 overexpression has been associated with gliomas, and head and neck, breast, bladder, prostate, kidney and non-small cell lung cancers, and mutations in HER1 have been associated with gliomas, as well as lung, breast and ovarian cancers. HER2 overexpression has been associated with breast, lung, pancreatic, colon, esophageal, endometrial and cervical cancers; HER3 has been associated with breast, colon, gastric, prostate and oral squamous cell cancers; and HER4 has been associated with breast and prostate cancers, as well as childhood medulloblastoma (see, e.g., Yarden et al. (2001) Nat. Rev. Mol. Cell Biol.
2:127-137).
The EGF family of ligands and receptors has been shown to play a role in the development of inflammatory arthritis. For example, the expression of HER2, and the presence of the EGFR ligands EGF, amphiregulin and TGF-a, have been detected in the RA synovium. Adenoviral delivery of the human EGFR family inhibitor herstatin, an alternative splice variant of HER2, has been shown to abrogate all clinical signs of collagen-induced arthritis (CIA) in mice. Herstatin disrupts dimerization, and acts as a natural inhibitor of native HER1, HER2 and HER3. A patient with long-standing RA, who had previously been treated with rituximab and adalimumab, experienced a significant reduction in joint pain following treatment with the anti-antibody cetuximab for head and neck cancer. These results indicate that HER-targeted treatments can be therapeutically useful in the treatment of autoimmune and inflammatory conditions, such as rheumatoid arthritis (RA) (see, e.g., Gompels et at.
(2011) Arthritis Research & Therapy 13 :R161).
Macrophages are a source of TNF in the chronically inflamed RA joint tissue.
Phenotypic analysis of macrophages from the synovial tissues of patients with RA
revealed an abundance of HBEGF+ (heparin binding EGF-like growth factor) inflammatory macrophages, that overexpress the proinflammatory genes NR43A
(nuclear receptor sub-family 4 group A member 3), PLAUR (plasminogen activator, urokinase receptor), and CXCL2, and the growth factors HB-EGF and epiregulin (EGFR family ligands). HBEGF+ inflammatory macrophages also produced the proinflammatory cytokine IL-1 and promoted synovial fibroblast invasiveness in an epidermal growth factor receptor-dependent manner. It was shown that the majority of medications used to treat RA targeted HBEGF+ macrophages in an ex vivo synovial tissue assay, and an EGFR inhibitor effectively blocked the macrophage-induced fibroblast response in RA tissue in an ex vivo assay, indicating that blockade of EGFR
responses can provide a non-immunosuppressive therapeutic approach for RA
(see, e.g., Kuo et at. (2019) Sci. Transl. Med. 11(491)). Such an approach is advantageous over the use of traditional anti-TNF therapies, which are immunosuppressive and are .. often associated with the development of serious infections, such as tuberculosis.
HER family signaling also has been associated with coronary atherosclerosis, which involves the migration of vascular smooth muscle cells in the arterial intima.
Activation of the thrombin receptor is required for smooth muscle cell migration and proliferation, and activation of this G-protein-coupled receptor relies on .. transactivation by HER1/EGFR in response to HB-EGF. EGFR expression also is associated with psoriasis; in normal skin, the expression of EGFR is limited to the basal layer, whereas in patients with psoriasis, EGFR and its ligand amphiregulin are highly expressed throughout the entire epidermal layer (see, e.g., Yarden et at. (2001) Nat. Rev. Mol. Cell Biol. 2:127-137).
Other HER-mediated diseases and conditions include neurodegenerative diseases and conditions, such as multiple sclerosis, Parkinson's disease, schizophrenia and Alzheimer's Disease. For example, several diseases and conditions are associated with, e.g., caused by, or aggravated by, exposure to one or more neuregulin (NRG) ligands, such as NRG1, including type I, II, and III, NRG2, NRG3, and/or NRG4.

Examples of NRG-associated diseases include neurological or neuromuscular diseases, including schizophrenia and Alzheimer's disease (see, e.g.,U U.S.
Publication No. 2010/0055093).
Due to their role in cancer and other proliferative diseases, rheumatoid arthritis, neurodegenerative diseases and autoimmune diseases, HERs are targets for therapeutic intervention. Anti-HER therapeutics include antibodies targeted to the extracellular domain (or ectodomain), referred to herein as the ECD, and small molecule tyrosine kinase inhibitors. Therapeutics approved for the treatment of cancers driven by the HER family of proteins include monoclonal antibodies, such as trastuzumab (directed at HER2), pertuzumab (directed at HER2), panitumumab (directed at HER1/EGFR) and cetuximab (directed at HER1/EGFR), and small molecule tyrosine kinase inhibitors, such as the HER1 kinase inhibitors gefitinib and erlotinib, and the dual HER2 kinase and HER1 kinase inhibitor lapatinib. For example, trastuzumab is used for the treatment of HER2-overexpressing node-positive or node negative breast cancer; cetuximab is used for the treatment of metastatic colorectal cancer, as well as head and neck cancer; panitumumab is used for the treatment of metastatic colorectal cancer; lapatinib is used as a frontline therapy for triple-positive breast cancer and as an adjuvant therapy for patients who have progressed on trastuzumab; and erlotinib is used to treat non-small cell lung cancer and pancreatic cancer.
Anti-HER therapeutics exhibit limited efficacy and limited duration of response. Trastuzamab (sold, for example, as Hercepting) is a humanized version of a murine monoclonal antibody, and targets the extracellular domain of HER2. The effectiveness of trastuzumab, however, requires high expression (at least 3-to 5-fold overexpression) of HER2, and, as a result, fewer than 25% of breast cancer patients qualify for treatment. Among this population, a large proportion fail to respond to treatment. In addition, small molecule tyrosine kinase inhibitors often lack specificity.
With the exception of patients that highly express HER2 and are treated with trastuzumab in combination with chemotherapy, the efficacy observed with single-targeted anti-HER antibodies or small molecule tyrosine kinase inhibitors is in the range of 10-15%. Treatments, particularly those directed at only one HER
family member, also suffer from intrinsic or acquired resistance, which is associated with the co-expression and ligand activation of other RTKs, particularly other HER
family members. For example, drug resistance is often associated with the up-regulation of, or compensation by, other HER family members, such as HER3 and HER4, or increased expression of HER1 or HER3 ligands by tumor cells. The homodimerization and heterodimerization among members of the HER family of receptors also has implications for therapies directed against a single HER
family receptor. Because of the limited effectiveness of the available therapies, alternative anti-HER therapies are required. Provided herein are alternative, more effective therapies for targeting the HER family of RTKs and their ligands.
2. Pan-Growth Factor Inhibition As described herein, resistance to single-targeted anti-HER therapies, such as trastuzumab cetuximab, gefitinib and erlotinib, often is associated with the co-expression and/or upregulation of other HER family members and/or the overexpression of their ligands. One strategy to reduce or overcome this resistance, and to improve the efficacy of HER-targeted therapies, is to inhibit multiple ligand-induced HER family members simultaneously. This can be achieved, for example, by a chimeric HER ligand-binding molecule that behaves like a receptor decoy and sequesters multiple HER family ligands, preventing ligand-dependent receptor activation and downregulating aberrant HER family activity.
a. RB242 Ligand Trap The antagonist designated RB242, which is a chimeric bi-specific ligand trap that is an Fc-mediated heterodimer of the EGFR (HER1) and HER3 ligand-binding domains, targets all four members of the EGFR/HER family. The EGFR and HER3 ligand-binding domains are dimerized by fusion of each ligand-binding domain with the Fc domain of human IgGl. In RB200, the C-termini of the extracellular domains (ECDs) of EGFR (corresponding to residues 1-621 of the mature EGFR protein, set forth in SEQ ID NO:41), and of HER3 (corresponding to residues 1-621 of the mature HER3 protein, set forth in SEQ ID NO:45), each are fused to the N-terminus of the Fc fragment of human IgG1 (corresponding to residues P100-K330 of SEQ ID NO:9), with a Gly-Arg-Met-Asp (GRMD) linker added to the N-terminus of the Fc fragment.
The HER3/Fc component of RB200 contains a 6xHis tag on the COOH terminus for purification.
RB200 has been shown to bind EGFR and HER3 ligands (including EGF, TGF-a, HB-EGF, amphiregulin, beta-cellulin, epiregulin, and epigen, and NRG1-a, NRG1-01 and NRG1-03, respectively) with high affinity, inhibit ligand-induced tyrosine phosphorylation of HER family members (HER1, HER2 and HER3), inhibit the proliferation of a diverse range of tumor cells in vitro, and suppress the growth of tumor xenografts (epidermoid carcinoma and non-small cell lung cancer) in nude mouse models. RB200 also exhibited synergy with tyrosine kinase inhibitors directed toward EGFR/HER1 and HER2 kinases, such as AG-825, erlotinib, gefitinib, or lapatinib, in the inhibition of tumor cell proliferation in vitro. The inhibition of ligand-stimulated phosphorylation of HER1, HER2 and HER3 was more effective by RB200 compared with monoclonal antibodies that target HER1 (C225) or HER2 (trastuzumab and 2C4) (see, e.g., Sarup et al. (2008) Mol. Cancer Ther.
7(10):3223-3236; Gompels et al. (2011) Arthritis Research & Therapy 13:R161).
To express the RB200 and RB242 heterodimeric chimeric fusion protein, vectors encoding the HER1/Fc and HER3/Fc constructs were co-transfected into HEK293T cells at a ratio of 1:3 (HER1/Fc:HER3/Fc). This results in the expression of HER1/Fc and HER3/Fc homodimers, in addition to the HER1/Fc:HER3/Fc heterodimer of interest. The expressed proteins were purified by a combination of Protein-A, Ni-Sepharose and EGFR-affibody column chromatography methods.
Analytic reverse-phase high performance liquid chromatography (HPLC) revealed that the RB242 heterodimer contained approximately 10% combined contamination with the two homodimers (see, e.g., Sarup et at. (2008) Mol. Cancer Ther.
7(10):3223-3236). Thus, improved methods are required to improve the yield and purity of the heterodimer.

b. RB200 and RB242 for the Treatment of Autoimmune Disease As discussed elsewhere herein, a significant proportion of RA patients do not respond, or stop responding, to treatment with anti-TNF therapies, such as anti-TNF
antibodies, which are associated with an increased risk of serious infections, including tuberculosis. Thus, alternative treatments are required. The increased expression of EGF ligands and receptors (HERs) has been documented in the synovium and synovial fluids of patients with rheumatoid arthritis (RA), indicating that therapies targeting EGFRs can be used to treat RA and other autoimmune and inflammatory diseases and disorders.
The bi-specific EGFR ligand trap RB200 (and its derivative RB242) displays a dose-dependent reduction in disease severity in collagen-induced arthritis (CIA). Mice with CIA were treated intraperitoneally with RB200 (or RB242), at a dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kg, on the day of disease onset (day 1), and on days 4 and 7 of disease. Treatment with 1 mg/kg or 10 mg/kg RB200 inhibited the increase in clinical score and paw swelling in a dose-dependent manner. EGF has been shown to promote angiogenesis, and RB200-treated mice showed a reduction in CD31-immunopositive staining, reflecting a reduction in synovial vessels, and inhibition of synovial angiogenesis. Joint sections of mice treated with PBS control showed high numbers of infiltrating cells in the inflamed synovium, as well as invasion and erosion of bone by the synovium, associated with significant CD31 expression. Joints from mice treated with 1 mg/kg or 10 mg/kg RB200 were protected, with normal appearance, well-preserved joint architecture, and few CD31-positive blood vessels.
These results indicate that the inhibition of EGFR-mediated responses can be for therapeutic use in the treatment of RA (see, e.g., Gompels et at. (2011) Arthritis Research & Therapy 13:R161).
The combination of TNF inhibition with an inhibitor of EGFR-mediated signaling can increase the therapeutic efficacy of anti-TNF therapies and be useful in the treatment of RA. It has been shown that the combined administration of a low dose of RB200 (0.5 mg/kg) with a sub-optimal dose of etanercept (1 mg/kg) inhibits the increase in clinical score and paw swelling, and completely abolishes CIA
with a similar effectiveness to that observed with the administration of an optimal dose of etanercept (5 mg/kg) alone. In comparison, the administration of low-dose alone or low-dose etanercept alone was ineffective. A fluorescently-labeled monoclonal antibody against E-selectin can be used to localize endothelial activation in inflamed tissues in vivo, and is a sensitive, specific and quantifiable molecular .. imaging technique for the evaluation of CIA. The combination of low-dose and low-dose etanercept decreased the amount of E-selectin detected in the paws to levels seen in healthy animals, whereas E-selectin was detected in the paws of CIA
mice that received low-dose RB200 alone, or low-dose etanercept alone. While there was a dose-dependent effect of RB200 alone and etanercept alone on joint architecture, with progressively fewer severely destroyed joints and more joints with mild or moderate destruction, the most pronounced effect was observed with the combination treatment, with 64% of j oints appearing normal, compared with 0%
in mice treated with either low-dose RB200 alone or low-dose etanercept alone.
The combination treatment also was more effective than high-dose etanercept alone, indicating the effectiveness of combining pan-EGFR and TNF-targeted therapies in promoting joint protection (see, e.g., Gompels et at. (2011) Arthritis Research &
Therapy 13:R161).
c. RB242 Ligand Trap The ligand trap designated RB242, derived from RB200, is an affinity optimized Fc-mediated triple mutant EGFR:HER3 heterodimer, comprising the mutations T15S and G564S in the EGFR ECD subdomains I and IV, respectively, with reference to the sequence of the mature EGFR protein (SEQ ID NO:41), and Y246A in the HER3 ECD subdomain II, with reference to sequence of the mature HER3 protein (SEQ ID NO:45). Compared to the parent molecule, RB200, RB242 displayed an average of 22-fold improvement in affinity for various ligands, including EGF, TGF-a, HB-EGF and NRG1-0, and demonstrated improved anti-proliferative activity against cultured monolayer BxPC3 pancreatic cancer cells and in a mouse model of human non-small cell lung cancer. RB242 also exhibited a 10- to 60-fold improvement in the inhibition of ligand-induced HER phosphorylation, compared to RB200 (see, e.g., Jin et al. (2009) Mol. Med. 15(1-2):11-20).

3. Optimized Multi-Specific, such as Bi-Specific, Growth Factor Trap Constructs Provided herein are multi-specific, such as bi-specific, growth factor trap constructs, that are designed to be pan cell surface receptor therapeutics by specifically targeting more than one cell surface receptor, such as by binding to ligands for one or more receptors and/or interacting with one or more cell surface receptors, as long as the activity of more than one cell surface receptor is modulated.
The constructs include those that target more than one HER family member, as well as those that target one or more HERs and additional receptors, such as a HER
that contributes to or participates in the development of resistance to anti-HER
therapies.
The growth factor trap constructs provided herein contain multiple, in particular, two, chimeric fusion polypeptides that each contain all or a portion of the extracellular domain (ECD) of one receptor, particularly a member of the HER family, such as EGFR/HER1, HER2, HER3 or HER4, that is fused to a multimerization domain, such as the Fc of a human immunoglobulin (Ig), such as the Fc of human IgG. The ECD
or portion thereof in the chimeric fusion polypeptide can be linked directly to the Fc, or indirectly, via a linker, such as a peptide linker. Typically, the C-terminus of the ECD
polypeptide is linked to the N-terminus of the multimerization domain, such as an IgG
Fc.
The growth factor trap constructs herein are expressed and purified as described, for example, in Sarup et al. (2008) Mol. Cancer Ther. 7(10):3223-3236;
Gompels et al. (2011) Arthritis Research & Therapy 13:R161; Jin et al. (2009) Mol.
Med. 15(1-2):11-20; and U.S. Patent Publication No. 2010/0055093. The following sections describe each portion of the multi-specific growth factor trap constructs provided herein.
a. The Extracellular Domain (ECD) Polypeptides Provided herein are multi-specific, such as bi-specific, growth factor trap constructs comprising the extracellular domains (ECDs) or portion(s) thereof, of two or more cell surface receptors (CSRs). In particular embodiments, the constructs are bi-specific, heterodimeric constructs, comprising two different cell surface receptors.
The constructs include a first ECD polypeptide and a second ECD polypeptide that each are linked directly or indirectly via a linker to a multimerization domain. In some embodiments, the first ECD polypeptide comprises the ECD of HER1/EGFR
(corresponding to residues 1-621 of SEQ ID NO:41), or a portion thereof, and the second ECD polypeptide comprises the ECD of HER2 (corresponding to residues 1-628 of SEQ ID NO:43), HER3 (corresponding to residues 1-621 of SEQ ID NO:45), or HER4 (corresponding to residues 1-625 of SEQ ID NO:47), or a portion thereof, particularly the ECD of HER3 or HER4, or a portion thereof In embodiments where the ECD polypeptide comprises less than the full-length ECD of a HER protein, it contains at least a sufficient portion of subdomains I, II and III for ligand binding and receptor dimerization. For example, the ECD can contain a sufficient portion of subdomains I and III for ligand binding, and/or can contain a sufficient portion of the ECD to dimerize with a cell surface receptor, including a sufficient portion of subdomain II. In some embodiments, the ECD contains subdomains I, II and III
and at least module 1 of domain IV.
In some examples, the multi-specific, such as bi-specific, growth factor trap constructs contain a first ECD polypeptide that contains all or a portion of the ECD of HER1/EGFR, HER2, HER3 or HER4, in particular, EGFR/HER1, and a second chimeric polypeptide that contains the ECD from a different CSR, such as, for example, HER2, HER3, HER4, an insulin growth factor-1 receptor (IGF1-R), a vascular endothelial growth factor receptor (VEGFR, e.g., VEGFR1), a fibroblast growth factor receptor (FGFR, e.g., FGFR2 or FGFR4), a TNFR, a platelet-derived growth factor receptor (PDGFR), a hepatocyte growth factor receptor (HGFR), a tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE, e.g., or TEK (TIE-2)), a receptor for advanced glycation end products (RAGE), an Eph receptor, or a T-cell receptor.
In a particular embodiment, the first ECD polypeptide comprises the full-length ECD of HER1/EGFR (corresponding to residues 1-621 of SEQ ID NO:41), or a portion thereof (e.g., residues 1-501 of SEQ ID NO:41, which correspond to subdomains I-III and module 1 of domain IV), and the second ECD polypeptide comprises the full-length ECD of HER3 (corresponding to residues 1-621 of SEQ
ID
NO:45), or a portion thereof (e.g., residues 1-500 of SEQ ID NO:45, which correspond to subdomains I-III and module 1 of domain IV), where the ECD
portion contains at least a sufficient portion of subdomains I and III to bind to a ligand of the HER receptor, and a sufficient portion of the ECD to dimerize with a cell surface receptor, including a sufficient portion of subdomain II. The first and second ECD
polypeptides form a multimer, e.g., a dimer, through interactions of their multimerization domains. The resulting multimeric construct provided herein binds to additional ligands as compared to the first or second chimeric polypeptide alone, or homodimers thereof, and/or dimerizes with more cell surface receptors than the first or second chimeric polypeptide alone, or homodimers thereof For example, the first and second ECD polypeptides form a heterodimer that binds to HER1 ligands and to HER3 ligands.
b. Modifications to the Extracellular Domains In some embodiments, at least one of the ECD domains or a portion thereof, includes a modification that alters ligand binding, specificity or other activity or property, compared to the unmodified ECD polypeptide. In such multimeric constructs, a second ECD portion can be the same ECD domain, wild-type or mutated form, or can be the ECD from any other cell surface receptor. The ECD or portion thereof of each monomer is linked to a multimerization domain directly or via a linker, or is linked to a second ECD or portion thereof directly, or via a linker. For example, the modification alters ligand binding, specificity or another activity or property of the ECD or full-length receptor containing such ECD, compared to the unmodified ECD or full-length receptor, whereby the heteromultimer exhibits the altered activity or property, such as altered ligand binding or specificity.
Such modifications include any that eliminate or add or enhance an activity, such as binding to an additional ligand. Exemplary of such multimeric constructs, are constructs that contain at least one HER1 ECD that contains a mutation in subdomain III that increases its affinity for a ligand other than EGF. Such increase in affinity is at least 2- to 10-fold, typically 100, 1000, 104, 105, 106 fold or more.
In particular embodiments, the growth factor trap construct is a heterodimer containing a HER1 (EGFR) chimeric fusion polypeptide and a HER3 chimeric fusion polypeptide, wherein each chimeric fusion polypeptide comprises the ECD of the receptor linked to the Fc of human IgGl, optionally via a peptide linker. Such chimeric fusion polypeptides are referred to herein as HER1/Fc and HER3/Fc.

Typically, the C-terminus of the ECD polypeptide is linked to the N-terminus of the multimerization domain, such as an IgG1 Fc.
In some examples, the HER1 portion has been enhanced for ligand binding and/or biological activity. In other examples, the HER3 portion has been enhanced for ligand binding and/or biological activity. In yet another example, both HER1 and HER3 portions have been enhanced for ligand binding and/or biological activity.
Exemplary modifications include, for example, S418F in HER1 (with reference to the sequence of the mature protein, set forth in SEQ ID NO:41), which allows the HER3 ligand NRG2-0 to stimulate RER1. The resulting ECD binds to or interacts with at least two ligands, one for HER1, such as EGF, and a second for HER3, such as NRG213. Other modifications include, for example, the mutations T155 and G5645 in the EGFR/HER1 ECD subdomains I and IV, respectively, with reference to the sequence of the mature EGFR protein (SEQ ID NO:41), and Y246A

in the HER3 ECD subdomain II, with reference to the sequence of the mature protein (SEQ ID NO:45), which, when combined, result in an average of 22-fold improvement in affinity for various ligands, including EGF, TGF-a, HB-EGF and NRG1-0. Additional mutations in the HER1 ECD include E330D/G5885, 5193N/E330D/G5885, and T43K/5193N/E330D/G5885, with reference to the sequence of precursor HER1 (including the signal peptide) set forth in SEQ ID
NO:40, and corresponding to E306D/G5645, 5169N/E306D/G5645 and T19K/5169N/E306D/G5645, with reference to the sequence of the mature HER1 polypeptide, set forth in SEQ ID NO:41. These mutations increase the HER1 binding affinity for the ligands EGF, HB-EGF, and TGF-a (see, e.g., U.S. Patent Publication No. 2010/0055093).
c. The Multimerization Domain In particular embodiments, the multimerization domain is an Fc domain, or a variant thereof, that effects multimerization. The Fc domain can be from any immunoglobulin (Ig) molecule, including from an IgG, IgM, or IgE. For example, the Fc domain can be from an IgGl, IgG2, IgG3 or IgG4, and includes the CH2 and domains, and optionally, all or a portion of the hinge region. In certain examples, the Fc portion is the Fc of human IgGl, optionally including all or a portion of the hinge region, and corresponding to, for example, residues 99-330, 100-330, 104-330, 330, 111-330, 113-330, or 114-330, of SEQ ID NO:9. Included also are the modified Fe domains as described in sections above, modified to have knobs-in-holes, and altered properties.
Each ECD polypeptide in the multi-specific growth factor trap construct is linked to the Fe directly, or indirectly via a linker, such as a chemical or a polypeptide linker, forming a chimeric fusion polypeptide (i.e., an ECD/Fc fusion polypeptide).
The multimerization domains, such as the Fe domains, of each chimeric fusion polypeptide, interact (via disulfide bonds in the case of Fe domains) to form a heteromultimer, such as a heterodimer.
The linker between the ECD and Fe portions of each chimeric fusion polypeptide can be a flexible peptide linker, such as, for example, a hinge region of an IgG, or other polypeptide linker comprised of small amino acids, such as glycine, serine, threonine, and/or alanine, at various lengths and combinations. For example, the linker can be (Gly),, (GGGGS)., (SSSSG),, or (AlaAlaProAla),, where n is 1-6, or can be GKSSGSGSESKS, GGSTSGSGKSSEGKG, GSTSGSGKSSSEGSGSTKG, GSTSGSGKPGSGEGSTKG, EGKSSGSGSESKEF, Gly-Arg-Met-Asp (GRMD), Ser-Cys-Asp-Lys-Thr (SCDKT), or Glu-Lys-Thr-Ile-Ser (EKTIS) (see, SEQ ID
NOs:816-834) or any other linker described elsewhere herein, or known in the art to be suitable for such purposes.
d. Modifications to the Fc Domains The Fe domains in the growth factor trap constructs provided herein are modified to improve or enhance protein expression and purity, as well as to improve the pharmacodynamic and pharmacokinetic properties, including, for example, by extending the in vivo half-life and/or altering immune effector functions, as described below, and to result in production of heterodimers as the predominant, or only, product.
i. Introduction of Knobs-in-Holes The Fe domain in the growth factor trap constructs provided herein can be engineered such that steric interactions promote stable interaction, and promote the formation of heterodimers over homodimers from a mixture of chimeric ECD
polypeptide monomers. As discussed elsewhere herein, the introduction of "knobs-in-holes" (KiH; also known as "knobs-into-holes") into the CH3 domains of an antibody (e.g. , IgG) heavy chain optimizes heterodimer production. The knobs-in-holes approach involves asymmetrically mutating interfacial residues in the CH3 domains of the two Fc monomers in a complementary manner. Generally, "knobs" or protuberances 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 the CH3 domains, and compensatory "holes" or cavities of identical or similar size to the knobs 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 of the Fc monomers heterodimerize by virtue of the knob inserting into a .. correspondingly designed hole on the partner CH3 domain. Knob-knob association is prevented due to steric repulsion, and hole-hole homodimers are destabilized.
In some embodiments, the Fc portions of the heterodimeric, growth factor trap constructs provided herein are engineered to contain knobs-in-holes. The knob mutation can be, for example, S354C, T366Y, T366W, or T394W, by EU numbering, which correspond to S237C, T249Y, T249W or T277W, respectively, with reference to the sequence of the human IgG1 heavy chain constant domain, set forth in SEQ ID
NO:9. The hole mutation can be Y349C, T3665, L368A, F405A, Y407T, Y407A, or Y407V, by EU numbering, which correspond to Y232C, T2495, L251A, F288A, Y290T, Y290A, or Y290V, respectively, with reference to the sequence of the human IgG1 heavy chain constant domain, set forth in SEQ ID NO:9. The introduction of knobs-in-holes increases the yield of the heterodimer of interest, reduces the amount of homodimer impurities, and facilitates the protein purification process for the bi-specific, heterodimeric growth factor trap constructs provided herein, for example, when compared to RB200 and RB242.
ii. Modifications that Enhance Neonatal Fc Receptor (FcRn) Recycling As described elsewhere herein, fusion with an IgG Fc increases the half-life of small protein therapeutics by taking advantage of neonatal Fc receptor (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. To improve the pharmacokinetics and overall pharmacology, residues within the Fc regions of the growth factor trap constructs provided herein can be mutated to increase the affinity for FcRn, generally by greater than 30-fold, further increasing the in vivo half-life.
In some embodiments, the Fc portions of the growth factor trap constructs herein are modified to enhance neonatal FcRn recycling, to increase the in vivo half-life. This can be effected by mutating residues at the interface of the CH2 and CH3 domains of the IgG Fc, which are responsible for binding to FcRn. Exemplary Fc modifications that increase binding to FcRn, and that can be introduced into the Fc portions of the growth factor trap constructs herein, 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, Y43 6H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V2591/V308F, V2591/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y, and combinations thereof (by EU numbering).
Corresponding mutations by Kabat numbering and sequential numbering, with reference to the sequence of the IgG1 heavy chain constant domain set forth in SEQ
ID NO:9, are set forth in Table 7 (IgG1 Fc Modifications that Enhance FcRn Binding) in the section describing Fc modifications. Other modifications, known in the art to confer enhanced or increased FcRn binding also are contemplated for use herein.
The modification of the Fc portions of the growth factor trap constructs provided herein to enhance FcRn binding and recycling, increases the in vivo half-life of the therapeutics, requiring the administration of lower doses and/or less frequent dosing, and improving the therapeutic efficacy, compared to RB200 and RB242.
iii. Effector Functions As described herein, immune effector functions mediated by IgG Fcs include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC; also called antibody-dependent cellular cytotoxicity), and antibody-dependent cell-mediated phagocytosis (ADCP; also called antibody-dependent cellular phagocytosis). The Fc regions of the growth factor trap constructs herein can be mutated or modified, as discussed below and elsewhere herein, to eliminate, reduce, or enhance, immune effector functions, including, for example, any one or more of CDC, ADCC and ADCP.

Since the growth factors targeted by the growth factor trap constructs are present as membrane proteins and as free (i.e., soluble) ligands, in certain embodiments, the immune effector functions, particularly ADCC, of the Fc portion in the ECD/Fc fusion polypeptides are retained. In alternative embodiments, in addition .. to human IgG1 Fc, other Fc regions also can be included in the ECD/Fc chimeric fusion polypeptides provided herein. For example, where effector functions mediated by Fc/FcyR interactions are to be minimized, fusion with IgG isotypes that poorly recruit complement or effector cells, and do not exhibit effector functions, such as, for example, the Fc of IgG2 or IgG4, is contemplated. This approach 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.

In certain examples, the Fc portion can be modified to enhance or increase immune effector functions. This can be achieved, for example, by modifications that increase binding to Clq (for CDC) and/or certain, activating FcyRs (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa and FcyRIIIb). Fc regions modified to have increased binding to Fc receptors can be more effective in facilitating the destruction of cancer cells in patients, even when linked with an ECD polypeptide. Antibodies destroy tumor cells via a number of possible mechanisms, including, for example, anti-proliferation via blockade of growth pathways, intracellular signaling leading to apoptosis, enhanced down-regulation and/or turnover of receptors, ADCC, ADCP, CDC, and promotion of the adaptive immune response. Thus, in embodiments where the growth factor trap constructs herein are used for the treatment of cancer, the Fc portions of the constructs can be modified to enhance or increase immune effector functions. Table 8 (IgG1 Fc Modifications that Enhance Immune Effector Functions) in Section F.4.d.i.c) (Enhancement of or Reduction/Elimination of Fc Immune Effector Functions) summarizes Fc modifications that increase binding to FcyRs or Clq, and thus, enhance immune effector functions, including ADCC, ADCP and CDC, and provides the corresponding modifications by Kabat numbering and by sequential numbering, with reference to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO:9. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fc portions of the growth factor trap constructs provided herein. Other modifications, known in the art to confer enhanced or increased immune effector functions, also are contemplated for use herein. These listing in the section above describing IgG1 Fc Modifications that Enhance Immune Effector Functions.
In alternative embodiments, the Fc portions of the growth factor trap constructs provided herein are modified to decrease or eliminate immune effector functions. This can be achieved, for example, by modifications that decrease or abrogate binding to Clq (for CDC) and/or certain, activating FcyRs (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa and FcyRIIIb). This is desirable, for example, where antagonism, but not killing of the cells bearing a target antigen is desired, or where the reduction of undesired or detrimental immune effector functions, such as unwanted pro-inflammatory cytokine release and off-target cytotoxicity, is necessary.
Thus, in embodiments where the growth factor trap constructs provided herein are used for the treatment of chronic inflammatory and autoimmune diseases and disorders, such as RA, the Fc portions of the constructs can be modified to reduce or eliminate immune effector functions.
Table 9 (IgG1 Fc Modifications that Reduce or Eliminate Immune Effector Functions) in Section F.4.d.i.c) (Enhancement of or Reduction/Elimination of Fc Immune Effector Functions) summarizes exemplary IgG1 Fc modifications that reduce or eliminate binding to activating FcyRs and/or Clq, and thus, reduce or .. eliminate immune effector functions, including ADCC, ADCP and CDC, and can be introduced into the Fc regions of the growth factor trap constructs herein.
The table provides the corresponding modifications by Kabat numbering and by sequential numbering, with reference to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO:9. Any one or more of these modifications, alone or in various combinations, can be introduced into the IgG1 Fc portions of the growth factor trap constructs provided herein. Other modifications, known in the art to reduce or eliminate immune effector functions, also are contemplated for use herein.
The Fc portions of the growth factor trap constructs provided herein also can be modified to increase binding to inhibitory FcyRs, which results in the suppression of the immune response. Therapeutic antibodies with immunosuppressive Fc modifications are advantageous for the treatment of inflammatory diseases.
These mutations can be incorporated into the Fc portions of the growth factor trap constructs herein that are intended for the treatment of diseases and conditions with an inflammatory component or etiology or involvement, such as, for example, RA, and other inflammatory and autoimmune diseases.
Modifications that increase binding to, or that confer selective binding to, inhibitory FcyRIIb, and/or FcyRI but not FcyRIIIa, can be engineered into the IgG1 Fc regions in the growth factor trap constructs provided herein. These modifications include, but are not limited to, one or more of S267E, N297A, L328F, L35 1S, T366R, L3 68H, P395K, 5267E/L328F, L351S/T366R/L368H/P395K, and combinations thereof, by EU numbering. Table 11 (IgG1 Fc Modifications that Increase Binding to Inhibitory FcyRIIb) in Section F.4.d.i.i shows the corresponding replacements by Kabat numbering, and by sequential numbering, with reference to the sequence of the IgG1 heavy chain constant domain, set forth in SEQ ID NO:9. These modifications were summarized in the section above describing IgG1 Fc Modifications that Increase Binding to Inhibitory FcyRIIb.
4. Compositions, Therapeutic Uses and Methods of Treatment Provided are nucleic acid molecules encoding the chimeric fusion polypeptides (i.e., ECD/Fc) and growth factor trap constructs, vectors containing the nucleic acid molecules. Also provided are cells containing a vector as described herein, and pharmaceutical compositions containing any of the growth factor trap constructs, encoding nucleic acid molecules, vectors or cells, described herein. The growth factor trap constructs herein are produced and purified as described previously, for example, in Sarup et al. (2008) Mol. Cancer Ther. 7(10):3223-3236;
Gompels et al. (2011) Arthritis Research & Therapy 13 :R161; Jin et al. (2009) Mol.
Med. 15(1-2):11-20; and U.S. Patent Publication No. 2010/0055093.
A multi-specific, including bi-specific, growth factor trap construct herein contains two or more, particularly two, chimeric proteins created by linking two or more, particularly two, of the same or different ECD polypeptides directly or indirectly to a multimerization domain. In some examples, where the multimerization domain is a polypeptide, such as an immunoglobulin Fc, a gene fusion encoding the ECD-multimerization domain chimeric polypeptide is inserted into an appropriate expression vector. The resulting ECD-multimerization domain chimeric proteins can be expressed in host cells, particularly mammalian cells (e.g., HEK293T or CHO

cells, or any other suitable mammalian cells described herein or known in the art), that are transformed with the recombinant expression vector(s), and allowed to assemble into multimers, such as dimers, where the multimerization domains interact to form multivalent polypeptides. The resulting chimeric polypeptides, and multimers formed therefrom, can be purified by any suitable method known in the art, such as, for example, by affinity chromatography over Protein A or Protein G columns.
Additionally or alternatively, other techniques for protein purification can be used, including, for example, gel electrophoresis, dialysis, ion-exchange chromatography, ethanol precipitation, HPLC, such as reverse phase HPLC, chromatography on silica, chromatography on heparin Sepharose, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation. Where two nucleic acid molecules encoding different ECD chimeric polypeptides are transformed into cells (e.g., HER1/Fc and HER3/Fc), the formation of homodimers and heterodimers will occur. Conditions for expression can be adjusted, such that heterodimer formation is favored over homodimer formation. For example, the ratios of the nucleic acid molecules encoding the different ECD chimeric polypeptides can be adjusted, such that an excess of one nucleic acid molecule results in the formation of less homodimers.
Additionally, as described above, the introduction of knobs-in-holes into the Fc monomers favors the formation of heterodimers over homodimers.
ECD chimeric polypeptides containing Fc regions also can be engineered to include a tag with metal chelates or other epitope, such as, for example, a 6xHis tag, a c-myc tag, a FLAG tag, maltose binding protein (MBP), glutathione-S-transferase (GST), or thioredoxin (TRX). The tagged domain can be used for rapid purification by metal-chelate chromatography, and/or by antibodies, and to allow for detection in Western blots, immunoprecipitation, or activity depletion/blocking in bioassays.
a. Pharmaceutical Compositions Provided herein are pharmaceutical compositions containing a multi-specific, such as a bi-specific, growth factor trap construct provided herein, or encoding nucleic acid molecule(s). Also provided are pharmaceutical compositions containing an isolated cell that contains a nucleic acid molecule or a vector provided herein. Such compositions contain a therapeutically effective amount of the growth factor trap construct. The pharmaceutical compositions can be formulated in any conventional manner, by mixing a selected amount of the growth factor trap construct, or nucleic acid molecule, with one or more physiologically acceptable carriers or excipients. The pharmaceutical composition can be used for therapeutic, prophylactic, and/or diagnostic applications. The concentration of active compound in the composition will depend on the absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and the amount administered, as well as other factors known to those of skill in the art.
Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. Selection of the carrier or excipient is within the skill of the administering professional, and can depend upon a number of parameters. These include, for example, the mode of administration (i.e., systemic, oral, nasal, pulmonary, local, topical, or any other mode), and the disorder treated. Pharmaceutical compositions that include a therapeutically effective amount of a multi-specific, such as a bi-specific, growth factor trap construct, or nucleic acid molecule described herein, also can be provided as a lyophilized powder that is reconstituted, such as with sterile water, immediately prior to administration.
The pharmaceutical compositions provided herein can be in various forms, e.g., in solid, semi-solid, liquid, powder, aqueous, or lyophilized form. The pharmaceutical compositions provided herein can be formulated for single dosage (direct) administration, or for dilution, or other modification. The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment. Typically, the compositions are formulated for single dosage administration. The compound can be suspended in micronized or other suitable form, or can be derivatized to produce a more soluble active product. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration, and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the targeted condition and can be empirically determined.
To formulate a composition, the weight fraction of compound is dissolved, suspended, dispersed, or otherwise mixed in a selected vehicle, at an effective concentration, such that the targeted condition is relieved or ameliorated.

Methods for the production of nucleic acids encoding the growth factor trap constructs provided herein include the methods described in Section H. Section H also describes vectors and cells that can be used, as well as methods for protein expression and purification. The compositions, formulations, dosages and administration methods described in Section I can be adapted for the production of compositions and formulations including the growth factor trap constructs and encoding nucleic acid molecules described herein. The dosages and administration methods can be determined by the administering professional, and are known in the art and described elsewhere herein.
b. Therapeutic Uses and Methods of Treatment The multi-specific, including bi-specific, growth factor trap constructs provided herein can be used for any purpose known to the skilled artisan for use of such molecules. For example, the growth factor trap constructs provided herein can be used for one or more of therapeutic, diagnostic, industrial and/or research purpose(s).
In particular, the multi-specific growth factor trap constructs provided herein can be used in the treatment of a variety of diseases and conditions involving CSRs, including RTKs, and, in particular, the HER family of proteins, including those described herein. HER signaling is involved in the etiology of a variety of diseases and disorders, and any such disease or disorder thereof is contemplated for treatment by a growth factor trap construct provided herein.
The growth factor trap constructs and the encoding nucleic acid molecules, as well as the pharmaceutical compositions, provided herein, can be used for the treatment of any condition for which anti-HER therapies (e.g., trastuzumab, cetuximab, gefitinib, erlotinib, and lapatinib, and others described herein and/or known in the art), are employed, including, but not limited to, cancer and other proliferative diseases and disorders, angiogenesis-related diseases and disorders, rheumatoid arthritis and other chronic inflammatory and autoimmune diseases and disorders, as well as neurodegenerative diseases and disorders of the central nervous system (CNS). For example, treatments using the growth factor trap constructs provided herein, include, but are not limited to, treatment of angiogenesis-related diseases and conditions, inflammatory diseases and conditions, autoimmune diseases and conditions, neurodegenerative diseases, and conditions associated with cell proliferation. Such diseases and conditions include, for example, ocular diseases, atherosclerosis, vascular injuries, Alzheimer's disease, cancers, smooth muscle cell-associated conditions, rheumatoid arthritis (RA), and various autoimmune diseases.
Dosage levels and regimens can be determined based upon known dosages and regimens, and, if necessary can be extrapolated based upon the changes in properties of the polypeptides and constructs provided herein, and/or can be determined empirically based on a variety of factors. Such factors include, for example, the body weight of the individual, as well as their general health, age, sex, and diet, and the activity of the specific compound employed, the time of administration, the rate of excretion, drug combinations, the severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician. The active ingredient typically is combined with a pharmaceutically effective carrier. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form or multi-dosage form can vary depending upon the host treated and the particular mode of administration.
Dosage depends upon the particular disorder, disease or condition that is treated, as well as the particular subject. Typical doses are similar to those of known anti-HER therapies, such as antibodies, including trastuzumab, cetuximab, pertuzumab, and panitumumab, and small molecule tyrosine kinase inhibitors, such gefitinib, erlotinib, and lapatinib. Exemplary doses, for a subject, including humans and other animals, range from about or 0.1 to 100 mg/kg, such as 1 mg/kg to about or mg/kg, such as 5 mg/kg to 25 mg/kg. Dose can be determined based on the assumption that an average human has a mass of about 75 kg. Doses can be adjusted for children, infants, and smaller adults.
25 Upon improvement of a patient's condition, a maintenance dose of a compound or composition can be administered, if necessary; and the dosage, the dosage form, or frequency of administration, or a combination thereof, can be modified. In some cases, a subject can require intermittent treatment on a long-term basis upon any recurrence of disease symptoms, or based upon scheduled dosages.
30 Treatment of diseases and conditions with the multi-specific growth factor trap constructs provided herein can be effected by any suitable route of administration, using suitable formulations as described herein, including, but not limited to, infusion, RECTIFIED SHEET (RULE 91) ISA/EP

subcutaneous injection, and inhalation, or intramuscular, intradermal, oral, topical and transdermal administration.
Provided herein is a method of treatment of a HER-mediated or HER-associated disease or condition, including testing a subject with the disease to identify which HER receptors are expressed or overexpressed, and, based on the results, selecting a multi-specific growth factor trap construct that targets at least one, typically two, of the HER receptors. In one embodiment, the disease is a cancer.
Exemplary of cancers for treatment herein include gliomas, as well as pancreatic, gastric, head and neck, cervical, lung, colorectal, endometrial, prostate, esophageal, ovarian, uterine, bladder or breast cancers. Cancers treatable with the growth factor (HER ligand) trap constructs herein are generally cancers expressing at least one HER
receptor, typically more than one HER receptor. Such cancers can be identified by any means known in the art for detecting HER expression. For example, HER2 expression can be assessed using a commercially available diagnostic/prognostic assay, such as HercepTestTm (Dako). Paraffin embedded tissue sections from a tumor biopsy are subjected to the immunohistochemistry (IHC) assay and accorded a protein staining intensity criteria. Tumors accorded with less than a threshold score are characterized as not overexpressing HER2, whereas those tumors with greater than, or equal to, a threshold score, are characterized as overexpressing HER2. In one example of treatment, HER2-overexpressing tumors are assessed as candidates for treatment with a multi-specific growth factor trap construct, such as any provided herein.
In another embodiment, the HER-mediated or HER-associated disease or condition is an inflammatory or autoimmune disorder, particularly rheumatoid arthritis. An animal model of arthritis, such as the collagen-induced arthritis (CIA) mouse model, can be used to test the growth factor trap constructs provided herein.
For example, mice treated with a growth factor trap construct herein, such as by local injection of protein, can be observed for reduction of arthritic symptoms, including paw swelling, erythema and ankylosis. Reduction in synovial angiogenesis and synovial inflammation also can be observed.
The multi-specific, including bi-specific, growth factor trap constructs, encoding nucleic acid molecules and pharmaceutical compositions provided herein, can be used in the treatment of HER (ErbB)-related diseases or HER receptor-mediated diseases, which are any diseases, conditions or disorders in which a HER
receptor and/or ligand is implicated in some aspect of the etiology, pathology or development thereof. HER-related diseases for treatment include cancers, such as, for example, glioma, or pancreatic, gastric, head and neck, cervical, lung, colorectal, endometrial, prostate, esophageal, ovarian, uterine, bladder, renal, or breast cancers.
Other diseases that can be treated include non-cancer proliferative diseases, such as, for example, those that involve proliferation and/or migration of smooth muscle cells, inflammatory or autoimmune diseases, skin disorders, and ophthalmic disorders.
Diseases and conditions for treatment include, for example, rheumatoid arthritis, a diabetic retinopathy, a disease of the anterior eye, psoriasis, restenosis, stenosis, atherosclerosis, hypertension from thickening of blood vessels, muscle thickening of the bladder, heart or other muscles, bladder diseases, endometriosis, and obstructive airway diseases, as well as diseases or conditions associated with (e.g., caused by, or aggravated by) exposure to one or more neuregulin (NRG) ligands, such as NRG1 (including type I, II, and III), NRG2, NRG3, and/or NRG4, or other HER family ligands. Examples of NRG-associated diseases, and diseases associated with other HER family ligands, include neurological or neuromuscular diseases, including schizophrenia, Parkinson's disease and Alzheimer's disease, cardiomyopathy, pre-eclampsia, nervous system disease, and heart failure.
Examples of cancers that can be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies, such as, for example, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, renal cell cancer, esophageal cancer, glioma, colorectal cancer, endometrial cancer, uterine cancer, salivary gland carcinoma, renal cancer, prostate cancer, thyroid cancer, hepatic carcinoma, as well as head and neck cancer.

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Claims (286)

WHAT IS CLAIMED:
1. A construct that is a tumor necrosis factor receptor 1 (TNFR1) antagonist construct of formula 1:
(TNFR1 inhibitor).¨linkerp¨ (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 the pharmacological property of the construct compared to the construct in the absence of the activity modifier; and 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.
2. The construct of claim 1, wherein the linker is selected from among a chemical linker, a polypeptide linker, and combinations thereof.
3. The construct of claim 1 or claim 2 that is a fusion protein.
4. The construct of any of claims 1-3, wherein the linker contains a plurality of linker components.
5. The construct of any of claims 1-4, wherein the TNFR1 inhibitor comprises a domain antibody (dAb).
6. The construct of any of claims 1-5, wherein, if the TNFR1 inhibitor is a domain antibody (dAb), the activity modifier is not an unmodified single Fc region or a human serum albumin antibody.
7. The construct of any of claims 1-6, wherein the TNFR1 inhibitor inhibits TNFR1 signaling.
8. The construct of any of claims 1-7, wherein the activity modifier increases serum half-life of the construct.
9. The construct of any of claims 1-8, wherein the activity modifier is albumin or an Fc that is modified to have reduced or no ADCC activity and/or reduced or no CDC activity.
10. The construct of any of claims 1-9, wherein the TNFR1 inhibitor inhibits a TNFR1 activity, but does not antagonize tumor necrosis factor receptor 2 (TNFR2) activity.
11. The construct of claim 10, where the TNFR1 inhibitor inhibits TNFR1 signaling.
12. A construct that is a multi-specific construct, 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.
13. The construct of claim 12 that is bi-specific for TFNR1 and a Treg expander.
14. The construct of claim 12 or claim 13, wherein the Treg expander is a TNFR2 agonist.
15. The construct of any of claims 12-14, comprising a linker to provide flexibility, increase solubility, or to relieve or reduce steric hindrance or Van der Waals interactions.
16. The construct of any of claims 12-15, further comprising an activity modifier to alter the activity of the construct.
17. The construct of any of claims 12-16 that has Formula 2:
(TNFR1 inhibitor),¨ (activity modifier)," ¨ (Linker (L))p ¨ (activity modifier),2 ¨
(TNFR2 agonist),,, or (TNFR1 inhibitor),¨ (activity modifier)," ¨ (Linker (L))p ¨ (activity modifier),2 ¨
(Treg expander)q, wherein:
n= 1, 2, or 3, p= 1, 2, or 3, q= 0, 1 or 2, and each of rl and r2 is independently .. 0, 1, or 2; and the components can be in the order specified or any other order as long as the construct interacts with TNFR1 and TNFR2 to antagonize TNFR1 and agonize TNFR2, or has Treg expander activity.
18. The construct of any of claims 1-17, wherein the TNFR1 inhibitor moiety inhibits binding of TNFa binding to TNFR1 and/or inhibits signaling.
19. A construct of formula 3a or 3b:

(TNFR2 agonist or Treg expander), ¨ linkerp¨ (activity modifier)q, formula 3a, Or (activity modifier)q ¨ linkern ¨ (TNFR2 agonist or Treg expander)n, formula 3b, wherein:
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 of the construct;
a TNFR2 agonist interacts with TNRFR2 resulting in TNFR2 activity;
a Treg expander, includes TNFR2 agonists, and is a 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.
20. The construct of any of claims 1-19, wherein the activity modifier is an Fc region or a modified Fc region or a short FcRnBP; and the linker comprises a hinge region, or is a linker comprising G and S
residues.
21. The construct of any of claims 1-20, wherein the linker has the sequence set forth in any of SEQ ID NOs: 812-834 or is a PEG moiety linker.
22. The construct of any of claims 1-21, comprising an activity modifier that is a modified Fc region or a peptide that increases serum half-life of the construct.
23. The construct of any of claims 1-22, comprising an Fc region or an Fc dimer.
24. The construct of any of claims 1-23 that comprises an Fc region that is/are modified to have reduced ADCC and/or CDC activity.
25. The construct of claim 24, wherein the Fc is modified to have reduced or no ADCC activity.
26. The construct of any of claims 1-25, wherein:
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, listed below, or known in the art;
the linker is any listed in the sequence listing or below or known in the art;
and RECTIFIED SHEET (RULE 9 1) ISA/EP

the activity modifier is any set forth in the sequence listing, known in the art, and/or set forth below.
27. A construct that is a TNFR1 antagonist construct, comprising a TNFR1 inhibitor that is a single chain antibody or antigen-binding portion thereof that specifically targets and inhibits TNFR1, but does not antagonize TNFR2, thereby preventing transient activation of TNFRI via receptor clustering.
28. The construct of claim 27, wherein the antibody or antigen-binding portion thereof, comprises a modification that improves a pharmacological property and/or structure of the construct.
29. The construct of any of claims 1-28 that agonizes TNFR2 signaling to thereby increase expression of regulatory T cells (Tregs).
30. The construct of any of claims 27-29, wherein the single chain antibody inhibits TNFR1 by inhibiting TNFR1 signaling.
31. The TNFRI antagonist construct of any of claims 27-30, wherein the antibody portion or antigen binding portion of the construct inhibits binding of TNFa to TNFR1.
32. The construct of any of claims 27-30, wherein the antibody or antigen binding portion of the construct does not inhibit binding of TNFa to TNFR I, but does inhibit TNFR1 signaling.
33. The construct of any of claims 28-32, wherein the pharmacological property is increased serum half-life.
34. The construct of any of claims 27-33, wherein the TNFR1 construct comprises an Fc modified to eliminate ADCC and/or CDC activity.
35. The construct of any of claims 27-34, wherein the TNFR1 construct comprises an Fc dimer.
36. The construct of claim 35, wherein one Fc monomer comprises holes, and the other comprises knobs, to form heterodimer.
37. The construct of claim 35, wherein:
the knob mutation(s) is/are selected from among S354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation(s) is/are selected from among Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering, RECTIFIED SHEET (RULE 9 1) ISA/EP

whereby the Fc monomers form the heterodimer.
38. The construct of any of claims 1-37 that comprises an Fc, wherein the Fc is from trastuzumab.
39. The construct of claim 38, comprising a linker that is a hinge region from an Fc region.
40. The construct of claim 39, wherein the hinge region is from trastuzumab, and is linked to the Fc region.
41. The construct of any of claims 27-40, comprising a linker that is linked to the anti-TNFR1 antagonist antibody or antigen-binding portion thereof.
42. The construct of claims 27-41, comprising a linker that is linked to the anti-TNFR1 antagonist antibody or antigen-binding portion thereof, directly or via a hinge region to an Fc region.
43. The construct of any of claims 1-42 that comprises an Fc region or modified Fc region that comprises the sequence of amino acids set forth in any of SEQ ID NOs:10, 12, 14, 16, 27, 30, 1469, and 1470.
44. The construct of any of claims 1-43, wherein the construct comprises a hinge region linked to the Fc portion.
45. The construct of any of claims 1-44, wherein the construct binds to neonatal Fc receptor (FcRn).
46. The construct of claim 45, wherein:
the TNFR1 construct comprises a short FcRn-binding peptide (FcRnBP); and 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.
47. The TNFR1 antagonist construct of claim 46, wherein the FcRnBP
contains 12-20 residues or 15 residues or 16 residues.
48. The TNFR1 antagonist construct of claim 46 or claim 47, wherein the FcRn-binding peptide (FcRnBP) comprises a peptide of any SEQ ID NOs:48-51.
49. The TNFR1 antagonist construct of any of claims 46-48, wherein the FcRn-binding peptide (FcRnBP) consists of a peptide of any SEQ ID NOs: 48-51.
50. The construct of any of claims 1-49, wherein the TNFR1 construct comprises an Fc heterodimer, wherein one Fc monomer comprises holes, and the other comprises knobs, whereby the Fc dimer that results is a heterodimer.
RECTIFIED SHEET (RULE 9 1) ISA/EP
51. The construct of any of claims 1-50 that is a TNFR1 antagonist construct, comprising:
a TNFR1 inhibitor;
an Fc dimer; and a Treg expander, wherein:
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.
52. The construct of claim 51, wherein the Treg expander is a TNFR2 agonist.
53. The construct of claim 51 or claim 52, further comprising a second Treg expander linked to the same Fc monomer as the TNFR1 inhibitor, wherein the first and second Treg expanders are the same or different.
54. The construct of claim 53, wherein the second Treg expander is a TNFR2 agonist.
55. The construct of claim 53, wherein the Treg expanders are the same.
56. The construct of any of claims 51-55, wherein the TNFR1 inhibitor inhibits or blocks TNFR1 signaling.
57. The construct of any of claims 51-56, wherein the TNFR1 inhibitor binds to TNFR1 and blocks or inhibits TNFa binding and TNFR1 signaling.
58. The construct of any of claims 51-56, wherein the TNFR1 inhibitor binds to TNFR1, does not or interfere with TNFa binding, and blocks or inhibits TNFR1 signaling.
59. The construct of any of claims 51-58, wherein the Treg expander is a TNFR2 agonist.
60. The construct of claim 59, wherein the TNFR2 agonist stimulates or induces TNFR2 signaling.
61. The construct of any of claims 51-60, wherein the Treg expander is a TNFR2 agonist that is an scFv, VHEI single domain antibody, or Fab of aTNFR2 agonist monoclonal antibody.
62. The construct of any of claims 1-61 that comprises all or a portion of trastuzumab, and is dimerized by N-terminal fusion with the C-terminus of trastuzumab.
63. The construct of any of claims 7-16, wherein the Treg expander is a TNFR2 agonist that is a small molecule, or a nucleic acid aptamer, or a peptide aptamer.
64. A construct of any of claims 1-63 that is a TNFR2 agonist construct of formula 3:
(Treg expander)n¨ linkerp ¨ (activity modifier)q, formula 3a, or (activity modifier)q ¨linkerp ¨ (Treg expander)n, formula 3b, wherein:
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 the 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.
65. The construct of any of claims 51-64, wherein the Treg expander is TNFR2 agonist.
66. The construct of claim 65, wherein the TNFR2 agonist stimulates or induces TNFR2 signaling.
67. The construct of any of claims 51-66 wherein the Treg expander is a TNFR2 agonist that is an scFv, VHH single domain antibody, or Fab of aTNFR2 agonist monoclonal antibody.
68. The construct of claim 67 that is dimerized by N-terminal fusion with the C-terminus of trastuzumab.
69. The TNFR1 antagonist construct of any of claims 51-68, wherein the Treg expander is a TNFR2 agonist that is a small molecule, or a nucleic acid, or peptide aptamer.
70. A construct, comprising a TNFR1 inhibitor moiety linked via a central PEG linker to one more Treg expanders, or comprising at least two TNFR1 inhibitors RECTIFIED SHEET (RULE 9 1) ISA/EP

that are the same or different, or comprising two Treg expanders that are the same or different.
71. The construct of claim 70, comprising a branched PEG moiety linking the TNFR1 inhibitor and one or more Treg expanders.
72. The construct of claim 70 or claim 71 that has a structure selected from among formulae 4A to 4D:
Formula 4A:
(CH2CH20)-0 In ________________________________________ I

I
0¨(OCH2CH2)-0¨CH2¨C ¨CH2-0 ¨(CH2CH2OKD
I

I

( i ¨ )7¨.
, or (CH2CH20 ,),-1.
I

I

I
0-(OCH2CH2)-0-CH2-C ¨R1 n I

I

, i CH2CH20 iµmv n is 1 to 5;
le is H or CH3, or CH2CH3 or other C1-05 alkyl 0 is aTNFR1 inhibitor (TNFR1 antagonist);
Ois a Treg expander; or Formula 4B:
H
gIr 0¨(CH2CH20)n 0 is a TNFR1 inhibitor (TNFR1 antagonist) is a Treg expander;
n is 1 to 5; or Formula 4C:
H õdm.
Ailli. AM
AMR H /NN moi Imo IMEN' 'OW N¨(CH2CH20)n AEI li.
/MI
IMO
'REIF is a TNFR1 inhibitor (TNFR1 antagonist), or is a Treg expander; and n is 1 to 5; or Formula 4D:
activity modifier _________ (CH2CH20)n or 0 (CH2CH20), wherein each 0 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.
73. The construct of any of claims 70-72, wherein the Treg expander is a TNFR2 agonist.
74. The construct of any claims 1-73, that comprises an activity modifier, wherein:
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.
75. The construct of claim 74, wherein the Fc or modified Fc is an IgG Fc or is an IgG1 or IgG4 Fc.
76. The construct of any of claims 1-75 that binds to neonatal Fc receptor (FcRn).
77. The construct of claim 76, wherein:
the 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.
78. The construct of claim 77, wherein the FcRnBP contains 12-20 residues or 15 residues or 16 residues.
79. The TNFR1 antagonist construct of claim 78, wherein the FcRn-binding peptide (FcRnBP) comprises a peptide of any SEQ ID NOs:48-51.
80. The TNFR1 antagonist construct of claim 78, wherein the FcRn-binding peptide (FcRnBP) consists of a peptide of any SEQ ID NOs:48-51.
81. A construct of any of claims 1-80 that is a TNFR1 antagonist construct, comprising:
a) a TNFR1 inhibitor moiety that is a TNFR1-selective;
b) optionally one or more linkers; and c) optionally a half-life extending moiety, wherein the antagonist construct comprises at least one of b) and c).
82. The construct of claim 81, wherein the TNFR1-selective antagonist selectively binds and inhibits TNFR1 signaling, but not TNFR2 signaling.
83. The construct of claim 81 or claim 82, wherein the TNFR1 inhibitor that is a selective antagonist comprises an antigen-binding fragment that selectively binds and inhibits TNFR1 signaling but not TNFR2 signaling.
84. The construct of claim 83, wherein the antigen-binding fragment that selectively binds and inhibits TNFR1 signaling but not TNFR2 signaling comprises a domain antibody (dAb), scFv, or Fab fragment.
85. The construct of any of claims 1-84, wherein the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody.
86. The construct of claim 85, wherein the human anti-TNFR1 antagonist monoclonal antibody is H398 that comprises SEQ ID NO:678, or is ATROSAB, or is an antigen binding portion thereof, or has a sequence having at least 95%
sequence identity to SEQ ID NO:31 or 32 or 673 or 678 or is an antigen-binding portion thereof that binds to TNFR1.
87. The construct of any of claims 1-86, wherein the TNFR1 inhibitor comprises 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.
88. The construct of any of claims 1-87 wherein the TNFR1 inhibitor comprises 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 inhibitor activity.
89. The construct of any of claims 1-88, wherein the TNFR1 inhibitor comprises 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.
90. The construct of any of claims 1-89 wherein the TNFR1 inhibitor 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 inhibitor or binding activity.
91. The construct of any of claims 1-90, wherein the TNFR1 inhibitor comprises a dominant-negative tumor necrosis factor (DN-TNF) or TNF mutein.
92. The construct of claim 91, wherein the 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:
V1M, L295, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A845, V85T, 586T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L295/R32W, L295/586T, R32W/586T, L295/R32W/586T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A845/V85T/586T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF, set forth in SEQ ID NO:2.
93. The construct of claim 91 or claim 92, wherein the TNFR1 inhibitor .. TNF mutein comprises the sequence of residues set forth in any one of SEQ
ID
NOs: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.
94. The construct of any of claims 1-93 that comprises a linker, wherein 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.
95. The construct of any of claims 1-94 that comprises a linker, wherein the linker comprises the sequence SCDKTH, corresponding to residues 222-227 of SEQ ID NO:26.
96. The construct of any of claims 1-95 that comprises a linker, wherein the linker comprises a glycine-serine (GS) linker.
97. The construct of claim 96, wherein the GS linker is selected from among (GlySer)n, where n= 1-10; (G1ySer2); (G1y4Ser)n, where n= 1-10;
(G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5;
GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS.
98. The construct of any of claims 1-97 that comprises a linker, wherein the linker comprises a GS linker and all or a portion of the hinge sequence of trastuzumab, corresponding to residues EPKSCDKTHTCPPCP (219-233 of SEQ ID
NO:26).
99. The construct of any of claims 1-98, wherein the linker comprises a GS
linker and comprises the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID NO:31.
100. The construct of any of claims 1-99, wherein the linker comprises a GS
linker and all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
101. The construct of any of claims 1-100, comprising an activity modifier, wherein the activity modifier is a half-life extending moiety that is an IgG
Fc, a polyethylene glycol (PEG) molecule, or human serum albumin (HSA).
102. The construct of claim 101, wherein the IgG Fc is an IgG1 or IgG4 Fc.
103. The construct of claim 101 or claim 102, wherein the IgG1 Fc is the Fc of trastuzumab, set forth in SEQ ID NO:27 or a sequence of amino acids having at least 95% sequence identity therewith.
104. The construct of claim 101 or claim 102, wherein the IgG4 Fc is the Fc of nivolumab, set forth in SEQ ID NO:30 or a sequence of amino acids having at least 95% sequence identity therewith.
105. The construct of any of claims 101-104, wherein the IgG1 Fc is the Fc of human IgG1 , set forth in SEQ ID NO:10.
106. The construct of any of claims 101-104, wherein the IgG4 Fc is the Fc of human IgG4, set forth in SEQ ID NO:16.
107. The construct of any of claims 1-106 that comprises a TNFR1 inhibitor, wherein the TNFR1 inhibitor is monovalent.
108. The construct of any of claims 81-107 that comprises a linker, wherein the linker comprises (G1y4Ser)3.
109. The construct of any of claims 81-107 that comprises a linker, wherein the linker comprises (G1y4Ser)3 and SCDKTH (residues 217-222 of SEQ ID NO:31).
110. The construct of any of claims 1-109 that comprises a linker, wherein the linker comprises (G1y4Ser)3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26.
111. The construct of any of claims 1-109 that comprises a linker, wherein the linker comprises (G1y4Ser)3 and the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
RECTIFIED SHEET (RULE 9 1) ISA/EP
112. The construct of any of claims 1-111, comprising the sequence of residues set forth in any one 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.
113. The construct of any of claims 1-112 that is a TNFR1 antagonist construct, wherein:
the TNFR1 construct comprises a short FcRn-binding peptide (FcRnBP); and a 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.
114. The construct of claim 113, wherein the FcRnBP contains 12-20 residues or 15 residues or 16 residues.
115. The construct of claim 113, wherein the FcRn-binding peptide (FcRnBP) comprises a peptide of any SEQ ID NOs:48-51.
116. The TNFR1 antagonist construct of claim 113, wherein the FcRn-binding peptide (FcRnBP) consists of a peptide of any SEQ ID NOs:48-51.
117. A construct of any of claims 1-116, comprising:
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.
118. The construct of claim 117, wherein the half-life extending moiety is not a human serum albumin antibody or an unmodified Fc.
119. A construct of any of claim 117 or claim 118 that is a TNFR1 antagonist, 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 GS linker selected from among (GlySer),, where n= 1-10; (G1ySer2);
.. (G1y4Ser),, where n= 1-10; (G1y3Ser),, where n= 1-5; (SerG1y4),, where n= 1-5;
(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;

GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) a half-life extending moiety that is an IgG Fc.
120. The TNFR1 antagonist construct of any of claims 117-119, wherein:
the GS linker is (GGGGS)3; and the IgG Fc is the Fc of trastuzumab or the Fc of nivolumab.
121. A construct of any of claims 1-120 that is a TNFR1 antagonist construct, 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.
122. The construct of claim 121, wherein:
the linker comprises all or a portion of the hinge sequence of trastuzumab;
and the IgG Fc is the Fc of trastuzumab.
123. The construct of claim 121, wherein:
the linker comprises all or a portion of the hinge sequence of nivolumab; and the IgG Fc is the Fc of nivolumab.
124. A construct of any of claims 1-123 that is a TNFR1 antagonist construct, 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 GS linker selected from among (GlySer)n, where n= 1-10; (G1ySer2);
(G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5;
.. (GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS;
RECTIFIED SHEET (RULE 9 1) ISA/EP

c) a second 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 d) a half-life extending moiety that is an IgG Fc.
125. The construct of claim 124, wherein:
the GS linker is (GGGGS)3;
the second linker comprises the sequence SCDKTH (residues 217-222 of SEQ
ID NO:31); and the IgG Fc is the Fc of trastuzumab.
126. The construct of claim 124, wherein:
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.
127. A construct that is a TNFR1 agonist, 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 GS linker selected from among (GlySer),, where n= 1-10; (G1ySer2);
(G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4),, where n= 1-5;
(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) a half-life extending moiety that is a PEG molecule.
128. The construct of claim 127, wherein the GS linker is (GGGGS)3.
129. The construct of claim 127 or claim 128, wherein the PEG molecule has a molecular weight of 30 kDa or more.
130. A construct of any of claims 1-129 that is a TNFR1 agonist construct, 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 GS linker selected from among (GlySer)n, where n= 1-10; (G1ySer2);
(G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5;
(GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) a half-life extending moiety that is human serum albumin.
131. The construct of claim 130, wherein the GS linker is (GGGGS)3.
132. The construct of any of claims 1-131 that is a TNFR1 antagonist, wherein the construct is optimized to eliminate immunogenic sequences or immunogenic epitopes.
133. The construct of any of claims 1-132, 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.
134. The TNFR1 antagonist construct of claim 133, wherein:
the knob mutation is selected from among 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T3665, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering.
135. The TNFR1 antagonist construct of claim 133 or claim 134, wherein the modification(s) to increase or enhance FcRn recycling is selected from among one or more of:
T250Q, T250R, M252F, M252W, M252Y, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering.
136. The TNFR1 antagonist construct of any of claims 133-135, wherein the immune effector function(s) 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).
137. The TNFR1 antagonist construct of claim 136, wherein the modification(s) to reduce or eliminate immune effector functions are selected from among one or more of:
in IgG1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, 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, 5228P/L235E, and 5228P/F234A/L235A, by EU numbering.
138. The construct of any of claims 1-137, that is a TNFR1 antagonist or multispecific or comprises a central PEG linker moiety, and the construct comprises a modified Fc region.
139. The construct of claim 138, wherein the 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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T3665, 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, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance one or more immune effector functions, wherein:
the immune effector function(s) is/are selected from among one or more of CDC, ADCC and ADCP; and the modification(s) to increase or enhance an immune effector function is/are selected from among one or more of:
in IgG1 : S239D, 1332E, 5239D/I332E, 5239D/A330L/I332E, 5298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/5298A in the first heavy chain and 5239D/A330L/I332E in the second heavy chain;
L234Y/L235Q/G236W/5239M/H268D/D270E/5298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain;
A327Q/P329A; D265A/5267A/H268A/D270A/K326A/5337A;
T256A/K290A/5298A/E333A/K334A; G236A; G236A/I332E;
G236A/5239D/I332E; G236A/5239D/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/5324T; 5267E;
H268F; 5324T; 5267E/H268F/5324T; G236A/I332E/5267E/H268F/5324T;
E345R; and E345R/E430G/5440Y; by EU numbering.
140. The construct of any of claims 1-139 that comprises an Fc region, comprising an IgG1 Fc that comprises one or more modifications to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb.
141. The TNFR1 antagonist construct of claim 140, wherein the modifications that increase binding to FcyRIIb are selected from among one or more of S267E, N297A, L328F, L3515, T366R, L368H, P395K, 5267E/L328F and L3515/T366R/L368H/P395K, by EU numbering.
142. A construct that is a Treg expander construct, 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.
143. The construct of claim 142 or any of claims 1-141 that comprises a Treg expander, wherein the Treg expander is a TNFR2 agonist.
144. The construct of claim 142 or claim 143 that further comprises a TNFR1-inhibitor.
145. The construct of claim 143 or claim 144, wherein the TNFR2 agonist is a TNFR2 selective agonist.
146. A construct of any of claims 1-145 that is TNFR2 agonist construct, 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.
147. The construct of claim 146, wherein the TNFR2 agonist is a TNFR2-selective agonist.
148. The construct of any of claims 142-147 that comprises activity modifier, wherein the activity modifier is a half-life extending moiety.
149. The construct of any of claims 142-148, wherein the TNFR2 agonist selectively activates or antagonizes TNFR2, without activating or antagonizing TNFR1.
150. The construct of any of claims 1-149 that comprises a TNFR2 agonist, wherein the TNFR2 agonist binds to one or more epitopes within TNFR2.
151. The construct of claim 150, wherein the TNFR2 is a human TNFR2.
152. The construct of 151, wherein the epitopes are 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.
153. The construct of any of claims 149-152, wherein 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.
154. The construct of claim 153, wherein the agonist anti-TNFR2 antibody is selected from 1VIR2-1 (also designated ab8161; U.S. Patent No. 9,821,010) or MAB2261 (U.S. Patent No. 9,821,010).
155. The construct of any of claims 149-154, wherein the TNFR2 agonist is an antigen-binding fragment selected from a dAb, scFv, or Fab fragment.
156. The construct of any of claims 149-155, wherein the TNFR2 agonist is a TNFR2-selective agonist.
157. The construct of any of claims 1-156 that comprises a TNFR2 agonist, wherein the TNFR2 agonist comprises a TNFR2 agonist TNF mutein.
158. The construct of claim 157, wherein the TNFR2 mutein 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/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A1451/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A145S, and combinations of any of the preceding, all with reference to SEQ ID NO:2.
159. The construct of claim 157, wherein the TNFR2 agonist is a TNF
mutein comprising the mutations D143N/A145R.
160. The construct of any of claims 142-159, wherein the linker comprises 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.
161. The construct of any of claims 1-160, comprising a linker, wherein the linker comprises the sequence SCDKTH, corresponding to residues 217-222 of SEQ
ID NO:31.
162. The construct of any of claims 161 that comprises a linker, wherein the linker comprises a glycine-serine (GS) linker.
163. The construct of claim 162, wherein the GS linker is selected from among (GlySer)n, where n= 1-10; (G1ySer2); (G1y4Ser)n, where n= 1-10;
(G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5;
GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS.
164. The construct of any of claims 146-163, wherein the linker 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.
165. The construct of any of claims 146-163, wherein the linker comprises a GS linker and the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID

NO:31.
166. The construct of any of claims 146-163, wherein the linker comprises a GS linker and all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
167. The construct of any of claims 1-166 that comprises a half-life extending moiety, wherein the half-life extending moiety is an IgG Fc, a polyethylene glycol (PEG) molecule, or human serum albumin (HSA).
168. The construct of claim 167, wherein the IgG Fc is an IgG1 or IgG4 Fc.
169. The construct of claim 168, wherein the IgG1 Fc is the Fc of trastuzumab, set forth in SEQ ID NO:27.
170. The construct of claim 168, wherein the IgG4 Fc is the Fc of nivolumab, set forth in SEQ ID NO:30.
171. The construct of claim 168, wherein the IgG1 Fc is the Fc of human IgGl, set forth in SEQ ID NO:10.
172. The construct of claim 168, wherein the IgG4 Fc is the Fc of human IgG4, set forth in SEQ ID NO:16.
173. The construct of any of claims 146-172, wherein the TNFR2 agonist is monovalent.
174. The construct of any of claims 1-173 that is a TNFR2 agonist construct wherein the TNFR2 agonist is bivalent.
175. The construct of any of claims 1-173 that is a TNFR2 agonist construct, wherein the TNFR2 is trivalent.
176. The construct of any of claims 1-175 that is a TNFR2 agonist construct, wherein the linker comprises (G1y4Ser)3.
177. The construct of any of claims 1-176 that is a TNFR2 agonist construct TNFR2, wherein the linker comprises (G1y4Ser)3 and SCDKTH (residues 217-222 of SEQ ID NO:31).
178. The construct of any of claims 1-177 that is a TNFR2 agonist construct, wherein the linker comprises (G1y4Ser)3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26.
179. The construct of any of claims 1-177 that is a TNFR2 agonist construct, wherein the linker comprises (G1y4Ser)3 and the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
180. The construct of any of claims 1-179 that comprises an activity modifier that is a half-life extending moiety.
181. The construct of claim 180, wherein the half-life extending moiety is a PEG.
182. The construct of claim 180, wherein the PEG has a molecular weight of at least or at least about 30 kDa.
183. The construct of claim 180, wherein the half-life extending moiety is human serum albumin (HSA).
184. A construct of any of claims 1-183 that is a TNFR2 agonist construct, comprising:
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 GS linker selected from among (GlySer),, where n= 1-10; (G1ySer2);
(G1y4Ser),, where n= 1-10; (G1y3Ser),, where n= 1-5; (SerG1y4),, where n= 1-5;

(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) an activity modifier that is a half-life extending moiety that is an IgG
Fc.
185. The construct of claim 184, wherein:
the GS linker is (GGGGS)3; and the IgG Fc is the Fc of trastuzumab or the Fc of nivolumab.
186. A construct of any of claims 1-185 that is a TNFR2 agonist construct, comprising:
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.
187. The construct of claim 186, wherein:
the linker comprises all or a portion of the hinge sequence of trastuzumab;
and the IgG Fc is the Fc of trastuzumab.
188. The construct of claim 187, wherein:
the linker comprises all or a portion of the hinge sequence of nivolumab; and the IgG Fc is the Fc of nivolumab.
189. A construct of any of claims 1-183 that is a TNFR2 construct, comprising:
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 GS linker selected from among (GlySer),, where n= 1-10; (G1ySer2);
(G1y4Ser),, where n= 1-10; (G1y3Ser),, where n= 1-5; (SerG1y4),, where n= 1-5;
(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;

GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS;
c) a second 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 d) an activity modifier that is a half-life extending moiety that is an IgG
Fc.
190. The construct of claim 189, wherein:
the GS linker is (GGGGS)3;
the second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO:31); and the IgG Fc is the Fc of trastuzumab.
191. The construct of claim 189, wherein:
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.
192. A construct of any of claims 1-191 that is a TNFR2 agonist construct, comprising:
a) the TNFR2 agonist that comprises an antigen-binding fragment of an agonist human anti-TNFR2 antibody selected from MR2-1 or MAB2261;
b) a linker comprising:
i) a GS linker selected from among (GlySer)n, where n= 1-10;
(G1ySer2); (G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5; GSGGSSGG;
GSSSGSGSGSSG; GSSSGSGSGSSGG; 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 IgG1 or IgG4 Fc, a PEG molecule, and human serum albumin (HSA), wherein:
the IgG1 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 30 kDa.
193. A construct of any of claims 1-192 that is or comprises 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/A1451/E146G/5147D, A145H/E146S/5147D, A145H/S147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A145S, S95C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2;
b) a linker comprising:
i) a GS linker selected from among (GlySer)n, where n= 1-10;
(G1ySer2); (G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5; GSGGSSGG;
GSSSGSGSGSSG; GSSSGSGSGSSGG; 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 IgG1 or IgG4 Fc, a PEG molecule, and human serum albumin (HSA), wherein:
the IgG1 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 30 kDa.
194. A construct of any of claims 1-193 that is or comprises 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.
195. A construct of any of claims 1-194 that 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 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.
196. A construct that 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.
197. A construct of any of claims 1-196 that is a TNFR2 agonist construct comprising:
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.
198. A construct of any of claims 1-197 that is or comprises a TNFR2 agonist construct comprising:
a) a TNFR2-selective TNF mutein comprising the mutations D143N/A145R;
b) a linker comprising all or a portion of the hinge sequence of nivolumab, 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.
199. The construct of any of claims 1-198 that is a TNFR1 antagonist construct or a TNFR2 agonist construct or both, wherein the IgG Fc is a monomer or a dimer.
200. The construct of any of claims 1-199, comprising a dAb.
201. The construct of claim 200, that comprises a Vhh single chain or double chain (nanobody) containing a dAb.
202. The construct of claim 201, comprising HSA linked to the dAb directly or via a linker.
203. The construct of claim 202, wherein the linker is a Gly-Ser (GS) linker and/or the HSA is linked to the C-terminus of the dAb via the linker or directly.
204. The construct of any of claims 1-203, comprising 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 and having TNFR1 antagonist activity.
205. The construct of claim 200 or 203 that comprises a dAb set forth in any of SEQ ID NOs: 52-83, 503-672, 1478 and 1479, and variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto, whereby the construct has TNFR1 antagonist activity.
206. The construct of claim 205, wherein the dAb 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.
207. The construct of claim 206, wherein the dAb is designated DOM1h-131-206 of SEQ ID NO:59 and variants thereof that have TNFR1 antagonist activity.
208. The construct of any of claims 200-207 wherein the sequence of the dAb or a necessary portion of the construct for administration to a human is humanized.
209. The construct of any of claims 1-208 that comprises a TNFR2 agonist or is a TNFR2 agonist construct.
210. A construct of any of claims 1-208 that is a or also is or comprises a TNFR2 agonist construct, wherein the TNFR2 agonist is modified to eliminate sequences of amino acids or epitopes that are immunogenic in the subject to be treated.
211. The construct of claim 200, wherein the subject is a human.
212. The construct of any of claims 189-211, wherein the TNFR2 agonist is a TNFR2-selective agonist.
213. The construct of any of claims 1-212 that is a TNFR2 agonist construct and comprises 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;
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).
214. The construct of claim 213, wherein:
a) 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, T3665, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering, whereby the Fc forms a dimer;
b) the modification(s) to increase or enhance FcRn recycling is selected from among one or more of T250Q, T250R, M252F, M252W, M252Y, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering; and c) the modification(s) to reduce or eliminate immune effector functions are selected from among one or more of:

in IgG1 : L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, 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, 5228P/L235E, and 5228P/F234A/L235A, by EU numbering.
215. A construct of construct of any of claims 1-213 that is a TNFR2 agonist construct that contains a modified IgG Fc, wherein 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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among Y349C, T3665, 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, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance 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 increase or enhance immune effector functions is selected from among one or more of:

in IgG1 : S239D, 1332E, S239D/I332E, 5239D/A330L/I332E, 5298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/5298A in the first heavy chain and 5239D/A330L/I332E in the second heavy chain;
L234Y/L235Q/G236W/5239M/H268D/D270E/5298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/5267A/H268A/D270A/K326A/5337A;
T256A/K290A/5298A/E333A/K334A; G236A; G236A/I332E;
G236A/5239D/I332E; G236A/5239D/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/5324T; 5267E; H268F;
5324T; 5267E/H268F/5324T; G236A/I332E/5267E/H268F/5324T;
E345R; and E345R/E430G/5440Y; by EU numbering.
216. The construct of any of claims 1-215 that is or comprises a TNFR2 agonist construct that comprises a modified IgG1 Fc, wherein the Fc is modified to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb.
217. The construct of claim 216, wherein the modifications that increase binding to FcyRIIb are selected from among one or more of 5267E, N297A, L328F, L3515, T366R, L368H, P395K, 5267E/L328F and L3515/T366R/L368H/P395K, by EU numbering.
218. A construct of any of claims 1-217 that is or comprises a TNFR2 agonist construct and that selectively activates or agonizesTNFR2, without activating or antagonizing TNFR1, comprising:
a) a TNFR2 agonist;
b) one or more linkers; and c) an activity modifier that is a half-life extending moiety, wherein:

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:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III), wherein MD is a multimerization domain that is/are the same or different;
TNFmut is a TNFR2-selective TNF mutein; and L1, L2 and L3 are linkers that can be the same or different.
219. The construct of claim 218, wherein the TNF muteins 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/A1451/E146G/5147D, A145H/E146S/5147D, A145H/S147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A145S, S95C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2.
220. The construct of claim 218, wherein the TNF muteins comprise the TNFR2-selective mutations D143N/A145R.
221. The construct of any of claims 218-220, wherein the multimerization domain 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).
222. The TNFR2 agonist construct of any of claims 218-221, wherein the multimerization domain is an IgG1 Fc or an IgG4 Fc and wherein the IgG1 Fc or IgG4 Fc also is the half-life extending moiety.
223. The TNFR2 agonist construct of any of claims 218-222, wherein the L1, L2 and/or L3 linkers are independently selected from among (GGGGS)n, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812).
224. The TNFR2 agonist construct of any of claims 218-223, wherein the linker between the TNFR2 agonist and the half-life extending moiety is:
a GS linker selected from among (GlySer),, where n= 1-10; (G1ySer2);
(G1y4Ser),, where n= 1-10; (G1y3Ser),, where n= 1-5; (SerG1y4),, where n= 1-5;
(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; or 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; or a combination thereof
225. The TNFR2 agonist construct of any of claims 218-224, wherein the half-life extending moiety is selected from among:
an IgG1 Fc that is the Fc of human IgG1, 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; and human serum albumin (HSA).
226. A construct of any of claims 1-217 that is or comprises a TNFR2 agonist construct, comprising:
a) the construct has the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III), wherein MD is a multimerization domain; TNFmut is a TNFR2-selective TNF
mutein; and L1, L2 and L3 are linkers that can be the same or different, wherein:
i) the 1VID 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);
ii) L1, L2 and L3 each are (GGGGS),, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a mixture thereof;
and iii) the TNF muteins comprise the TNFR2-selective mutations D143N/A145R;
b) a half-life extending moiety selected from among:
an IgG1 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; and human serum albumin (HSA); and c) a linker between the TNFR2-selective agonist and the half-life extending moiety, wherein the linker comprises:
a GS linker selected from among (GlySer),, where n= 1-10; (G1ySer2);
(G1y4Ser),, where n= 1-10; (G1y3Ser),, where n= 1-5; (SerG1y4),, where n= 1-5;

(GlySerSerGly)., where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; or 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; or a combination thereof
227. A construct of any of claims 1-217 that is TNFR2 agonist construct, comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III), wherein MD is a multimerization domain; TNFmut is a TNFR2-selective TNF
mutein; and L1, L2 and L3 are linkers that can be the same or different, and wherein:
i) the 1VID is selected from an IgG1 Fc or an IgG4 Fc;
ii) L2 and L3 in Formula II, and L1 and L2 in Formula III each independently is (GGGGS)., where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a combination thereof;
iii) each of Ll in Formula II and L3 in Formula III is independently selected from among:

a GS linker selected from among (GlySer),, where n= 1-10;
(G1ySer2); (G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5;
(SerG1y4),, where n= 1-5; (GlySerSerGly),, where n= 1-5;
GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG;
GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG;
GGSSGGSSGGGSSGGSSG; and GSSSGS; or 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; or a combination thereof; and iv) the TNF muteins comprise the TNFR2-selective mutations D143N/A145R.
228. The construct of claim 227, wherein the MD is selected from:
an IgG1 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.
229. The construct of claim 227 or claim 228, wherein the MD is the IgG1 Fc of trastuzumab, and the linker between the 1VID and the adjacent TNF mutein is all or a portion of the hinge sequence of trastuzumab, corresponding to residues of SEQ ID NO:26.
230. The construct of claim 227 or claim 228, wherein the MD is the IgG1 Fc of trastuzumab, and the linker between the 1VID and the adjacent TNF mutein comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO:31).
231. The construct of any of claims 227-230 wherein the MD is the IgG1 Fc of trastuzumab, and the linker between the MD and the adjacent TNF mutein comprises (G1y4Ser)3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26.
232. The construct of claim 227 or claim 228, wherein the MD is the IgG1 Fc of trastuzumab, and the linker between the 1VID and the adjacent TNF mutein comprises (G1y4Ser)3 and SCDKTH (residues 222-227 of SEQ ID NO:31).
233. The construct of claim 227 or claim 228 wherein the 1VID is the IgG4 Fc of nivolumab, and the linker between the 1VID 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.
234. The construct of claim 227 or claim 228, wherein the MD is the IgG4 Fc of nivolumab, and the linker between the 1VID and the adjacent TNF mutein comprises (G1y4Ser)3 and all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29.
235. The construct of any of claims 1-234 that is an agonist construct, wherein the TNFR2 agonist is modified to eliminate immunogenic sequences or epitopes that are immunogenic in the subject.
236. The construct of claim 235, wherein the subject is a human.
237. The construct of any of claims 1-236, that is a TNFR2 agonist construct and that comprises 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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T3665, 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, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/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 IgG1 : L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, 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, 5228P/L235E, and 5228P/F234A/L235A, by EU numbering.
238. The construct of any of claims 1-237 that is TNFR2 agonist construct that comprises 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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T3665, 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, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance 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 increase or enhance immune effector functions is selected from among one or more of:
in IgG1 : S239D, 1332E, 5239D/I332E, 5239D/A330L/I332E, 5298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/5298A in the first heavy chain and 5239D/A330L/I332E in the second heavy chain;
L234Y/L235Q/G236W/5239M/H268D/D270E/5298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/5267A/H268A/D270A/K326A/5337A;
T256A/K290A/5298A/E333A/K334A; G236A; G236A/I332E;
G236A/5239D/I332E; G236A/5239D/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/5324T; 5267E; H268F;
5324T; 5267E/H268F/5324T; G236A/I332E/5267E/H268F/5324T;
E345R; and E345R/E430G/5440Y; by EU numbering.
239. The TNFR2 agonist construct of any of claims 1-238 that is a TNFR2 agonist construct that comprises an IgG1 Fc that is modified to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb.
240. The TNFR2 agonist construct of claim 239, wherein the modifications that increase binding to FcyRIIb are selected from among one or more of 5267E, N297A, L328F, L3515, T366R, L368H, P395K, 5267E/L328F and L3515/T366R/L368H/P395K, by EU numbering.
241. A construct of any of claims 1-240 that is a multi-specific TNFR1 inhibitor/TNFR2 agonist construct, and is of formula:
(TNFR1 inhibitor)n ¨ Linker (L)p ¨ (TNFR2 agonist)q (Formula I), or (TNFR1 inhibitor)n ¨ Linker (L)p ¨(TNFR2 agonist)q, or (TNFR1 inhibitor), ¨ (TNFR2 agonist)q¨ Linker (L)p, or (TNFR2 agonist)q¨ (TNFR1 inhibitor), ¨ Linker (L)p, or any of the above, comprising an optional activity modifier, wherein:
n= 1 or 2, p= 1, 2, or 3, and q= 1 or 2;
the TNFR1 inhibitor interacts with TNFR1 to inhibit its activity;
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; and the linker increases solubility of the construct, or increases flexibility, or alters steric effects of the construct.
242. The construct of any of claims 1-241 that is a multi-specific TNFR1 inhibitor/TNFR2 agonist construct, wherein:
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.
243. The construct of claim 241 or claim 242, wherein:
a) the TNFR1 inhibitor is selected from among:
i) an antigen-binding fragment of a human anti-TNFR1 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 NOs: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
mutein comprising a soluble TNF molecule, with one or more amino acid replacements that confer selective inhibition of TNFR1 and are selected from among:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A845, V85T, 586T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L295/R32W, L295/586T, R32W/586T, L295/R32W/586T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A845/V85T/586T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF, set forth in SEQ ID NO:2;
b) the linker is selected from:
i) a GS linker selected from (GlySer)n, where n= 1-10; (G1ySer2);
(G1y4Ser)n, where n= 1-10; (G1y3Ser)n, where n= 1-5; (SerG1y4)n, where n= 1-5; (GlySerSerGly)n, where n= 1-5; GSGGSSGG; GSSSGSGSGSSG;
GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG;
GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS;
and/or ii) all or a portion of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26, or all or a portion of the hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO:29;
and iii) an IgG1 or IgG4 Fc, wherein:
the IgG1 Fc is selected from the IgG1 Fc of human IgGl, set forth in SEQ ID NO:10, or the IgG1 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 modifications to introduce knobs-into-holes, and/or increase or enhance neonatal Fc receptor (FcRn) recycling, and/or reduce or eliminate immune effector functions; and c) the TNFR2 agonist is selected from:
i) an antigen-binding fragment 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; or ii) an antigen-binding fragment of an agonistic human anti-TNFR2 antibody selected from 1VIR2-1 or MAB2261; or iii) 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/5147T, Q88N/T895/A145S/E146A/5147D, Q88N/A1451/E146G/5147D, A145H/E146S/5147D, A145H/S147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/5147D, D143V/F144L/A145S, S95C/G148C, and D143V/A1455, with reference to SEQ ID NO:2; or iv) a single-chain TNFR2-selective TNF mutein trimer, comprising the mutations D143N/A145R, wherein the TNF muteins are linked by (GGGGS)n, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID
NO:812); or v) a TNFR2-selective agonist comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III);
whereby 1VID is a multimerization domain; TNFmut is a TNFR2-selective TNF mutein; and L1, L2 and L3 are linkers that can be the same or different, and wherein:
the MD is selected from EHD2 (SEQ ID NO:808), MEID2 (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);
L1, L2 and L3 each are (GGGGS),, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a mixture thereof; and the TNF muteins comprise the TNFR2-selective mutations D143N/A145R.
244. The construct of claim 241 or claim 242 that is a multi-specific TNFR1 antagonist/TNFR2 agonist construct, wherein:
a) 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 NOs: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-selective mutations selected from among K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A1451/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2.
245. The construct of claim 241 or claim 242, wherein:
a) 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 NOs: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, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A145S/E146A/5147D, Q88N/A1451/E146G/5147D, .. A145H/E146S/5147D, A145H/S147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2.
246. The construct of claim 241 or claim 242 wherein:
a) 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 NOs: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, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A1451/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A145S, S95C/G148C, and D143V/A1455, with reference to SEQ ID
NO:2.
247. The construct of claim 241 or claim 242, wherein:
a) 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 NOs: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, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A1451/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/5147D, A145K/E146D/5147T, A145R/E146T/5147D, A145R/5147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, and any combination of .. the preceding mutations, with reference to SEQ ID NO:2.
248. The construct of any of claims 241-247 that comprises 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.
249. The construct of claim 248, wherein the Fc comprises knobs-into-holes modifications:
the knob mutation is selected from among one or more of 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T3665, L368A, F405A, Y407T, Y407A, and Y407V by EU numbering.
250. The construct of claim 248, wherein the 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, N4345, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering.
251. The construct of claim 248, wherein the Fc comprises 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).
252. The construct of claim 248 that comprises modification(s) to reduce or eliminate immune effector functions that are selected from among one or more of:
in IgG1: L235E, L234A/L235A, L234E/L235F/P3315, L234F/L235E/P3315, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, 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, 5228P/L235E, and 5228P/F234A/L235A, by EU numbering.
253. The construct of any of claims 241-247, 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 5354C, T366Y, T366W, and T394W by EU numbering; and the hole mutation is selected from among one or more of Y349C, T3665, 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, 5254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/5254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N4345, V2591/V308F, V2591/V308F/M428L, E294de1/T307P/N434Y, and T256N/A378V/5383N/N434Y, by EU numbering; and c) a modification(s) to increase or enhance 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 increase or enhance immune effector functions is selected from among one or more of:
in IgG1 : 5239D, 1332E, 5239D/I332E, 5239D/A330L/I332E, 5298A/E333A/K334A; F243L/R292P/Y300L/V3051/P396L;
L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L;
L234Y/G236W/5298A in the first heavy chain and 5239D/A330L/I332E in the second heavy chain;
L234Y/L235Q/G236W/5239M/H268D/D270E/5298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/5267A/H268A/D270A/K326A/5337A;
T256A/K290A/5298A/E333A/K334A; G236A; G236A/I332E;
G236A/5239D/I332E; G236A/5239D/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/5324T; 5267E; H268F;
5324T; 5267E/H268F/5324T; G236A/I332E/5267E/H268F/5324T;
E345R; and E345R/E430G/5440Y; by EU numbering.
254. The construct of any of claims 241-253, wherein the construct that comprises an IgG1 Fc that is modified to increase binding to the inhibitory Fcy receptor (FcyR) FcyRIIb.
255. The construct of claim 254, wherein the modifications that increase binding to FcyRIlb are selected from among one or more of S267E, N297A, L328F, L3515, T366R, L368H, P395K, 5267E/L328F and L3515/T366R/L368H/P395K, by EU numbering.
256. The construct of any of claims 1-255 that is a multi-specific TNFR1 antagonist/TNFR2 agonist, wherein:
the TNFR1 antagonist is monovalent; and the TNFR2 agonist is monovalent.
257. The construct of any of claims 1-255 that is a multi-specific TNFR1 antagonist/TNFR2 agonist, wherein:
the TNFR1 antagonist is monovalent; and the TNFR2 agonist is bivalent.
258. The construct of any of claims 241-257 that is a multi-specific TNFR1 antagonist/TNFR2 agonist, wherein:
a) the TNFR1 antagonist is selected from:
i) an antigen-binding fragment of a human anti-TNFR1 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 NOs: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 TNFR1 and are selected from among:
V1M, L295, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A845, V85T, 586T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L295/R32W, L295/586T, R32W/586T, L295/R32W/586T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF, set forth in SEQ ID NO:2;
b) the linker is a branched chain PEG molecule that is at least or at least about 30 kDa in size; and c) the TNFR2 agonist is selected from:
i) an antigen-binding fragment 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; or ii) an antigen-binding fragment of an agonistic human anti-TNFR2 antibody selected from 1VIR2-1 or MAB2261; or iii) 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/5147T, Q88N/T895/A1455/E146A/5147D, Q88N/A1451/E146G/5147D, A145H/E1465/5147D, A145H/5147D, L29V/A145D/E146D/5147D, A145N/E146D/5147D, A145T/E1465/5147D, A145Q/E146D/5147D, A145T/E146D/5147D, A145D/E146G/5147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/5147D, D143V/F144L/A1455, 595C/G148C, and D143V/A1455, with reference to SEQ ID NO:2; or iv) a single-chain TNFR2-selective TNF mutein trimer, comprising the mutations D143N/A145R, wherein the TNF muteins are linked by (GGGGS)n, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID
NO:812); or v) a TNFR2-selective agonist comprising the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III);
whereby 1VID is a multimerization domain; TNFmut is a TNFR2-selective TNF mutein; and L1, L2 and L3 are linkers that can be the same or different, and wherein:

the MD is selected from EHD2 (SEQ ID NO:808), IVIEID2 (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);
L1, L2 and L3 each are (GGGGS),, where n = 1-5, or all or a portion of the stalk region of TNF (SEQ ID NO:812), or a mixture thereof; and the TNF muteins comprise the TNFR2-selective mutations D143N/A145R.
259. The construct of claim 258 wherein each of the TNFR1 antagonist and TNFR2 agonist is monovalent.
260. The construct of claim 258 wherein the TNFR1 antagonist is monovalent, and the TNFR2 agonist is bivalent.
261. The construct of any of claims 1-260 that is a 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.
262. Use of the construct of any of claims 1-260 that is a multi-specific TNFR1 antagonist/TNFR2 agonist, 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.
263. A composition, comprising a construct of any of claims 1-260 in a pharmaceutically acceptable carrier or vehicle.
264. The composition of claim 263, 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.
265. The construct of any of claims 1-261 or the use of claim 262, or the composition of claim 263 or claim 264, wherein the chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or the disease, condition or disorder characterized by overexpression of TNF or deregulated TNFR1 signaling in its etiology is selected from:
rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), spondyloarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), uveitis, fibrotic diseases, endometriosis, lupus, multiple sclerosis (MS), congestive heart failure, cardiovascular disease, myocardial infarction (MI), atherosclerosis, metabolic diseases, cytokine release syndrome, septic shock, sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), SARS-CoV-2, influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, Alzheimer's disease, Parkinson's disease, Behcet's disease, Dupuytren's disease, Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS), pancreatitis, type I diabetes, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, graft rejection, graft versus host disease (GvHD), lung inflammation, pulmonary diseases and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, genetically inherited diseases with TNF/TNFR1 as the causative pathologic mediator, periodic fever syndrome, or cancer.
266. The construct of any of claims 1-261 or the use of claim 262, or the composition of claim 263 or claim 264, for use in the treatment of rheumatoid arthritis.
267. Use of the construct of any of claims 1-261 or the use of claim 262, or the composition of claim 263 or claim 264 for the treatment of rheumatoid arthritis.
268. A construct that is a TNFR2 antagonist construct that comprises a TNFR2 antagonist, and optionally a linker and optionally an activity modifier.
269. The construct of claim 268 that comprises formula 5:
(TNFR2 antagonist).¨linkerp¨ (activity modifier)q, or linkup¨ (activity modifier)q_(TNFR2 antagonist)., 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 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; and 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.
270. The construct of claim 268 or claim 269, wherein each of the activity modifier and linker is as defined and described for the constructs in any of claims 1-250.
271. The construct of any of claims 268-270, wherein TNFR2 antagonist:
reduces and/or inhibits the proliferation of myeloid-derived suppressor cells (MDSCs); and/or induces apoptosis within MDSCs, by binding TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment; and/or induces the expansion of T effector cells, including cytotoxic CD8+ T cells, via the inhibition of Treg expansion and activity.
272. The construct of any of claims 268-271, wherein the TNRF2 antagonist is an antibody, antigen-binding fragment thereof, or single chain antibody that binds 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 VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO:4), and/or an RECTIFIED SHEET (RULE 9 1) ISA/EP

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.
273. The construct of any of claims 268-272, wherein 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).
274. The construct of any of claims 268-273, wherein 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. 2019/0144556 for descriptions of these antibodies);
antibodies and antibody fragments and single chain forms 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, 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.
275. The construct of any of claims 268-274, wherein 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).
276. The construct of any of claims 268-275, wherein the TNFR2 antagonist is an antibody, fragment thereof, or single chain form thereof that contains one 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-L2 sequence of TNFRAB1, corresponding to residues 49-55 of SEQ ID NO:1213; and/or the CDR-L3 sequences set forth in any of SEQ ID NOs: 1221, 1222, and 1241-1244, or the CDR-L3 sequence of TNFRAB1, corresponding to residues 88-96 of SEQ ID NO:1213;
and/or CDR-H1 and CDR-H2 sequences of the consensus sequence of a human antibody heavy chain variable domain of SEQ ID NO:1245 replaced with the corresponding CDR sequences of a phenotype-neutral, TNFR2-specific antibody, and/or the CDR-L1, CDR-L2 and CDR-L3 sequences of the sequence of a human antibody light chain variable domain of SEQ ID NO:1246 replaced with the corresponding CDR sequences of a phenotype-neutral, TNFR2-specific antibody, to produce humanized, antagonistic TNFR2 antibodies.
277. The construct of any of claims 268-275, wherein the TNFR2 antagonist specifically binds to an epitopes within TNFR2 set forth in any one of SEQ
ID NOs:1247-1464.
278. The construct of any of claims claim 268-277, wherein TNFR2 antagonist specifically binds to an epitope(s) selected from among:
(a) one or more 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, and do not bind to the epitope containing residues KCSPG corresponding to residues 56-60 of SEQ ID NO:4;
and/or (b) one or more TNFR2 epitopes comprising the sequence of amino acids comprising:
RECTIFIED SHEET (RULE 9 1) ISA/EP

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 NO:4.
279. The construct of any of claims 268-278 that comprises a TNFR2 .. antagonist that is a small molecule.
280. The construct of claim 279, wherein the TNFR2 antagonist is thalidomide or an analog thereof.
281. The construct of claim 280, wherein the thalidomide analog is lenalidomide and pomalidomide.
282. The construct of any of claims 268-281, comprising a TNFR2 antagonist that that reduces FoxP3 expression and inhibits the suppressive activity of Tregs.
283. The construct of claim 282, wherein the TNFR2 antagonist is a histone deacetylase inhibitor that can reduce FoxP3 expression and inhibit the suppressive activity of Tregs.
284. The construct of claim 282 or claim 283, wherein the inhibitor is panobinostat or cyclophosphamide or Triptolide.
285. The construct of any of claims 268-282 for use for treatment of infectious diseases, and cancers that express TNFR2.
286. The construct of claim 285, wherein the cancer 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.
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